Chemical probes for left-handed DNA and chiral metal complexes as Z-specific anti-tumor agents

This invention concerns a coordination complex and salts and optically resolved enantiomers thereof, of the formula (R).sub.3 --M, wherein R comprises 1,10-phenanthroline or a substituted derivative thereof, M comprises a suitable transition metal, e.g. ruthenium(II) or cobalt(III), and R is bonded by M by a coordination bond. The complexes of this invention are useful in methods for labeling, nicking and cleaving DNA. The lambda enantiomer of complexes of this invention is useful in methods for specifically labeling, detecting, nicking and cleaving Z-DNA. The complexes may also be used in a method for killing tumor cells and may be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition for the treatment of tumor cells in a subject. The invention further concerns methods for treating a subject afflicted with tumor cells.

BACKGROUND OF THE INVENTION 
Much of the information set forth herein has been published. See Barton, J. 
K. et al., J. Am. Chem. Soc., 1984, 106: 2172-2176 (Apr. 6, 1984); Barton, 
J. K. and Raphael, A., J. Am. Chem. Soc., 1984, 106: 2466-2468 (Apr. 18, 
1984); Barton, J. K. et al., Proc. Natl. Acad. Sci. USA, 1984, 
81:1961-1965 (Apr. 27, 1984); and Barton, J. K., J. Biom. Structure and 
Dynam., 1983, 1: 621-632 (Jan. 18, 1984). The above-mentioned papers were 
distributed by the respective publishers on the dates provided in 
parentheses. 
Throughout this application various publications are referenced by arabic 
numerals within parentheses. Full citations for these publications may be 
found at the end of the specification immediately preceding the claims. 
The disclosures of these publications in their entireties are hereby 
incorporated by reference into this application in order to more fully 
describe the state of the art to which this invention pertains. 
The binding of heterocyclic compounds to DNA by intercalation, where the 
planar aromatic cation stacks between adjacent base pairs of the duplex 
(1), has been the subjected of considerable investigation (2-4). 
Intercalative drugs can be strongly mutagenic, and some, as adriamycin and 
daunomycin, serve as potent chemotherapeutic agents (5). The small 
intercalators such as ethidium or proflavine in addition provide useful 
chemical probes of nucleic acid structure (6). Metallointercalators have 
been particularly useful in probing DNA structure and the intercalation 
process itself, because the ligands or metal may be varied in an easily 
controlled manner to facilitate the individual application (7). 
The aromatic chromophore of the intercalative cation can provide a 
sensitive handle to monitor the conformation and flexibility of the helix. 
Many intercalators show antibacterial or anticancer activity and, because 
the inserted residue often resembles a base pair in shape and thickness, 
intercalators are commonly frame shaft mutagens. (3) Intercalation appears 
to require, simply, a planar heterocyclic residue, (4) and in fact 
cationic metal complexes which contain aromatic ligands bind to DNA by 
intercalation as well. (5) Platinum intercalators have uniquely provided 
electron dense probes for x-ray diffraction experiments. (6) Moreover the 
metallointercalation reagents have offered particular experimental 
flexibility in that both the metal and accessory ligands may be varied for 
individual applications. Comparisons of the binding of the intercalative 
2,2'-bipyridylplatinum(II) reagent with the analogous nonintercalating 
bis(pyridine)platinum(II) species by fiber X-ray diffraction methods, for 
example, demonstrated quite simply the requirement for ligand planarity in 
the intercalation process (8). 
The original studies of metallointercalators centered on square-planar 
platinum(II) complexes containing aromatic terpyridyl or phenanthroline 
ligands (9), and single-crystal studies of terpyridylplatinum(II) species 
stacked with nucleotides showed the platinum complex to insert almost 
fully between the base pairs (10, 11). More recently the reagent 
methidiumpropyl-Fe(II)EDTA, which contains a redox-active metal center 
tethered to an organic intercalator, has been applied in "footprinting" 
experiments to determine the sequence specificities of small drugs bound 
to DNA (12). Bis(phenanthroline)-cuprous ion (13) has similarly been 
employed in DNA cleavage experiments (14), and this reagent also 
presumably binds initially to the DNA by intercalation. 
(3,5,6,8-tetramethyl-1,10-phenanthroline).sub.3 Ru(II) has been reported 
and its use against influenza virus, fungus, yeast and leukemia 
investigated (98). The ability of the tris tetramethyl complex to bind to 
or cleave DNA has not been reported. Furthermore, enantiomers of that 
complex have not been separated and their respective properties compared. 
Reagents of high specificity and even stereoselectivity would be desirable 
in the design both of potent drugs and of structural probes. For the 
chiral complex (phen).sub.3 Zn.sup.2+ (phen=1,10-phenanthroline) an 
enantiomeric preference in binding to B-DNA has been observed (15). As for 
the tetrahedral (phen).sub.2 Cu.sup.+ complex, and in contrast to the 
square-planar platinum intercalators, the octahedral coordination in the 
tris(phenanthroline) metal cations can permit a partial insertion of only 
one coordinated ligand. Thus while one ligand is stacked between base 
pairs, the remaining nonintercalated phenanthroline ligands should be 
available to direct the enantiomeric selection. 
The left-handed DNA helix has received considerable attention since the 
original crystallographic study of the Z-DNA fragment [d(CpG)].sub.6 (16). 
Solution conditions that include high ionic strength (17), hydrophobic 
solvents (18), the presence of certain trivalent cations (19), or covalent 
modification with bulky alkylating agents (19-23) all facilitate the 
transition of a right-handed double helix into a left-handed form. This 
striking conformational transition was first observed for poly[d(G-C)] 
(17). More recently, the alternating purine-pyrimidine sequence 
[d(G-T)].sub.n .multidot.[d(C-A].sub.n has been shown to form Z-helices as 
well (24, 25). Methylation of cytosine residues at carbon-5 lends 
stability to Z-form DNA (19, 26) and, under physiological conditions, 
transitions to a left-handed structure can occur to relieve the torsional 
strain in underwound negatively supercoiled DNA (27-29). These latter 
findings suggest mechanisms for left-handed DNA formation in the cell, 
where such structures could be important in controlling gene expression. 
Negatively supercoiled simian virus 40 DNA, for example, has been found to 
contain potentially Z-DNA-forming alternate purine-pyrimidine regions 
within transcriptional enhancer sequences (30). 
To explore any biological role for left-handed DNA, sensitive and selective 
probes are required. Assays of superhelix unwinding, NMR experiments and 
circular dichroism have been used in detecting Z-DNA. These methods, 
however, are indirect, do not assay for helix handedness, and require 
large quantitites of material. More recently antibodies to Z-DNA have been 
elicited. The antibodies provide a more sensitive means of detection. 
Z-DNA appears to be a strong immunogen. Anti-Z-DNA antibodies have been 
elicited with both brominated poly[d(G-C)] (31) and poly[d(G-C)] modified 
with diethylenetriamineplatinum(II) (32) as antigens. The structures of 
Z-DNA and in particular of a modified Z-form provide a multitude of 
antigenic characteristics: the left-handed helicity, the zigzag 
dinucleotide phosphate repeat, the protruding purine substituents in the 
shallow major groove. It is not surprising then that the various 
antibodies obtained appear specific for different localized features of 
Z-DNA (33). The development of a specific chemical probe, so designed as 
to recognize a known structural element of Z-DNA, e.g. the helix 
handedness, would offer a simple complementary approach but has not 
heretofore been reported. 
Enantiomeric selectivity has been observed in the interactions of 
tris(phenanthroline) metal complexes with B-DNA (15, 35-35). Experiments 
with tris(phenanthroline)zinc(II) have indicated stereoselectivity (15); 
dialysis of B-DNA against the racemic mixture leads to the optical 
enrichment in the .LAMBDA. enantiomer. Subsequent luminescene, 
electrophoretic, and equilibrium dialysis studies of the 
well-characterized ruthenium(II) analogues have shown that the 
tris(phenanthroline) metal isomers bind to DNA by intercalation and it is 
the .DELTA. enantiomer that binds preferentially to a right-handed duplex 
(34, 35). The enantiomeric selectively is based on steric interactions 
between the nonintercalated phenanthroline ligands and the phosphate 
backbone. Although the right-handed propeller-like isomer intercalates 
with facility into a right-handed helix, steric repulsions interfere with 
a similar intercalation of the .LAMBDA. enantiomer. 
Based on this premise, tris(phenanthroline) metal complexes appear useful 
in the design of probes to distinguish left-handed and right-handed DNA 
duplexes. The design flexibility inherent in metallointercalation 
reagents, in which both ligand and metal may be varied easily, makes the 
coordination complexes attractive probes (7, 8, 35). We have concentrated 
here on phenanthroline complexes of ruthenium(II) because of the high 
luminescence associated with their intense metal-to-ligand charge-transfer 
band (37, 38) and because the exchange-inert character of the low-spin 
d.sup.6 complexes limits racemization (20). 
Furthermore, there has been considerable interest in DNA endonucleolytic 
cleavage reactions that are activated by metal ions, (39, 40) both for the 
preparation of "footprinting" reagents (41) and as models for the 
reactivity of some antitumor antibiotics, notably bleomycin (42) and 
streptonigrin. (43) The features common to these complexes are that the 
molecule has a high affinity for double-stranded DNA and that the molecule 
binds a redox-active metal ion cofactor. The delivery of high 
concentrations of metal ion to the helix, in locally generating oxygen or 
hydroxide radicals, yields an efficient DNA cleavage reaction. 
Additionally, cobalt(III) bleomycin cleaves DNA in the presence of light. 
(44) 
SUMMARY OF THE INVENTION 
This invention concerns a coordination complex or salt thereof having the 
formula (R).sub.3 --Co(III), wherein R comprises 1,10-phenanthroline or a 
substituted derivative thereof and R is bound to Co by a coordination 
bond. 
One embodiment concerns a method for labeling DNA with a complex of the 
formula (R).sub.3 --M, wherein R comprises 1,10-phenanthroline or a 
substituted derivative thereof, M comprises a suitable transition metal, 
and R is bonded to M by a coordination bond. In this and other embodiments 
a suitable transition metal is one which is capable of forming an 
octahedral complex with R, such as ruthenium (II) or cobalt (III). The 
invention also concerns a DNA molecule labeled with a complex of the 
formula (R).sub.3 --M, as defined above, wherein the complex is bound to 
the DNA by intercalation. 
Another embodiment of this invention is a method for selectively labeling 
Z-DNA with the lambda enantiomer of a complex of the formula (R).sub.3 
--M, as defined above. This method comprises contacting the lambda 
enantiomer of the complex under suitable conditions such that the complex 
binds to the Z-DNA. The invention further involves a labeled DNA molecule 
comprising Z-DNA and the lambda enantiomer of a complex of the formula 
(R).sub.3 --M, as defined above, the complex being bound to the Z-DNA. 
Another embodiment of this invention concerns a method for detecting the 
presence of Z-DNA. This method involves selectively labeling Z-DNA 
according to the above-mentioned method and detecting the presence of the 
complex bound to the Z-DNA. 
Still another embodiment of this invention is a method for nicking 
double-stranded DNA by effecting breakage of at least one phosphodiester 
bond along the DNA. The method involves contacting the DNA with a cobalt 
(III)-containing complex of this invention under suitable conditions such 
that the complex intercalates into the DNA to form an adduct and 
irradiating the adduct so formed with a sufficient dose of ultraviolet 
radiation of an appropriate wavelength so as to nick the DNA at the 
site(s) of intercalation. This invention further involves a method for 
cleaving double-stranded DNA which comprises nicking the DNA by the 
above-mentioned method and treating the nicked DNA so produced with a 
suitable enzyme capable of cleaving single-stranded DNA under effective 
conditions to cleave the nicked DNA at the site of the nick(s). 
An additional embodiment of this invention is a method for selectively 
nicking Z-DNA by effecting breakage of at least one phosphodiester bond 
along the Z-DNA. The method involves contacting a DNA molecule containing 
a Z-DNA sequence with a lambda enantiomer of a cobalt (III)-containing 
complex of this invention under suitable conditions such that the complex 
binds to the Z-DNA to form an adduct and irradiating the adduct so formed 
with a sufficient dose of ultraviolet radiation of an appropriate 
wavelength so as to nick the DNA at the binding site(s). Double-stranded 
Z-DNA may be selectively cleaved by selectively nicking the Z-DNA by the 
above-mentioned method and treating the nicked DNA so produced with a 
suitable enzyme capable of cleaving single-stranded DNA under effective 
conditions to cleave the nicked double-stranded DNA at the site of the 
nick(s). 
This invention further concerns a method for killing a portion of a 
population of appropriate tumor cells. The method involves contacting the 
tumor cells under suitable conditions with an effective amount of the 
lambda enantiomer of a coordination complex of the formula (R).sup.3 --M, 
as previously defined, to kill the tumor cells. In another embodiment a 
racemic cobalt(III)-containing complex of the invention may similarly be 
used to kill tumor cells. When a cobalt (III)-containing complex is used, 
the method may further comprise irradiating the tumor cells with a 
suitable dose of ultraviolet radiation of an appropriate wavelength at a 
suitable time after the tumor cells have been contacted with the complex, 
permitting the complex to nick DNA. 
Still another embodiment of this invention is a pharmaceutical composition 
for the treatment of tumor cells in a subject. The pharmaceutical 
composition comprises a pharmaceutically acceptable carrier and an 
effective anti-tumor amount of a cobalt(III)-containing complex of the 
invention or of the lambda enantiomer of a complex of the formula 
(R).sub.3 --M, as defined previously. Such a composition may be used in a 
method for treating a subject afflicted with tumor cells so as to cause 
regression of the tumor cells. This method involves administering to the 
subject by a suitable route the pharmaceutical composition in an amount 
sufficient to cause regression of the tumor cells.

DETAILED DESCRIPTION OF THE INVENTION 
As mentioned above, this invention concerns a coordination complex or salt 
thereof having the formula (R).sub.3 --Co(III), wherein R comprises 
1,10-phenanthroline or a substituted derivative thereof and R is bound to 
Co by a coordination bond. A "substituted derivative" as the phrase is 
used herein is a compound obtained by replacing one or more hydrogen atoms 
present in 1,10-phenanthroline with one or more moieties having the 
characteristic that the complex containing the resulting compound binds to 
DNA. Merely by way of example, the substituted derivative of 
1,10-phenanthroline may be 4,7-diamino-1,10-phenanthroline; 
3,8-diamino-1,10-phenanthroline; 
4,7-dimethylenediamine-1,10-phenanthroline; 
3,8-diethylenediamine-1,10-phenanthroline; 
4,7-dihydroxyl-1,10-phenanthroline; 3,8-dihydroxyl-1,10-phenanthroline; 
4,7-dinitro-1,10-phenanthroline; 3,8-dinitro-1,10-phenanthroline; 
4,7-diphenyl-1,10-phenanthroline; 3,8-diphenyl-1,10-phenanthroline; 
4,7-dispermine-1,10-phenanthroline, or 3,8-dispermine-1,10-phenanthroline. 
Unless otherwise specified the complex of this invention is a racemic 
mixture of enantiomers. The invention also concerns the optically resolved 
delta and lamda isomers of the complex. 
One embodiment concerns a method for labeling DNA with a complex of the 
formula (R).sub.3 --M, wherein R comprises 1,10-phenanthroline or a 
substituted derivative thereof as defined above, M comprises a suitable 
transition metal, and R is bonded to M by a coordination bond. In this and 
other embodiments a suitable transition metal is one which is capable of 
forming an octahedral complex with R, such a ruthenium (II) or cobalt 
(III). The labeling method involves contacting the DNA with the complex 
under suitable conditions such that the complex binds to the DNA, e.g. by 
intercalation. 
The invention also concerns a DNA molecule labeled with a complex of the 
formula (R).sub.3 --M, as defined above, and the complex is bound to the 
DNA, e.g by intercalation. Preferably the labeled DNA molecule is produced 
by the above-described method. 
A further embodiment is a method for selectively labeling Z-DNA with the 
lambda enantiomer of a complex of the formula (R).sub.3 --M, as defined 
above. This method comprises contacting the DNA with the lambda enantiomer 
of the complex under suitable conditions such that the complex binds to 
the Z-DNA. The invention further involves a labeled DNA molecule 
comprising Z-DNA and the lambda enantiomer of a complex of the formula 
(R).sub.3 --M, as defined above, the complex being bound to the Z-DNA. 
Preferably the labeled DNA molecule is produced by the above-described 
method. 
Another embodiment of this invention concerns a method for detecting the 
presence of Z-DNA. This method involves selectively labeling Z-DMA 
according to the above-mentioned method and detecting the presence of the 
complex bound to the Z-DNA, e.g. by spectroscopic methods (See Experiments 
hereinafter). 
Still another embodiment of this invention is a method for nicking 
double-stranded DNA by effecting single-stranded scission, i.e., breakage 
of at least one phosphodiester bond along the DNA. The method involves 
contacting the DNA with a cobalt (III)-containing complex of this 
invention under suitable conditions such that the complex binds to the 
DNA, e.g. by intercalation, to form an adduct and irradiating the adduct 
so formed with a sufficient dose of ultraviolet radiation of an 
appropriate wavelength so as to nick the DNA at the site(s) of 
intercalation. An appropriate ultraviolet wavelength in this an other 
embodiments is a wavelength which is absorbed by the ligand bands of the 
complex used. As described hereinafter, the ligand band absorption of a 
complex of this invention may be determined spectroscopically by 
conventional methods. 
This invention further involves a method for cleaving double-stranded DNA 
which comprises nicking the DNA by the above-mentioned method and treating 
the nicked DNA so produced with a suitable enzyme capable of cleaving 
single-stranded DNA under effective conditions to cleave the nicked 
double-stranded DNA at the site of the nick(s). By this method 
double-stranded scission of the DNA is effected. Suitable enzymes for 
effecting double-stranded cleavage of nicked DNA in this and other 
embodiments included those which are not deactivated in the presence of 
the complex used for DNA nicking, e.g. S1 nuclease. 
An additional embodiment of this invention is a method for selectivity 
nicking Z-DNA by effecting breakage of at least one phosphodiester bond 
along the Z-DNA. The method involves contacting a DNA molecule containing 
a Z-DNA sequence with the lambda enantiomer of a cobalt (III)-containing 
complex of this invention under suitable conditions such that the complex 
binds to the Z-DNA to form an adduct and irradiating the adduct so formed 
with a sufficient dose of ultraviolet radiation of an appropriate 
wavelength, as previously defined, so as to nick the DNA at the binding 
site(s). Double-stranded Z-DNA may be selectively cleaved by selectively 
nicking the Z-DNA by the above-mentioned method and treating the nicked 
DNA so produced with a suitable enzyme capable of cleaving single-stranded 
DNA under effective conditions to cleave the nicked DNA at the site of the 
nick(s). 
This invention further concerns a method for killing a portion of a 
population of appropriate tumor cells. The method involves contacting the 
tumor cells under suitable conditions with an effective amount of the 
lambda enantiomer of a coordination complex of the formula (R).sup.3 --M, 
as previously defined, to kill the tumor cells. Alternatively, a racemic 
cobalt(III)-containing complex of this invention may be similarly used. 
When a cobalt (III)-containing complex is used, the method may further 
comprise irradiating the tumor cells with a suitable dose of ultraviolet 
radiation of an appropriate wavelength at a suitable time after the tumor 
cells have been contacted with the complex, permitting the complex to nick 
DNA. 
Still another embodiment of this invention is a pharmaceutical composition 
for the treatment of tumor cells in a subject e.g. a human or animal. The 
pharmaceutical composition comprises an effective anti-tumor amount of the 
lambda enantiomer of a complex of the formula (R).sub.3 --M, as defined 
previously, and a pharmaceutically acceptable carrier. Again, a racemic 
cobalt(III)-containing complex may be used alternatively. Preferably the 
complex is a cobalt(III)-containing complex. Suitable carriers include 
sterile saline or buffer-containing solutions or other carriers known in 
the art such as those used with cisplatin. 
Such a composition may be used in a method for treating a subject, e.g. a 
human or animal, afflicted with tumor cells so as to cause regression of 
the tumor cells. This method involves administering to the subject by a 
suitable route the pharmaceutical composition in an amount sufficient to 
cause regression of the tumor cells. Suitable routes of administration 
include parenteral administration and topical administration, e.g. in 
cases such as skin cancers where the tumor cells are located on or near an 
exposed surface of the subject. Furthermore, if the complex used is a 
cobalt(III)-containing complex, the method may additionally involve 
irradiating the tumor cells with a suitable dose of ultraviolet radiation 
of an appropriate wavelength permitting the complex to nick DNA. In this 
method the irradiation should be conducted at a suitable time after 
administration of the compositon to the subject, i.e. to permit the 
complex to interact with the DNA. It should also be noted that optically 
resolved entantiomers of the complexes of this invention, specifically the 
lambda isomer, may provide superior results both in killing tumor cells 
and in treating subjects afflicted with tumor cells. 
EXPERIMENTAL DETAILS 
Octahedral complexes with three bidentate ligands like phenanthroline do 
not contain an inversion center, and therefore, as shown below, two 
enantiomeric forms are present. Note that the intercalating portion of the 
molecule, the phenanthroline ligand, is coordinated directly to the 
asymmetric center of the cation, the metal. Because this chiral metal 
center is really proximal to the site of intercalation, the interaction of 
these complexes with DNA provides a clear illustration of stereospecific 
drug binding to a similarly asymmetric DNA helix. Furthermore this 
stereospecific binding mode provides a means for designing probes for DNA 
helicity. 
##STR1## 
Ruthenium(II) complexes have been found useful because of (i) the 
kinetically inert character of the low-spin d.sup.6 species, (ii) their 
intense metal to ligand charge-transfer (MLCT) band in the visible 
spectrum and since (iii) many chemical and spectroscopic properties of the 
poly(pyridine) complexes have been established. The electronic structure 
of the ground and excited states of tris(bipyridine)ruthenium(II) has been 
examined in detail. (45) The strong visible absorption band, distinct from 
the absorption of DNA, in (phen).sub.3 Ru.sup.2+ as well as its high 
luminescence provide spectroscopic tools to monitor the intercalative 
process. (38, 46) Most importantly, in contrast to (phen).sub.3 Zn.sup.2+, 
which is somewhat labile, (47) the ruthenium(II) complexes are essentially 
inert to racemization. (48) 
Optical isomers of (phen).sub.3 Ru.sup.2+ may be isolated in pure form, 
(48, 49) and the absolute configurations have been assigned (50). Electric 
dichroism measurements of (phen).sub.3 Ru.sup.2+ bound to DNA have been 
conducted (51) and support the findings of enantiomeric selectivity. 
Although a preference in binding is found between enantiomers in the 
phenanthroline series, both isomers do in fact intercalate into the 
right-handed helix as discussed above. To amplify the chiral 
discrimination and hence improves the sensitivity of the chiral probe, 
suitable substituents, e.g. amino-, ethylenediamino-, hydroxyl-, nitro-, 
phenyl- or spermine-substituents, may be added to each phenanthroline 
ligand at appropriate ring sites, e.g. 3,8- or 4,7. Bulky substituents at 
the distal sites on the cation can block completely the intercalation of 
the isomer into a right-handed helix, and thus provides selective 
spectroscopic probes for the handedness of the DNA duplex. The structure 
of the left-handed enantiomer of a preferred complex, 
.LAMBDA.-tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) (RuDIP), 
which binds to left-handed Z-DNA but not to right-handed B-DNA, is shown 
below. 
##STR2## 
As for the smaller tris(phenanthroline) metal complexes, DNA binding is 
evident in the presence of racemic RuDIP. (52) The similarity to 
(phen).sub.3 Ru.sup.2+ in spectral characteristics on binding to DNA, i.e. 
hydrochromicty and luminescence enhancements, suggested that RuDIP also 
bonds to the duplex by intercalation. RuDIP is however substantially more 
bulky and hydrophobic than the parent (phen).sub.3 Ru.sup.2+ cation. 
Substitution of phenyl groups at the distal 4- and 7-positions leads to 
several significant perturbations. First, the solubility of the ruthenium 
complex in aqueous solution is diminished appreciably, which is a 
practical consideration. Second, stacking with the base pairs in the helix 
now requires that the phenyl groups rotate into the plane of the 
phenanthroline. The rotation to planarity can be accomplished with minimal 
steric interactions of the neighboring hydrogen atoms by lengthening the 
carbon-carbon bond between the phenyl and phenanthroline moieties; 
precedence for this type of structural distortion is found in the case of 
biphenyl which is planar when stacked in a solid state lattice. (53) The 
phenyl groups once rotated into the plane of the phenanthroline increase 
the surface area for base pair overlap substantially as compared with 
(phen).sub.3 Ru.sup.2+, stabilizing the intercalatively bound metal-DNA 
complex. An increased affinity for the helix is indeed apparent in gel 
electrophoresis experiments where changes in supercoiled DNA mobility are 
first evident at an order of magnitude lower total ruthenium concentration 
than for (phen).sub.3 Ru.sup.2+. Hydrophobic interactions of the 
non-intercalated ligands abutting the helical groove may also account for 
some increase in affinity for the duplex. Third, and perhaps most 
importantly, the added phenyl substituents increase the chiral 
discrimination markedly. While the delta isomer can still bind closely 
into the right-handed helix, intercalation of the lambda isomer is now 
completely blocked. 
Because the steric constraints are governed by the helicity of the duplex, 
RuDIP enantiomers can offer a specific chemical probe to distinguish 
right-handed and left-handed DNAs. While for the lambda isomer steric 
interactions between the non-intercalated phenyl groups and the 
DNA-phosphate backbone prevent close association to B-DNA, no similar 
repulsive interactions limit binding to the left-handed Z-DNA. (16) Hence 
an assay of duplex binding by the ruthenium enantiomers equivalently 
assays the DNA conformation. It is necessary at this point to note that 
RuDIP is indeed a probe for helical structure and does not itself promote 
a conformatioion transition between B- and Z-DNA. The circular dichroic 
spectra of poly(dGC) in the B-form or Z-form without ruthenium present are 
identical to those of DNA solutions containing ruthenium at an added ratio 
of 0.05 per nucleotide. Unlike other smaller intercalators which in 
binding at high drug/DNA ratios causes Z--B conformational transition (54, 
55, 56), racemic RuDIP does not interconvert the helical forms. The 
enantiomers of RuDIP provide, therefore, a chemical means to examine DNA 
conformation and specifically, the handedness of the helix. DNAs of 
particular repetitive sequence or those covalently bound by particular 
drugs will be intriguing to study. Because of the high luminescence of the 
ruthenium complexes, some intersecting applications become feasible. FIG. 
1 shows, for example, fluorescent micrographs (57) of Drosophila polytene 
chromosomes stained with racemic RuDIP. These samples were prepared in 
collaboration with Dr. O. J. Miller and Dr. D. Miller. Centers of high 
RuDIP concentration correlate closely with the band regions seen by phase 
contrast and no staining is evident in either interband regions or puffs. 
Experiments using the individual enantiomers can be conducted to determine 
whether a high local concentration of a particular conformation is 
present. These micrographs should provide a useful addition to those 
obtained through staining with fluorescent antibodies, (31) with the 
particular advantage of pointing out specific structural features 
depending upon the stereoselectivity observed. 
Metallointercalation reagents also offer flexibility in the design of a 
stereospecific DNA nicking agent. Such agents have been prepared by using 
tris-phenanthroline complexes with a suitable choice of redox-active 
metal, e.g. cobalt. Tris(phenanthroline)cobalt(III) (58), for example, at 
low concentrations cleaves DNA when irradiated at 254 nm. Furthermore the 
high stereospecificity of the tris(diphenylphenanthroline) (DIP) metal 
isomers (59) with DNA helices is preserved in these cleavages reactions. 
Numerous spectroscopic and x-ray crystallographic studies have shown that 
DNA may adopt a range of conformations, from the right-handed A- and 
B-forms to the striking left-handed Z-DNA helix (60, 61). Regions of 
conformational heterogeneity along the strand, such as cruciform 
structures, single-stranded loops, and left-handed segments, have been 
detected using DNA enzymes (62), and it has been suggested that local DNA 
conformation might play some role in regulating gene expression. Chiral 
metal complexes of this invention can intercalate into the helix and are 
therefore particularly advantageous in probing local DNA conformation (15, 
34, 52, 64). Tris(diphenylphenanthroline) (DIP) ruthenium(II) complexes 
provide a spectroscopic probe for helix handedness; the lambda isomer, 
which does not bind B-DNA owing to steric constraints, binds avidly to 
Z-DNA (63). Upon photoactivation, the analogous cobalt isomers, 
Co(DIP).sub.3.sup.3+, furthermore cleave DNA stereospecifically, providing 
a sensitive assay for local regions in the Z-form (64). The specific 
left-handed sites have now been mapped where .LAMBDA.-Co(DIP).sub.3.sup.3+ 
cleaves in the plasmids pLP32 (29), containing a d(CG).sub.32 insert, and 
pBR322 (65). In pLP32 a primary cleavage occurs at the insert, and in 
native pBR322 cleavage occurs at four discrete sites: 1.45 kb, 2.3 kb, 3.3 
kb, and 4.2 kb. 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 that Z-DNA might serve as a conformational punctuation mark to 
demarcate the ends of genes. 
.LAMBDA.-Co(DIP).sub.3.sup.3+, the photoactivated DNA cleaving agent used 
here to detect Z-DNA segments is shown below. 
##STR3## 
The possible application of the complexes of this invention in 
chemotherapy has also been investigated. Beyond the antimicrobial and 
antitumor activities common to intercalating agents, if Z-DNA is important 
in gene regulation, the stereospecific intercalators of this invention 
could display a unique potency in vivo. Preliminary experiments in tissue 
cultures have shown high toxicity without irradiation, and phototherapy 
may provide greater potency and tissue specificity. 
EXPERIMENTS 
Materials and Methods 
I. Tris(phenanthroline)ruthenium II 
Ruthenium Complexes 
[(phen).sub.3 Ru]Cl.sub.2 :2H.sub.2 O was prepared as follows: to a 
solution of 0.5 g K.sub.2 RuCl.sub.5 in 50 ml hot water containing 1 drop 
6N HCl was added 0.81 g phenanthroline monohydrate (Aldrich). The 
resulting mixture was boiled for 15 minutes to fully dissolve the ligand 
and thereafter 0.75 ml of 50% hypophosphorous acid neutralized with 2N 
NaOH was added. The solution was refluxed for 30 minutes and filtered hot 
to remove solid material. To the filtrate was added 10 ml 6N HCl, the 
volume reduced to about 20 ml and cooled at 0.degree. overnight. Single 
orange crystals of [(phen).sub.3 Ru]Cl.sub.2 were obtained. See also (38). 
Enantiomers were obtained by successive diastereomeric recrystallizations 
with antimony D-tartrate anion. (49) At most, two recrystallizations were 
required to achieve [.alpha.].sub.D 1317, after which point additional 
purification did not yield increased optical activity. Several samples of 
the .DELTA. and .LAMBDA. isomers were used in the course of the various 
binding studies, and, on the basis of comparison to literature values for 
the specific rotation, all showed a level of optical purity given by 
[(C(.DELTA.-Ru)-C(.LAMBDA.-Ru)/(C(.DELTA.-Ru)+C(.LAMBDA.-Ru))].gtoreq.0.92 
. The steroisomers were isolated as perchlorate salts; and elemental 
analyses (performed by Galbraith Lab., TN) were as follows: %C, 49.34; %H, 
3.29; N, 9.52; calculated for [(phen).sub.3 Ru](CIO.sub.4).sub.2.2H.sub.2 
O, %C, 49.32; %H, 3.22; %N, 9.59. Spectrophotometric and luminescence 
titrations of racemic Ru(phen).sub.3 Cl.sub.2 and equimolar mixtures of 
.DELTA.- and .LAMBDA.-Ru(phen).sub.3 (CIO.sub.4) with DNA agreed closely, 
indicating that the presence of perchlorate (.ltoreq.50M) was without 
effect. Stock ruthenium solutions were either freshly prepared or kept in 
the dark. Concentrations of (phen).sub.3 Ru.sup.2+ were determined 
spectrophotometrically by using .epsilon..sub.447 1900M.sup.-1 cm.sup.1. 
(38) 
BUFFERS AND CHEMICALS 
Experiments were carried out at pH 7.1 in buffer 1 (5 mM Tris, 50 nM NaCl), 
buffer 2 (5 mM Tris, 4.0M NaCl), or buffer 3 (50 mM Tris acetate, 20 mM 
sodium acetate, 18 mM NaCl pH 7.0). Solutions were prepared with distilled 
deionized water. Plasticware was used throughout and was cleaned by 
soaking overnight in 10% HNO.sub.3 followed by exhaustive rinsing. 
Dialysis membranes were prepared by the following protocol: After they 
were boiled successively in sodium carbonate, 1% EDTA, and 1% SDS and 
exhaustively rinsed in deionized water, the membranes were heated to 
80.degree. C. in 0.3% sodium sulfite, acidified at 60.degree. C. with 2% 
sulfuric acid, and thereafter rinsed again with deionized water and 1% 
EDTA. This procedure serves to minimize metal binding to the membranes. 
Nucleic Acid 
Calf thymus DNA, obtained from Sigma Chemical Co., was purified by phenol 
extraction as described previously. (9c) Poly(dGC).poly(dGC)from P. L. 
Biochemicals Inc. and plasmid ColE1 from Sigma Chemical Co. were 
extensively dialyzed in buffer before use. DNA concentrations per 
nucleotide were determined spectrophotometrically by assuming 
.epsilon..sub.260 6000M.sup.-1 cm.sup.-1 for calf thymus DNA and 
.epsilon..sub.260 8400M.sup.-1 cm.sup.-1 for poly(dGC). 
Spectroscopic Measurements 
Absorption spectra were recorded on a Cary 219 spectrophotometer. 
Absorbance titrations of racemic, .DELTA.- and .LAMBDA.-(phen).sub.3 
Ru.sup.2+ in buffer 1 were performed by using a fixed ruthenium 
concentration to which increments of the DNA stock solution were added. 
Ruthenium was also added to the DNA stock to keep the total dye 
concentration constant. Luminesence measurements were conducted on a 
Perkin-Elmer LS-5 flourescence spectrophotometer at ambient temperature. 
Samples were excited at 447 nm, and emission was observed between 500 and 
700 nm. All experiments were carried out in buffer 1 with (phen).sub.3 
Ru.sup.2+ concentrations typically of 10 .mu.M and DNA phosphate/ruthenium 
ratios varying from 1 to 50. Lifetime measurements were performed on an 
Ortec 776 single-photon counter and timer in line with an Apple Computer. 
The samples were excited with a PRA 510A nanosecond lamp, and emission was 
observed at 593 nm. Reproducible lifetimes for the bound species in the 
presence of free ruthenium were obtained by neglecting the first 1.2 
.mu.s(2.tau.[Ru(phen).sub.3.sup.2+.sub.free ]), of the decay curve. 
Electrophoresis 
Dye gel electrophoresis of supercoiled DNA in 1% agarose was performed in 
buffer 3 by using the method of Espejo and Lebowitz (6) modified as 
described previously.(15) Ruthenium concentrations in the gels were 
carefully determined on the basis of several absorbance readings of the 
stock concentrations for the enantiomers. Because of the high background 
luminescence of (phen).sub.3 Ru.sup.2+, gels were destained for 24 hours 
in buffer prior to staining with ethidium. 
Equilibrium Dialysis 
Binding isotherms were obtained on the basis of dialysis of calf thymus DNA 
in buffer 1 against (phen).sub.3 Ru.sup.2+ at 22.degree. C. The DNA was 
dialyzed first exhaustively in buffer to remove small fragments. 
Thereafter, dialysis against ruthenium was allowed to continue for at 
least 24 hours after which time equilibration was achieved. Each sample 
consisted of 2 mL of dialysate containing .DELTA.-, .LAMBDA.-, or 
rac-(phen).sub.3 Ru.sup.2+, varying in concentration between 50 and 1,000 
.mu.M, and, within the dialysis bag, 1 mL of 1 mM DNA phosphate. To 
determine bound and free concentrations, absorbance spectra were taken of 
dilutions (3-50 .mu.M). Free ruthenium concentrations outside the bag were 
determined on the basis of absorbance readings at 447 nm. For 
concentrations of ruthenium inside the bag, in the presence of DNA, 
readings were obtained at the isosbestic point, where .epsilon..sub.464 
13630 M.sup.-1 cm.sup.-1 (vide infra). Equilibrium dialysis of poly (dGC) 
was conducted similarly in buffer 2. Measurements of circular dichroism 
were obtained on a Jasco J-40 automatic recording spectropolarimeter. 
Because of the irregular baseline of the instrument, all spectra were 
digitized and replotted after base-line subtraction. Data analyses were 
performed on a IBM PC and a Digital VAX 11/780. 
II. Tris(4,7-diphenyl-1,10-phenanthroline)rutheniumII 
Nucleic Acids 
Calf thymus DNA (Sigma) was purified by phenol extraction (66). 
Poly[d(G-C)] (P-L Biochemicals) was dialyzed at least three times before 
use. Experiments were conducted at pH 7.2 in buffer 1 [4.5 mM Tris.HCl/45 
mM NaCl/150 .mu.M Co(NH.sub.3).sub.6 Cl.sub.3 /10% dimethyl sulfoxide], 
buffer 2 [5 mM Tris.HCl/50 mM NaCl/150 .mu.M Co(NH.sub.3).sub.6 Cl.sub.3 
], or buffer 3 [5 mM Tris.HCl/4.0M NaCl]. DNA concentrations per 
nucleotide were determined spectrophotometrically assuming 
.epsilon..sub.260 =6600 M.sup.-1.cm.sup.-1 for calf thymus DNA (67) and 
.epsilon..sub.260 =8400 M.sup.-1.cm.sup.-1 for poly[d(G-C)] (68). In 
preparing Z-DNA, poly[d(G-C)] stock solutions were incubated in the cobalt 
hexammine buffer for 2-18 hr to ensure both a complete transition to the Z 
conformation and minimal aggregation. Stock solutions were examined 
spectrophotometrically and by CD before use. 
Ruthenium Complexes 
The synthesis of RuDIP trihydrate was carried out as described above, 
substituting 4,7-diphenyl-1,10-phenanthroline for the unsubstituted 
1,10-phenanthroline. See also (38). Concentrations were determined 
spectrophotometrically using .epsilon..sub.460 =2.95.times.10.sup.4 
M.sup.-1.cm.sup.-1. Elemental analyses were consistent with literature 
values. The .DELTA. and .LAMBDA. isomers were either separated by 
successive recrystallizations with the antimony tartrate anion in 50% 
ethanol or prepared by asymmetric synthesis in the presence of antimony 
tartrate and then recrystallized. .LAMBDA.-RuDIP forms the less soluble 
diastereomeric salt with antimonyl D-tartrate. The separated isomers were 
isolated finally as perchlorate salts. The assignments of absolute 
configuration have been made on the basis of the relative binding 
affinities of these enantiomers for B-DNA (see below). Many rounds of 
recrystallization yielded a small quantity of .LAMBDA.-RuDIP having 
[.theta.].sub.283 =-4.0.times.10.sup.3 deg.M.sup.-1.cm. This assignment is 
consistent with both the UV CD for tris(1,10-phenanthroline)ruthenium(II) 
[(phen).sub.3 Ru.sup.2+ ],assigned previously (31), and studies of the 
enantiomeric preference of (phen).sub.3 Ru.sup.2+ for B-DNA (34, 35, 51). 
The optical purities of the .DELTA.- and .LAMBDA.-RuDIP samples used below 
were 41% and 70%, respectively. Therefore the sample designated 
.DELTA.-RuDIP contains 70.5% .DELTA. isomer and 29.5% .LAMBDA. isomer, and 
that designated .LAMBDA.-RuDIP is composed of 14% .DELTA.- and 86% 
.LAMBDA.-RuDIP. 
Spectroscopic Measurements 
Absorbance spectra were recorded using a Varian Cary 219 UV/visible 
spectrophotometer and luminescence spectra, with a Perkin-Elmer LS-5 
fluorescene spectrophotometer. Titrations were carried out using a 
constant ruthenium concentration (4-6 .mu.M) to which increments of either 
calf thymus DNA or poly[d(G-C)] were added. Because RuDIP has limited 
solubility in aqueous solution (.ltoreq.10 .mu.M), dimethyl sulfoxide was 
included in buffer 1. CD spectra of B-DNA or Z-poly[d(G-C)] with 150 .mu.M 
Co(NH.sub.3).sub.6.sup.3+ were unaffected by the presence of the dimethyl 
sulfoxide. Although more difficult, titrations in buffer 2 and buffer 3 
were also conducted. 
III. CobaltIII Complexes 
Tris(4,7-diphenyl-1,10-phenanthroline)cobalt(III) (Co(DIP).sub.3.sup.3+) 
tri-tartrate was prepared as follows: 4,7-diphenyl-1,10-phenanthroline 
(Aldrich) was dissolved in a minimum volume of ethanol to which one third 
stoichiometric CoCl.sub.2.6H.sub.2 O was added. The green brown solution 
was oxidized by using Br.sub.2 /H.sub.2 0, and a heavy orange precipitate 
formed immediately. The solution was refluxed for 1 h, and concentrated 
hydrochloride was added. The bromine oxidation was then repeated. The 
crude chloride salt was used directly for enantiomeric separations. With 
either 1- or d-tartaric acid (Aldrich), the deep red tartrate (Tar) 
diastereomeric salts [.LAMBDA.-Co(DIP).sub.3 ].(L-Tar).sub.3 and 
[.DELTA.-Co(DIP).sub.3.(d-Tar).sub.3, were prepared by successive 
recrystallizations in 50% ethanol, pH 7.0. 
Chemical and spectroscopic data for these complexes are as follows: Anal. 
Calcd for [Co(DIP).sub.3 ](Tar).sub.3.H.sub.2 O(CoC.sub.84 N.sub.6 
O.sub.19 H.sub.65) C, 66.32; H, 4.32; N, 5.52; Found: C, 65.87; H, 4.46; 
N, 5.78. Absorption spectra showed .lambda..sub.max at 278 and 312 nm 
(shoulder). The circular dichroic spectra resemble those of enantiomers of 
Ru(DIP).sub.3.sup.2+, and absolute configurations have been assigned on 
that basis. 
IV. Cleavage Methods 
[Co(DIP).sub.3 ](tartrate).sub.3 (10 .mu.M) was added to pBR322 DNA (100 
.mu.M nucleotides) in 50 mM tris-acetate buffer containing 18 mM NaCl, ph 
7.0. The 20 .mu.l sample was then irradiated at 315 nm (with a 1000 W 
Hg/xenon lamp narrowed to 315.+-.5 nm with a monochrometer) for 90 seconds 
and ethanol precipitated. The ethanol wash removes unreacted 
Co(DIP).sub.3.sup.3+ as well as the metal and ligand products of the 
reaction. After resuspension in trisacetate buffer containing 50 mM NaCl 
and 10 mM MgCl.sub.2, pH 7.0, restriction enzyme was added (either EcoRI, 
BamHI, AvaI or NdeI) using at least a threefold excess to insure complete 
linearization. This was incubated at 37.degree. C. for 45 min. The pH of 
the reaction mixture was then lowered to 5.0 and 10 mM Zn(N0.sub.3).sub.2 
added along with 4 units of Sl nuclease, and the samples were incubated 
for 5 min at 37.degree. C. This step causes cleavage of the DNA by Sl 
opposite the site nicked by Co(DIP).sub.3.sup.3+. Electrophoresis on 1% 
agarose gels followed (50 mM tris-acetate, 18 mM NaCl, pH 7.0) to resolve 
the double stranded fragments produced. In these experiments pBR322 
sequences are numbered beginning at the EcoRI site according to Sutcliffe 
(65). Gels were stained with 5 .mu.g/ml ethidium bromide for 0.5 hr then 
destained in buffer for 2 hr. Gels were photographed using a Polaroid 600 
camera with a red filter and 615 positive/negative film and irradiated 
from below. 
V. In vitro Screening 
For cell culture studies, a modification of the techniques of Fischer (69) 
was used. The cells were incubated in McCoy's Medium 5A with 15% fetal 
calf serum. The initial inoculum was 40,000 to 60,000 leukemic cells/ml. 
For studies of the inhibition of cell growth, 0.1 ml of a 20-fold 
concentration of the drug in question was added to 2 ml of media 
containing 4.times.10.sup.4 cells/ml in Linbro tissue culture multiwell 
plates and allowed to incubate at 37.degree. C. in 5% CO.sub.2 for 96 hr. 
By these times, growth to approximately 10.sup.6 cells/ml occurred in the 
control wells. The contents of each well were agitated to resuspend the 
cells and counted on a Coulter Counter. The percentage of inhibition of 
growth and the concentrations inhibiting cell growth by 50% were 
calculated. Cell culture experiments were conducted with mouse leukemia 
cell lines L1210 and P815. The cell lines and growth medium may be 
obtained from the American Type Culture Collection (ATCC), Rockville, Md. 
RESULTS 
I. Tris(1,10-phenanthroline)ruthenium (II) 
Spectroscopic Studies 
The binding of .LAMBDA.- and .DELTA.-(phen).sub.3 Ru.sup.2+ to duplex DNA 
leads to a decrease and small shift in the visible absorption of the 
ruthenium species and a corresponding increase and shift in luminescence. 
FIG. 2 shows both the absorption spectra and luminescence spectra of 
(phen).sub.3 Ru.sup.2 + in the presence and absence of calf thymus DNA. 
The spectral changes observed here are often characteristic of 
intercalation. 
The hypochromic shift in the broad charge-transfer band of (phen).sub.3 
Ru.sup.2+ as a result of binding to the polynucleotide can be seen in FIG. 
2A. A decrease of 12% in absorbance at 447 nm is found for the saturating 
DNA levels employed. Since at these concentrations 70% (phen).sub.3 
Ru.sup.2+ is in the bound form, .epsilon.(bound)/.epsilon.(free)=0.83 at 
447 nm. The hypochromic effect is small compared with that found for other 
intercalators, which may indicate that the charge is not being 
preferentially localized onto the intercalated ligand. Also for the free 
ruthenium complex the predominant polarization of the charge-transfer band 
is perpendicular to the molecular C3 axis (46) rather than parallel to the 
intercalative plane. Shown in the figure is the change in absorbance for 
the racemic mixture; also because the observable hypochromic effect is 
small, significant differences between enantiomers were not obtained. For 
both isomers a spectral shift of 2 nm to lower energy is found, which 
supports an electronic stacking interaction of the phenanthroline ligand 
with the base pairs of the helix. Isosbestic points at 355 and 464 nm are 
also apparent. 
An enhancement in the luminescence of (phen).sub.3 Ru.sup.2+ on binding to 
duplex DNA parallels the observed hypochromicity. FIG. 2B shows the 
emission spectra of free (phen).sub.3 Ru.sup.2+ and of both lambda and 
delta isomers bound to DNA. These spectra also reveal a shift of 2 nm to 
longer wavelength with DNA binding. Moreover, in the presence of 0.25 mM 
DNA phosphate, emission increases of 48% and 87% are observed respectively 
for .LAMBDA.- and .DELTA.-(phen).sub.3 Ru.sup.2+ (10 .mu.M). Note that a 
significant fraction of the ruthenium is free in the presence of the DNA 
at these concentrations, but the associated increase in solution viscosity 
for higher DNA concentrations precluded studies at saturating binding 
levels. The greater increase in luminescence seen for the .DELTA. isomer 
in the presence of DNA over that for the .LAMBDA. isomer indicates simply 
that a higher proportion of the .DELTA. isomer is bound, rather than that 
their modes of association with the helix differ. Measurements of the 
excited-state lifetimes of enantiomers in the absence and presence of the 
DNA yielded results consistent with this interpretation. For .DELTA.- and 
.LAMBDA.-(phen).sub.3 Ru.sup.2+, determined separately and as a racemic 
mixture, identical experimental lifetimes of 2.0 and 0.6 .mu.s were found 
respectively in the presence and absence of DNA. Both isomers therefore 
bind to the helix in a similar fashion, and indeed, if fully bound, would 
display similar enhancements in luminescence. Substantial increases in 
fluorescent lifetimes with intercalation have been observed in several 
instances (2-4, 70), notably for ethidium, and may be explained by the 
greater rigidity and lower collisional frequency of the molecule when 
stacked within the helix. 
Measurements of Helical Unwinding 
Both .LAMBDA.- and .DELTA.-(phen).sub.3 Ru.sup.2+ reversibly unwind and 
rewind supercoiled DNA as a function of increasing concentration of 
ruthenium(II), and for a given total concentration, a greater unwinding 
effect is evident for the .DELTA. isomer. FIG. 3 illustrates the migration 
of pColE1 DNA through 1% agarose gels containing increasing levels of 
(phen).sub.3 Ru.sup.2+. Mobilities are plotted relative to the supercoiled 
DNA control to permit the inclusion of data from several gel 
electrophoresis trails. As can be seen in the figure, both isomers unwind 
the helix. With increasing levels of ruthenium bound, the duplex unwinds, 
and for a closed circle this unwinding results in first a release of 
negative supercoils at low levels bound and then the introduction of 
positive supercoils; the nicked DNA, without similar topological 
constraints, displays no variation in mobility. The bars in the figure 
indicate the width of the DNA bands, which vary because of the 
distribution of topoisomers in the sample. The observed duplex unwinding 
provides a strong indication of intercalative binding. Control experiments 
also show the unwinding to be reversible; preincubation of the DNA with 
ruthenium complex has no effect on gel mobility. It is interesting to note 
that no DNA cleavage is observed as a result of binding (phen).sub.3 
Ru.sup.2+, even after irradiation with ultraviolet light (short 
wavelength) for 1 h. 
For a given level of total ruthenium, a higher amount of duplex unwinding 
is found in the presence of .DELTA.-(phen).sub.3 Ru.sup.2+, respectively. 
The comigration of nicked and closed circular DNAs occurs in the presence 
of 90 and 120 .mu.M .DELTA.-(phen).sub.3 Ru.sup.2+. This comigration point 
represents a fixed amount of helical unwinding. A lower added 
concentration of .DELTA.-(phen).sub.3 Ru.sup.2+ is needed to unwind all 
the negative supercoils in the pColE1 DNA. These results therefore also 
reflect the higher affinity of the .DELTA. isomer over the .LAMBDA. isomer 
for the right-handed helix. At a given total concentration of ruthenium, 
more of the .DELTA. isomer is bound and greater helical unwinding is 
evident. The alternative explanation for the lower concentration of the 
.DELTA. isomer at the comigration point would be that the .DELTA. isomer 
has a larger unwinding angle than the .LAMBDA. isomer, so that the .DELTA. 
isomer unwinds the duplex more per drug bound. A particularly large 
difference between unwinding angles (about 30%) would be needed to account 
for the electrophoresis results, however, and only small variations in 
unwinding angles are generally observed (4). Moreover larger, if any, 
structural perturbations should accompany binding of the .LAMBDA. isomer 
to the right-handed helix rather than the .DELTA. isomer. Here then, as 
well, the results show that the .DELTA. isomer possesses a greater 
affinity for the DNA duplex. 
These data may be used to estimate the intercalative unwinding angle. If we 
assume for the racemic mixture that the average comigration point of 
nicked and closed forms occurs with 100 .mu.M ruthenium, then, on the 
basis of our determination of the binding constant provide (in-fra), a 
binding ratio of 0.089 per nucleotide is required to unwind fully the 
supercoils in the plasmid. Interestingly this value is identical with that 
calculated for ethidium, since in buffer 3 the comigration of nicked and 
closed pColE1 species in the presence of 5.times.10.sup.-7 M dye was 
observed. Therefore the unwinding angle for (phen).sub.3 Ru.sup.2+ is 
estimated to be the same as that for ethidium (6). 
Equilibrium Dialysis Experiments 
The results of dialysis of calf thymus DNA with racemic (phen).sub.3 
Ru.sup.2+ at 22.degree. C. in buffer 1 are shown in FIG. 4 in the form of 
Scatchard plat (71). The data have been fit by nonlinear least-squares 
analysis to the following equation governing noncooperative binding to the 
helix, as derived by McGhee and von Hippel (72): 
##EQU1## 
where r is the ratio of the bound concentration of ruthenium to the 
concentration of DNA phospate, C.sub.F is the concentration of ruthenium 
free in solution, K(O) is the intrinsic binding constant, and the integer 
I, which measures the degree of anticooperativity, is the size of a 
binding site in base pairs. In fitting the data, the binding parameter 
K(O) was varied for several integer values of L. The best fit, shown as 
the solid curve in FIG. 4, yielded a binding constant 
K(O)=6.2.times.10.sup.3 M.sup.-1 (.+-.2%) and an exclusion site size (L) 
of four base pairs. Data from luminescence titrations were consistent with 
this curve. The binding constant is quite low in comparison to values of 
3.times.10.sup.5 and 5.times.10.sup.4 M.sup.-1 (extrapolated to the ionic 
strength of our buffer) for ethidium and [(phen)Pt-(en)].sup.2+, 
respectively (73). The lower affinity of (phen).sub.3 Ru.sup.2+ is not 
surprising since only partial stacking of the phenanthroline ligand is 
feasible in this octahedral complex; greater overlap of the phenanthroline 
with the base pairs may be achieved in the square-planar platinum(II) 
species. The steric bulk of the nonintercalated ligands determines also 
the large four base-pair site size compared to a two base-pair (neighbor 
excluded) site for basically planar reagents (9, 74). Inspection of 
space-filling models show that the perpendicular phenanthroline ligands 
each span two base pairs either above or below the intercalatively bound 
phenanthroline, which is consistent with the binding isotherm. 
In these equilibrium dialysis experiments of the racemic mixture, the 
relative binding of the two enantiomers to the polynucleotide may be 
determined sensitively on the basis of the degree of optical enrichment on 
the unbound enantiomer in the dialysate. FIG. 5 shows the circular 
dichroism (1.5.times.10.sup.-5 M) of the dialysate after equilibration of 
calf thymus DNA (1 mM) with racemic (phen).sub.3 Ru.sup.2+ 
(2.times.10.sup.-4 M). Also shown for comparison is the circular dichroism 
of .DELTA.-(phen).sub.3 Ru.sup.2+ (0.2 .mu.M). The spectra show clearly 
that the dialysate has been optically enriched in the less favored isomer. 
The .DELTA. enantiomer binds preferentially to the right-handed helix. The 
degree of chiral discrimination may be made more quantitative by comparing 
the level of optical enrichment (2% for the sample shown) with the overall 
amount of ruthenium bound. On the basis of a simple competition between 
the enantiomers for sites along the helix with no cooperativity and, for 
simplification, describing the binding by each enantiomer in terms of the 
familiar Scatchard equation, X, the ratio of binding constants 
K(.DELTA.)/K(.LAMBDA.) may be calculated as follows: 
##EQU2## 
where P is the concentration of DNA phosphate, n is the ratio of drug to 
DNA phosphate bound at saturation, taken as 0.125, C.sub.B is the total 
concentration of ruthenium bound, and .DELTA.C is the difference in free 
concentrations between .DELTA. and .LAMBDA. isomers as measured by the 
intensity in the circular dichroism. Measurements of several samples 
yielded values of 1.1-1.3 for X. Thus the binding affinity of 
.DELTA.-(phen).sub.3 Ru.sup.2+ is found to be 10-30% greater than 
.LAMBDA.-(phen).sub.3 Ru.sup.2+ for calf thymus DNA by this method. This 
value is comparable to the differences seen in luminescence and unwinding 
experiments. Since the overall binding of (phen).sub.3 Ru.sup.2+ is small, 
binding isotherms obtained through equilibrium dialysis tended to show 
some scatter. A direct comparison of the binding isotherms for the 
enantiomers in equilibrium dialysis experiments using the pure isomers 
therefore could not be achieved; significant differences were not evident. 
Interestingly it appears that the method of optial enrichment yields the 
most sensitive assay for the differential binding. 
Since the enrichment experiment provides the most sensitive method to 
examine enantiometric discrimination, poly(dGC) in 4M NaCl was also 
dialyzed against rac(phen).sub.3 Ru.sup.2+ to test for any enantiomeric 
preferences in binding to a left-handed DNA helix (16, 17). At the low 
binding levels examined, the circular dichroism of the polymer remains 
inverted, indicating that racemic (phen).sub.3 Ru.sup.2+ did not induce a 
Z-- B transition. After equilibrium dialysis with bound concentrations 
comparable to those in earlier experiments using calf thymus DNA, e.g., 
under conditions where low levels of enrichment could be detected, no 
optical activity was observed in the dialysate. Therefore, although 
intercalative binding had occurred, given similar spectral characteristics 
as in binding to the right-handed helix, no preference in binding was 
evident. In FIG. 5 the essentially base line spectrum of a solution after 
dialysis against Z-form poly(dGC) has also been included. This lack of 
discrimination for (phen).sub.3 Ru.sup.2+ is understandable in view of the 
shallow, almost grooveless character of the left-handed Z-DNA helix. 
II. Tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) 
In spectroscopic studies of racemic RuDIP with B- and Z-DNA, changes are 
seen in both the visible absorption and luminescence spectra of RuDIP on 
addition of either B- or Z-form DNA. Hence, binding may be monitored 
sensitively using either spectroscopic technique. Visible absorption 
titrations of racemic RuDIP in buffer 1 (described below) with calf thymus 
DNA and Z-form poly[d(G-C)] are shown in FIG. 6. The overall similarity of 
these titrations is apparent. Binding of either duplex DNA leads to 
hypochromicity in the intense metal-to-ligand charge-transfer band of the 
ruthenium complex. A small red shift (about 2 nm) in the spectrum of the 
bound complex and an isosbestic point at 485 nm can be seen. That spectral 
changes occur as a function of addition of either DNA form suggests that 
racemic RuDIP binds to both B- and Z-DNA. The similarity in spectral 
changes most likely reflects a similar mode of association of the 
ruthenium complex with either the right-handed B-DNA helix or the 
left-handed Z-DNA helix. 
Differences in binding to the two forms are evident, however. A greater 
reduction in the absorption intensity of Ru-DIP accompanies binding to 
Z-form poly[d(G-C)] than to the B-DNA helix. In FIG. 6, for example, the 
apparent reduction in intensity with the addition of a 13:1 ration of calf 
thymus DNA-phosphate/ruthenium is only 9% whereas, for the left-handed 
helix, the reduction occurring at a nucleotide/ruthenium ratio of 5:1 is 
17%. The greater hypchromicity in binding to Z-DNA is explained in part by 
the different stereoselectivities governing binding to each helix. 
Although both enantiomers bind to Z-DNA, only the .DELTA.enantiomer may 
bind easily to right-handed B-DNA. The differences in stereoselectivity 
cannot fully account for the difference in hypochromicity, however, 
because the hypochromicity in spectra of racemic RuDIP with Z-DNA is more 
than twice that observed with calf thymus DNA. If one assumes that the 
extinction coefficients for RuDIP when bound to each helix are the same, 
which seems reasonable based on the equal isosbestic points observed, then 
the larger hypochromic effect with Z-DNA suggests that both RuDIP 
enantiomers possess a greater affinity for Z-form poly[d-(G-C)] than for 
calf thymus DNA. Equilibrium dialysis experiments support this conclusion. 
The luminescence of RuDIP is also enhanced on binding to the DNA duplex. 
FIG. 7 shows the emission spectrum of racemic RuDIP (3 .mu.M) in the 
absence and presence of calf thymus DNA and Z-form poly[d(G-C)] (15 .mu.M 
nucleotide). The shift in the spectrum to lower energy is particularly 
pronounced despite the broad nature of the transition; the maximum shifts 
10 nm to longer wavelength in the presence of DNA. Greater luminescence is 
seen here on binding to B-DNA, despite the lower apparent affinity for 
this helix. In buffer 2, at a DNA-phosphate/ruthenium ratio of 5:1, the 
emission intensity of racemic RuDIP increases by 30% and 47% in the 
presence of Z-form poly[d(G-C)] and calf thymus DNA, respectively. The 
different enhancements may depend in part on nucleic acid composition as 
well as duplex conformation, RuDIP bound to B-form poly[d(G-C)] yields 
less luminescence than when bound to calf thymus DNA, despite having an 
equal affinity for these polynucleotides. 
Racemic RuDIP appears to bind to both B- and Z-DNA rather than promoting a 
transition from one conformation to the other. CD spectra of Z-form 
poly[d(G-C)] in Co(NH.sub.3).sup.3+ (buffer 1) or in 4M NaCl (buffer 3) 
are unaltered by the addition of racemic RuDIP at a nucleotide/ruthenium 
ratio of 10. Conversion of the Z-form to B-form with RuDIP is inconsistent 
also with the differential hypochromism and luminescence observed. If 
RuDIP promoted a Z to B transition, albeit inefficiently, rather than 
binding to both B- and Z-helices, then both the reduction in absorbance 
and the enhancement in luminescence observed on addition of Z-DNA would be 
less than or equal to that found with B-DNA, i.e., in proportion to the 
fraction of DNA converted. Instead significantly greater hypochromism is 
found when Z-DNA rather than B-DNA is added to the racemic mixture or 
indeed to each enantiomer individually. Therefore racemic RuDIP must bind 
to both DNA conformations. Consistent with these results, a conformational 
transition from Z-DNA to B-DNA would not be expected if the affinity of 
the metal cation for the Z-form were greater than that for the B-form. The 
ethidium cation, which binds to B-DNA by intercalation, is known to 
promote a Z to B transition at high binding ratios (75, 76) and presumably 
possesses a greater affinity for the B-form helix. The substantially 
larger RuDIP cation cannot saturate the DNA to comparable levels, which 
may be an important distinction. Moreover, although the ethidium ion can 
fully intercalate into B-DNA, RuDIP cannot and the nonintercalating 
ligands of RuDIP may dominate its interactions with the duplex. 
The utility of the RuDIP enantiomers as a probe for helical conformation 
becomes apparent when the binding characteristics of each enantiomer to B- 
and Z-DNAs are compared. Plots of the relative absorbance at 460 nm of the 
individual enantiomers as a function of the addition of either B-form calf 
thymus DNA or Z-form poly[d(G-C)] in buffer 1 are shown in FIG. 8. Based 
on the presence or absence of hypochromicity, it is clear that although 
RuDIP binds to B-DNA, the .LAMBDA. isomer does not. .DELTA. RuDIP does 
however bind to Z-DNA. Indeed, with Z-DNA no stereospecificity is 
observed. Hence the assay of duplex binding by the .LAMBDA. isomer yields 
a sensitive assay for the Z-DNA conformation. 
Strong enantiomeric selectivity governs the interaction of RuDIP with the 
right-handed D-DNA helix. The decrease in absorbance with increasing DNA 
concentration observed for the .LAMBDA., racemic mixture, and .DELTA. 
samples can be fully accounted for based on the percentage of the .DELTA. 
enantiomer present in the particular preparation (see Experimental). The 
pure .LAMBDA. enantiomer does not bind to B-DNA. The presence of the 
phenyl groups at the 4 and 7 positions of the nonintercalated 
phenanthroline ligand has served to amplify the chiral discrimination. In 
comparison, differences in binding of (phen).sub.3 Ru.sup.2+ enantiomers 
had been seen only in spectrophotometric titrations at high DNA/ruthenium 
levels; the ratio of the affinities for B-DNA of .DELTA. to .LAMBDA. 
isomers is about 1.3(34, 35). For RuDIP, with hydrogen atoms now replaced 
by phenyl groups, instead of simple interference with the DNA phosphate 
oxygen atoms, one finds that the steric bulk of the phenyl groups 
completely blocks interactions of the isomer in the right-handed gove. 
.DELTA.-RuDIP, however, binds with facility to a right-handed helix, 
indeed more avidly than .DELTA.-(phen).sub.3 Ru.sup.2+. This striking 
amplification in enantiomeric selectivity for RuDIP compared with 
(phen).sub.3 Ru.sup.2+ strongly supports our model for stereospecific 
intercalation. 
Z-DNA serves as a poor template to discriminate between the enantiomers; 
identical reductions in absorbance intensity are found for the .DELTA. and 
.LAMBDA. isomers (FIG. 8). Because of the shallow and very wide character 
of the major groove in Z-DNA, there are no steric constraints comparable 
with that found with B-DNA. Hence, if the binding modes are equivalent, no 
chiral specificity would be expected. The similarity in spectral 
characteristics of RuDIP in binding to the different DNA duplexes points 
to this similarity in binding modes. However, the lack of chiral 
specificity in binding to Z-DNA does limit what can be said at present 
about the interaction of RuDIP enantiomers with a Z-form helix. Based on 
relative hypochromicities, it appears that both .LAMBDA. and .DELTA.-RuDIP 
possess greater affinities for Z-form poly[d(G-C)] than for B-DNA. 
Hydrophobic interactions with the helical surface may lend some stability 
to the bound complex(77). The difference in affinity furthermore does not 
reflect a preference for base composition. Titrations of racemic RuDIP 
with B-form poly[d(G-C)] in buffer 1 lacking cobalt hexammine showed 
hypochromicity equal to that seen with calf thymus DNA. Also, cobalt 
hexammine itself does not appear to alter binding to the helix. RuDIP 
titrations using calf thymus DNA with and without 
Co(NH.sub.3).sub.6.sup.3+ were identical. In addition, the interaction 
cannot be explained purely be electrostatic interactions. Although 
smaller, hypochromic effects, approximately one-third of that shown here, 
are found in titrations in 4M NaCl (buffer 3) with either poly[d(G-C)] or 
calf thymus DNA. Partial intercalation into the DNA by both RuDIP 
enantiomers would be consistent with these results. It is finally 
important to note that the similar titrations of both enantiomers that are 
seen with Z-DNA but not with B-DNA suggest that neither enantiomer 
converts the Z-form helix to the B-DNA conformation. If that were the 
case, selectivity between the enantiomers would become apparent. 
III. Cobalt(III) Complexes 
FIG. 9 shows gel electrophoretic separations of plasmid ColE1 DNA after 
incubation with cobalt complexes and irradiation for variable times. DNA 
cleavage is followed by monitoring the conversion of supercoiled (form I) 
closed circular plasmid DNA to the nicked circular form (form II) and 
linear (form III) species. (The original ColE1 pareparation contained 60% 
form I and 40% form II molecules). FIG. 9A reveals the complete conversion 
of form I to II after a 1-h irradiation in the presence of 10 .mu.M 
(phen).sub.3 CO.sup.3+. Neither irradiation of the DNA at these low 
intensities without cobalt nor incubation with cobalt without light 
yielded significant strand scission. (Irradiation at 310 nm where there 
are strong ligand transitions also leads to cleavage). It is likely that 
the reduction of Co(III) is the important step leading to DNA cleavage and 
not that irradiation provides a means to generate cobalt(II) in situ. DNA 
incubation with the tris(phenanthroline) complex initially in the 
cobaltous form yielded no reaction unless irradiated. Presumably the 
cobaltous complex is oxidized in solution to the cobaltic species, since 
it is the + 3 oxidation state in cobalt polyamine complexes that is 
photochemically active. Also dithiothreitol inhibits activity of 
Co(phen).sub.3.sup.3+, perhaps by precluding regeneration of an active 
cobalt(III) species. This finding is in contrast to the iron and copper 
systems whose thiols are thought to stimulate activity by generating the 
metal species in the reduced form (39-43). Interestingly, electrophoresis 
also reveals with increasing irradiation a small reproducible increase in 
the mobility of form II; this may reflect some short-range radical-induced 
DNA cross-linking (78). 
The cleavage reaction is furthermore strongly stereospecific. FIG. 1B shows 
pColEl DNA of low superhelical density after incubation with either 
.LAMBDA.-Co(DIP).sub.3.sup.3+ or .DELTA.-Co(DIP).sub.3.sup.3+ (17, 18) 
and irradiation with ultraviolet light. Incubation of pColE1 DNA of low 
superhelical density with the .LAMBDA. isomer, which cannot bind to a 
right-handed duplex owing to steric constraints, yields no appreciable 
reaction (19), whereas incubation with .DELTA.-Co(DIP).sub.3.sup.3+, which 
is able to associate closely with right-handed B-DNA, shows efficient 
nicking activity comparable to that seen with Co(phen).sub.3.sup.3+. 
Nicking was observed, however, upon titration of pColE1 of increasing 
superhelical density with .LAMBDA.Co(DIP).sub.3.sup.2+. This different 
cleavage efficiency by each enantiomer is consistent with the earlier 
finding (7) of conformational discrimination by the ruthenium(II) isomers; 
one enantiomer of Ru(DIP).sub.3.sup.2+ binds to B-DNA, but both .DELTA.- 
and .LAMBDA.-Ru(DIP).sub.3.sup.2+ binds to the left-handed Z-DNA helix. 
These results underscore the importance of an intimate association of the 
metal with the duplex. 
In FIG. 9B the overall concentrations of the cobalt isomers are equal, yet 
the .LAMBDA. trication, if it cannot intercalate, does not yield DNA 
strand scission. 
The .LAMBDA.-tris(phenanthroline) metal complexes do, however, bind to 
left-handed Z-DNA (21, 22). We examined the plasmid pBR322 containing a 
42-base pair alternating guanine-cytosine insert (pLP42) (29, 79) and 
which was shown (28, 17) to adopt the Z-conformation in 4M NaCl. Under 
these conditions, cleavage by both Co(DIP).sub.3.sup.3+ enantiomers is 
obtained. Hence the isomer may recognize and cleave left-handed helices. 
More interesting, however, is the finding, plotted in FIG. 10 as percent 
loss of supercoiled form, that the plasmid pBR322 at physiological salt 
concentrations and without extreme superhelix underwinding also is 
significantly cleaved by the isomer. Given our other results of 
differential binding based on DNA helicity (35, 63) and the differential 
cleavage of ColE1 of low superhelical density described above, it appears 
that .LAMBDA.-Co(DIP).sub.3.sup.3+ might bind to and cleave a natural 
left-handed segment in pBR322 DNA of low superhelical density in normal 
salt concentrations. These observations support the findings by Rich and 
co-workers (80) of anti-Z antibody binding to the 14-base pair alternating 
purine-pyrimidine segment in pBR322). The statistically significant 14 
base pair sequence (CACGGGTGCGCATG) in pBR322 shows alternation of purine 
and pyrimidine with one base out of register. Alternating 
purine-pyrimidine sequences tend to adopt the Z conformation. The plasmid 
pColE1 sequence contains no comparable stretch of alternation. 
IV. Cleavage Site Mapping 
Irradiation at 315 nm of .LAMBDA.-Co(DIP).sub.3.sup.3+ (10 .mu.M) solutions 
containing supercoiled pLP32 or pBR322 yields nicked circular form II 
DNAs. Photoreduction of Co(DIP).sub.3.sup.3+ enantiomers bound to DNA 
leads to oxidative single-strand scission at the DNA binding site (64). In 
order to establish that cleavage and therefore binding occurs at discrete 
sites, the scheme outlined in FIG. 11 was employed. Following irradiation 
of Co(DIP).sub.3.sup.3+ -DNA samples and the production of nicked circles, 
the DNAs were linearized using a restriction enzyme which is known to 
cleave the plasmid at only one site along the strand. Subsequent treatment 
with Sl nuclease, which is specific for single-stranded regions, cleaves 
the DNA only opposite to the cobalt-induced nick producing a pair of 
linear fragments. From the sizes of these fragments, determined based upon 
their gel electrophoretic mobilities relative to markers, the distance of 
the cleavage site from the restriction site origin may be obtained. In 
order to distinguish whether the site is clockwise or counterclockwise to 
the origin, at least two restriction enzymes which cut at sufficiently 
distinct locations were examined. It is important to notice that this 
procedure yields distinct fragments only if binding and subsequent 
cleavage occurs at discrete sites. Non-specific cleavage produces 
fragments of all sizes and hence a smear on the gel; thus the presence of 
some contaminating form II DNA just alters the background intensity. 
Control experiments of sample irradiated without cobalt or cobalt binding 
but without irradiation yielded no distinct bands. Non-specific DNA damage 
as a result of irradiation was negligible. Controls showed that full 
linearization of the plasmid was essential, however, to avoid mapping Sl 
hypersensitive sites (62). Some restriction enzymes did not yield complete 
linear digests, either because of thymine dimer formation at the 
restriction site or inhibition due to Co(DIP).sub.3.sup.3+ reaction, and 
these were not used. Finally, samples were irradiated only for short times 
so that no more than one nick per plasmid would occur. The fact that the 
sizes of pairs of fragments must sum to 4363 base pairs provided a useful 
experimental redundancy. By this general procedure the coarse map of 
.LAMBDA.-Co(DIP).sub.3.sup.3+ cleavage sites in any plasmid may be 
obtained. 
The plasmid pLP32 which contains a Z-DNA segment at a well-defined location 
(29, 81) was examined first. The plasmid had been constructed by inserting 
a d(CG).sub.32 fragment into the filled-in BamHI site (position 375) of 
pBR322 (29). The densitometric scan of the AvaI digest after reaction with 
.LAMBDA.-Co(DIP).sub.3.sup.3+ is shown in FIG. 12. In addition to the 
linear form several bands and shoulders are evident; their sizes in 
kilobase pairs (kb) are indicated in the figure. The appearance of the 
pair of fragments at 3.3 kg and 1.1 kg from the AvaI site (position 1424) 
shows that a major cleavage point is indeed at the Z site. Parallel 
digestion with Ndel established this position uniquely. More interesting 
perhaps is the comparison to the AvaI digest of pBR322, the same plasmid 
but lacking the insert. The pattern here is identical except that it lacks 
the 3.3 kb and 1.1 kb fragments. In pLP32, then, the cobalt complex must 
recognize and cleave a site not present in pBR322, the Z-form d(CG).sub.32 
insert. The result demonstrates that the complex can cleave specifically 
at a left-handed site. 
Other conformations are not similarly accessible to the chiral cobalt 
complex. .LAMBDA.-tris(diphenylphenanthroline) complexes or ruthenium(II) 
and cobalt(III) do not react as assayed spectrophotometrically (34, 63) 
(for ruthenium) and by cleavage assays (64) (for cobalt) with B-form 
helices. The delta isomer in contrast can bind both B- and Z-forms and 
photolysis experiments using .DELTA.-Co(DIP).sub.3.sup.3+ show 
non-specific cleavage of the linear DNA but with some specific band 
formation. It has also been found that (phen).sub.3 Ru.sup.2+ complexes do 
not bind significantly to double-stranded RNA and hence it is unlikely 
that .LAMBDA.-Co(DIP).sub.3.sup.3+ would recognize an A-form helical 
conformation. Additionally, racemic Co(DIP).sub.3.sup.3+ cleavage of 
single-stranded phage DNA X174 was examined. Here after photolysis about 
12% cleavage was observed, less than the 15% double-stranded content in 
the X174 sample, calculated based upon hypochromicity. It is unlikely then 
that Co(DIP).sub.3.sup.3+ enantiomers could recognize open looped regions 
of a plasmid. Instead the only DNA conformation for which appreciable 
binding and cleavage by .LAMBDA.-Co(DIP).sub.3.sup.3+ has been found is 
Z-DNA. 
Photolysis and digestion of both pLP32 and pBR322 actually yields several 
distinct fragments, seen in FIG. 12, and therefore additional cleavage 
sites for .LAMBDA.-Co(DIP).sub.3.sup.3+, similar structurally to the 
lefthanded d(CG).sub.32 insert must be present in these plasmids. Based 
upon numerous trials using either EcoRI, BamHI, AvaI, or NdeI for 
linearization, there appears to be four discrete cleavage sites in pBR322, 
given in order of intensity 1.45.+-.0.05 kb, 3.3.+-.0.1 kb&gt;4.24.+-.0.02 
kb&gt;2.25.+-.0.07 kb. The standard deviations are based upon averaging at 
least seven experiments. The plasmid pLP32 shows cleavage at these same 
positions in addition to cleavage at the insert. FIG. 13A shows a typical 
EcoRI digest. Fragment pairs are evident and, since EcoRI linearizes at 
the origin, the lengths of one fragment of the pair shows the position in 
kilobases of the site. The relative intensities, weighted by the fragment 
molecular weight, reflects either the relative site affinity for 
.LAMBDA.-Co(DIP).sub.3.sup.3+ or relative cleavage efficiency at a site. 
Variations in relative site intensities as a function of irradiation time 
and also as a function of salt concentration in the incubation mixture was 
observed. The influence of salt and superhelical density on the relative 
expression of these sites is currently being examined. The weakest site 
recognized is consistently at 2.3 kb. Table 1 summarizes the specific 
sites in pBR322 found with cleavage by .LAMBDA.-Co(DIP).sub.3.sup.3+. 
TABLE 1 
______________________________________ 
.LAMBDA.-Co(DIP).sub.3.sup.3+ Cleavage Sites 
Alternating 
Purine-Pyrimidine 
Sequences 
______________________________________ 
1.45 .+-. 0.05 kb 
1447-1460 
CACG .sub.--GGTGCGCATG 
2.25 .+-. 0.07 kb 
2315-2328 
CGCACA .sub.--GATGCGTA 
3.32 .+-. 0.11 kb 
3265-3277 
GTATATATG .sub.--AGTA 
4.24 .+-. 0.02 kb 
4254-64 
T .sub.--CCGCGCACAT 
______________________________________ 
V. In vitro Screening 
Diphenyl tris complexes of this inventions were screened for cytotoxic 
activity against mouse leukemia cells as previously described. The results 
of two of the complexes are set forth in Table 2. 
TABLE 2 
______________________________________ 
Cytotoxicity of Cobalt and Ruthenium Complexes 
Compound Cell Line.sup.1 
ID.sub.50 (.mu.g)/ml) 
______________________________________ 
Ru(DIP).sub.3 Cl.sub.2 
L 1210 4.49 
P 815 5.42 
Co(DIP).sub.3.(d-tartrate).sub.3 
L 1210 0.51 
P 815 0.52 
______________________________________ 
.sup.1 L 1210 and P 815 are mouse leukemia cell lines, see Burchenal, J. 
H. et al., CANCER RESEARCH, 42:2598-6000 (July 1982) 
DISCUSSION 
I. Tris(phenanthroline)ruthenium(II) 
Results of experiments described above indicate that 
tris(phenanthroline)ruthenium(II) binds to DNA by intercalation. The 
optical changes on binding to DNA agree with those seen for other 
intercalators. Hypochromicity in the metal to ligand charge-transfer 
(MLCT) band is observed and represents an overall 17% decrease in 
intensity. Stacking interactions with the base pairs lead to hypochromic 
shifts in the II.fwdarw.II* transitions of organic intercalating dyes, and 
it is interesting that the II symmetry of the MLCT preserves the 
hypochromic effect. Substantial increases in the luminescence of 
(phen).sub.3 Ru.sup.2+ also accompany binding to the duplex. The 
enhancement in emmission and corresponding increased luminescent lifetimes 
may simply reflect the decreased mobility of the complex when sandwiched 
into the helix. Emission lifetimes are comparable to those found for 
(phen).sub.3 Ru.sup.2+ in sodium lauryl sulfate micelles.(82) In addition 
to perturbations in the electronic structure of the bound reagent, 
intercalation leads to hydrodynamic changes in the DNA duplex. With 
increasing concentrations, (phen).sub.3 Ru.sup.2+ reversibly unwinds and 
rewinds superhelical DNA. Although not absolutely definitive,(4) this 
result provides a very strong indication of intercalation. Surely helical 
unwinding and lengthening accompany the binding of (phen).sub.3 Ru.sup.2+. 
Finally the binding isotherms obtained by equlibrium dialysis yield 
parameters that are reasonable for the intercalative mode of association. 
The complex binds to duplex DNA with relatively low affinity and, when 
bound, encompasses a four base-pair site. The octahedral coordination 
around the metal precludes effective stacking of the complex between base 
pairs. if the complex is viewed with one of the three phenanthroline 
ligands inserted into the helix, then the other two ligands actually 
protrude above and below the face of this phenanthroline and decrease the 
effective area of overlap. Hence only partial insertion is possible, which 
accounts for the low binding constant. The fact that so small a region of 
overlap with only partial insertion is necessary for a stabilizing 
interaction with the duplex is interesting to consider with respect to the 
binding of aromatic amino acid residues to DNA. The four base-pair site 
size is similarly consistent with the structural model for the bound 
complex, presented herein, where one ligand intercalates and the remaining 
two ligands span the groove of the helix. A site size of four base pairs 
is understandable since the internuclear distance of 10.4 A between distal 
hydrogen atoms on the ligands not only exceeds the 10.2 A of a single 
interbase pair site but must result in partial blockage of the next 
neighboring base pair both above and below the plane of insertion. 
Furthermore the direct comparison between enantiomers of spectroscopic 
features, binding properties, and structural parameters establishes that 
the .DELTA. enantiomer possesses the greater affinity for a right-handed 
helix. Intercalation of tris(phenanthroline)ruthenium(II) into the duplex 
imposes different steric constraints on .DELTA. and .LAMBDA. isomers, and 
it is this difference that determines the enantiomeric selectivity. 
Perhaps the strongest evidence in support of intercalation is the observed 
chiral discrimination. The .DELTA. enantiomer, a right-handed 
propeller-like structure, displays a greater affinity than 
.LAMBDA.-(phen).sub.3 Ru.sup.2+ for the right-handed DNA helix. FIG. 14 
illustrates the basis for the enantiomeric selectivity. With one 
phenanthroline ligand intercalated, the two nonintercalated ligands of the 
.DELTA. isomer fit closely along the right-hand helical groove. The 
nonintercalated ligands of the .LAMBDA. enantiomer, in contrast, are 
repelled sterically by the phosphate backbone of the duplex. The 
disposition of the left-handed enantiomer is opposed to the right-handed 
helical groove. The stereoselectivity seen here is in the direction 
proposed originally for (phen).sub.3 Zn.sup.2+ and supports the assignment 
of the absolute configurations for the zinc enantiomers. No 
stereoselectivity is apparent in the association of (phen).sub.3 Ru.sup.2+ 
with Z-DNA. This left-handed helix does not contain a groove of size and 
depth comparable to that in B-DNA, and therefore comparable or actually 
mirror image steric constraints are not expected. Instead the base pairs 
in the Z-DNA helix are pushed outward toward the solvent, resulting in at 
most a very wide and shallow major "groove". Hence Z-DNA provides a poor 
template for this discrimination. 
Although it is .DELTA.-(phen).sub.3 Ru.sup.2+ that binds preferentially to 
B-DNA, the .LAMBDA. enantiomer does intercalate into the right-handed 
helix. The ratio of affinities of .DELTA. and .LAMBDA. isomers for B-DNA 
is 1.1-1.5, depending upon the method of analysis. Luminescence 
enhancements and unwinding experiments with supercoiled DNA suggest the 
.DELTA. isomer to bind 30-50% more strongly. It is interesting that 
supercoiling does not alter the selectivity. Optical enrichment assays, 
which can be extremely sensitive and reflect a direct competition between 
enantiomers for the helix, yield values of 10-30% greater affinity of 
.DELTA.-(phen).sub.3 Ru.sup.2+ for calf thymus DNA. A more precise 
determination of relative affinities is difficult because the binding 
constant of either enantiomer for the helix is low. In fact, then, the 
enantiomer can bind to the right-handed helix, although the phosphate 
backbone limits access. The addition of bulky substituents onto the 
phenanthroline rings, in severly blocking interactions of the left-handed 
enantiomer with the duplex, is necessary to prevent completely 
intercalation of the .LAMBDA. isomer. 
These results provide an example of stereospecific interactions with DNA. 
The stereoselectivity observed is governed by the handedness of the DNA 
helix. The asymmetric duplex structure serves as a template which 
discriminates in binding the small molecules on the basis of their 
chirality. It is interesting that the change in symmetry of the metal 
complex alone yields a significant difference in its recognition by the 
helix. The comparison of spectroscopic and binding characteristics of 
isomers of (phen).sub.3 Ru.sup.2+ has afforded a detailed description of 
the structural basis for the enantiomeric selectivity observed first for 
(phen).sub.3 Zn.sup.2+ (15). The difference in biological activities of 
tris-(phenanthroline)metal enantomers is, perhaps, also a function of this 
stereoselectivity.(83) Indeed the interaction of (phen).sub.3 Ru.sup.2+ 
with DNA illustrates how stereospecificity may be incorporated into the 
design of drugs that bind to the duplex and provides a means to design 
reagents that can distinguish the handedness of the DNA helix.(35, 63, 64) 
Certainly these stereospecific interactions underscore the ability of 
small intercalating drugs to recognize differences in nucleic acid 
structure. 
II. Tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) 
The chiral DIP complexes, as shown by the experiments described above serve 
as specific chemical probes for the handedness of the DNA helix in 
solution. Spectrophotometrics titrations have shown that, although one 
RuDIP enantiomer, assigned as .LAMBDA.-RuDIP, does not bind at all to the 
B-DNA helix, the bulky asymmetric cation can bind to Z-DNA. Monitoring the 
binding of this isomer to DNA by any means therefore equivalently assays 
the helical conformation. The intense metal-to-ligand charge-transfer band 
in the ruthenium complexes provides a particularly sensitive handle with 
which to examine the binding, either spectrophotometrically or through its 
accompanying luminescence. 
Striking enantiometic selectivity is found in the interactions of the RuDIP 
cations with right-handed B-DNA, and this chiral discrimination is 
consistent with an intercalative model. The changes in the visible 
spectrum of RuDIP, i.e., the hypochromic shift and luminescence 
enhancement observed in the presence of duplex DNA parallel in detail 
those seen in spectra of (phen).sub.3 Ru.sup.2+ as a function of DNA 
addition. It has been shown that (phen).sub.3 Ru.sup.2+ binds to B-DNA by 
partial intercalation of the phenanthroline ligand between the base pairs 
(34, 35). Given that generally the ruthenium-metal-to-ligand 
charge-transfer transition shows little sensitivity to solvent or 
environment (84, 85), the close resemblance of properties of RuDIP to 
(phen).sub.3 Ru.sup.2+ suggests that the cations bind the DNA in a similar 
fashion. Intercalation of the diphenylphenanthroline ligand between the 
helix base pairs requires that the phenyl groups rotate into the plane of 
the phenanthroline ligand. This rotation to a planar structure with 
minimal steric interactions between nearby hydrogen atoms can be 
accomplished by legthening the carbon-carbon bond between the phenyl and 
phenanthroline moieties. Equivalent structural distortions are seen in 
biphenyl, which is planar in the stacked solid lattice (86). Also, the 
extremely bulky tetrapyridyl-porphyrin cations, which require extensive 
distortion, are thought to bind to the DNA duplex by intercalation (87, 
88). Importantly, once rotated into the plane of the phenanthroline, the 
phenyl groups in RuDIP add substantially to the surface area available for 
overlap with the base pairs as compared with (phen).sub.3 Ru.sup.2+, and 
therefore greater stability of the bound ruthenium-DNA complex is 
expected. In fact, binding of RuDIP to DNA assayed by any method becomes 
evident at 10% of the concentration of (phen).sub.3 Ru.sup.2+, which 
reflects the increased affinity of RuDIP over (phen).sub.3 Ru.sup.2+ for 
B-DNA. Perhaps the clearest support for the intercalation model rests in 
the dramatic enhancement in stereoselectivity observed for RuDIP in 
comparison with (phen).sub.3 Ru.sup.2+. The phenyl groups, while 
facilitating intercalation of .DELTA.-RuDIP into the right-handed helix, 
completely preclude binding by the .LAMBDA. enantiomer. 
Corey-Pauling-Kolton space-filling models of the RuDIP complexes with a 
B-DNA helix are shown in FIG. 15. The orientations with respect to the 
helix are indicated in the accompanying sketches. The .LAMBDA. enantiomer, 
with one diphenylphenanthroline ligand intercalated, can fit very closely 
along the helical groove. The two nonintercalating ligands, with a 
disposition in line with the right-handed helix, abut the helical groove. 
These close hydrophobic interactions of the nonintercalated ligands are 
not possible with the mirror-image enantiomer. In contrast, as presented 
in FIG. 14, if one ligand (not visible) is oriented perpendicular to the 
helix axis, then the two remaining ligands of the .LAMBDA. enantiomer are 
disposed contrary to the right-handed groove. The ruthenium model must 
therefore be shown in front of the DNA helix in the figure, rather than 
intercalated, because the interaction of the phenyl groups with the 
DNA-phosphate backbone at the positions indicated by the arrows completely 
blocks access. Thus, the stereoselectivity that we see is determined by 
the steric constraints imposed by the asymmetry in the helix, its 
handedness. 
Just as the helix asymmetry can serve as a template to discriminate between 
RuDIP enantiomers, differential binding by the enantiomers may be used 
advantageously in determining the chirality of the helix. Table 3 
indicates a general scheme to probe helical conformations using RuDIP 
cations. Although .LAMBDA.-RuDIP does not bind to the right-handed B-DNA 
duplex, spectrophotometric titrations have shown significant binding to 
Z-DNA and therefore hypochromism of .LAMBDA.-RuDIP on addition of a test 
DNA sample may be used as an indication of the Z-conformation. It was 
particularly interesting to us to find that no stereoselectivity governs 
binding to the Z-form helix. The bulky cation likely avoids the very 
narrow helical crevice in the Z-DNA structure, and intercalative binding 
to the more shallow hydrophobic surface in Z-DNA, the equivalent of the 
major groove in the B-form, would not be expected to yield any chiral 
discrimination. Z-DNA does not mirror B-DNA in solution. Instead we 
predict that a left-handed but more B-like conformation (18, 89, 90) would 
yield a mirror-image selectivity. 
TABLE 3 
______________________________________ 
Scheme for probing DNA conformation with RuDIP enantiomers 
Reactivity 
With the With the 
.DELTA.isomer 
.LAMBDA.isomer 
DNA duplex conformation 
______________________________________ 
+ - Right-handed B-like 
+ + Left-handed Z-like or lacking a 
groove 
- - Unstacked or with base pairs 
inaccesible 
- + Left-handed B-like 
______________________________________ 
The chiral tris(diphenylphenanthroline) metal complexes may therefore be 
used in solution to examine DNA helical conformations: those of naturally 
occurring sequences, in the presence of drugs, and in protein-bound 
complexes. Furthermore, the reagents represent a new route for 
conformation-specific drug design. 
III. Cobalt(III) Complexes 
The DNA cleavage experiments described above are important in several 
respects. The photoactivated DNA cleavage reaction with 
Co(phen).sub.3.sup.3+ illustrates with a simple inorganic complex the 
notion of DNA strand scission mediated by a locally generated redox 
reaction. Reduction of Co(III) with perhaps concomitant hydroxide 
oxidation may be responsible for cleavage. With regard to applications, 
this photoactivated reaction should make possible "footprinting" as a 
function of time. Most importantly, the differential cleavage of ColE1 DNA 
by enantiomers of Co(DIP).sub.3.sup.3+ represents a clear example of a 
conformation-specific DNA cleaving molecule. This molecule will be useful 
in determining regions of Z-DNA conformation within long segments of 
native DNA. Moreover the high level of recognition of DNA conformation by 
these chiral inorganic complexes suggests the powerful application of 
stereospecificity in DNA drug design. 
IV. Cleavage Site Mapping 
Features unique to the plasmid recognition sites determined above with 
.LAMBDA.-Co(DIP).sub.3.sup.3+ were examined. A 
.LAMBDA.-Co(DIP).sub.3.sup.3+ is not a sequence-specific reagent and there 
is no sequence homology evident at these positions in pBR322. Instead 
.LAMBDA.-Co(DIP).sub.3.sup.3+ is a conformation-specific cleavage agent 
and it is the common left-handed conformation at these locations that is 
likely to be recognized by the cobalt complex. Alternating 
purine-pyrimidine sequences have been shown to adopt the Z-DNA 
conformation most readily, because alternative residues in Z-DNA have 
bases in the syn conformation. Inspection of the pBR322 sequence revealed 
that the .LAMBDA.-Co(DIP).sub.3.sup.3+ recognition sites included the 
longest runs of alternating purines and pyrimidines allowing for one base 
out of alternation. Table 1 shows also the alternating sequences that 
appear within one standard deviation of each measured recognition site. At 
positions 1447, 2315, 3265, and 4254 begin respectively 14, 14, 13, and 11 
base pair regions with alternating purine and pyrimidine residues having 
one mistake. These regions correspond essentially to one helical turn in a 
Z-DNA conformation and are the longest of such conformation homology 
within the plasmid. Sequences not recognized by 
.LAMBDA.-Co(DIP).sub.3.sup.3+ were then considered. There are several 
other sequences, beginning at 1171, 1533, and 1709, that also constitute 
11 base pair segments with one mistake that are not significantly cleaved 
by .LAMBDA.-Co(DIP).sub.3.sup.3+, and the longest sequence of alternation 
in the plasmid with no mistakes, 10 bp beginning at position 2785, is also 
not cleaved. At this stage it is not known whether the flanking sequences 
at these sites are affecting Z-DNA formation or 
.LAMBDA.-Co(DIP).sub.3.sup.3+ recognition. The sequences within the 
recognition sites detected do have a range of GC contents. It is 
interesting that the Z-DNA conformation in pBR322 has been detected at the 
1447 site in equally low salt buffers by a completely different route, 
crosslinking studies (80) with anti-Z-DNA antibodies, which lends 
confirmation to the Z-form assignment. Thus it is proposed that these four 
sites of alternating purine-pyrimidine residues adopt the Z-conformation 
under physiological conditions in native supercoiled pBR322 and are 
specifically recognized and cleaved by .LAMBDA.-Co(DIP).sub.3.sup.3+. 
It is interesting, finally, to ask whether these Z-DNA segments share some 
common biological function in this plasmid. pBR322, assembled from these 
naturally occurring plasmids, contains three genetically distinct coding 
regions, the tetracycline resistance genes, the .beta.-lactamase gene 
conferring ampicillin resistance, and the origin of replication. FIG. 13B 
shows the map of these genes in pBR322. It is curious to notice the 
correspondence in position between the ends of these discrete coding 
elements and the Z-DNA recognition sites. A single polypeptide in pBR322 
appears to be necessary for tetracycline resistance (91). The 3'-end of 
the region encoding this peptide is thought to be near the AvaI site at 
1425 bp; sequences upstream from the tetracycline resistance promotor 
(which begin at 45 bp) were lost in construction from pSC101. The 
.beta.-lactamase gene is defined upstream by the start site at 4201 with 
the -35 consensus region for the promoter ending at 4236, 18 bp away from 
the Z-form cleavage site (92). The 3'-end of the region encoding 
.beta.-lactamase is found at position 3295, which is 22 bp upstream of the 
Z-form alternating purine-pyrimidine site. Lastly the essential region 
constituting the origin of replication in pBR322 extends from the RNA/DNA 
junction at 2536 to position 2360, 32 base pairs from the weak Z-form site 
detected with .LAMBDA.-Co(DIP).sub.3.sup.3+ (65, 93). Thus there appears 
to be a remarkable correspondence between Z-DNA sites recognized by the 
cobalt complex and the ends of genetic coding elements. It is tempting to 
suggest that the Z-DNA conformation might provide a general structural 
signal or punction mark which demarcates the ends of these genes. 
Consistent with this idea, the Z-conformation has been shown to provide a 
poor template for transcriptional activity with E. coli RNA polymerase 
(94). This notion is consistent also with the location of alternating 
purine-pyrimidine tracts in SV40 DNA enhancer sequences which bind 
anti-Z-DNA antibodies (30), to the d(GT).sub.n tracts at the ends of yeast 
chromosomes (95), and to the alternating purine-pyrimidine long terminal 
repeats in mouse mammary tumor virus (96). Moreover recent experiments 
with mung bean nuclease in the malaria parasite Plasmodium have 
demonstrated that a particular conformation rather than a sequence appears 
to be shared by gene termination sites (97). These correlations of 
location with conformation are intriguing and support the notion that DNA 
polymorphism may be involved in gene expression, that specific sequences 
may adopt specific local conformation which contain information, and that 
chromosomal regulation may involve DNA conformation-specific signals. 
In sum, the results presented herein indicate that several discrete Z-DNA 
sites exist under native physiological conditions in pBR322. The positions 
of these sites mark the ends of genetically distinct coding elements in 
the plasmid. A cobalt complex of this invention, e.g. 
.LAMBDA.-Co(DIP).sub.3.sup.3+ thus provides a photoactivated site-specific 
cleaving agent that is useful to map these sites and should be helpful in 
establishing a relationship between the locations of Z-DNA segments and 
its biological function. 
V. Anti-tumor Activity 
Complexes of this invention showed high potency against leukemia cells in 
the previously described screen. These complexes should be useful as 
anti-tumor agents, and should be active in vivo as well, e.g. in a 
composition containing a pharmaceutically acceptable carrier. 
The results set forth in Table 2, above also indicate a synergistic effect 
when use of a cobalt complex is combined with ultraviolet radiation. A 
decrease in the amount of cobalt required for cell death of greater than 
about 10-fold, and in some cases up to 50-fold has thereby been observed. 
Isolation of chromatin showed extensive DNA cleavage. Hence it appears that 
the complexes can pass into the cell, remain intact therein and interact 
with DNA as a cellular target. 
Since these complexes contain large, planar ligands, selective 
intercalation is optimized. Individual enantiomers and mixed-ligand 
complexes should also be useful in this and other embodiments of the 
invention. The .LAMBDA. Z-specific enantiomers should be especially useful 
in anti-tumor compositions and uses. 
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