Triheterocyclic peptides capable of binding the minor and major grooves of DNA

The present invention provides a triheterocyclic peptide having first, second, and third 5-member heterocyclic moieties having the following formula CR.sub.3 CONH--Q.sup.1 --CONH--Q.sup.2 --CONH--Q.sup.3 -CONH--(CR.sub.3).sub.S --B, wherein Q.sup.1 is selected from a group consisting of: ##STR1## wherein Q.sup.2 is selected from a group consisting of: ##STR2## wherein Q.sup.3 is selected from a group consisting of: ##STR3## wherein at least one of A and Z is other than C; wherein A is C, N, O, or S; wherein B is N(CR.sub.3).sub.n or C(NH.sub.2).sub.2 ; wherein n is an integer from 2 to 10; wherein P is H, a lower alkyl or a halogen; wherein Q1, Q2, and Q3 are the same or different; wherein R is H, a lower alkyl group; wherein S is an integer from 1 to 10; wherein X' represents CR.sub.3, (CR.sub.2).sub.n --NRY, or (CR.sub.2).sub.n --CR.sub.2 Y; wherein X" represents CR.sub.3, (CR.sub.2).sub.n --NRY, or (CR.sub.2).sub.n --CR.sub.2 Y; wherein X'" represents CR.sub.3, (CR.sub.2).sub.n --NRY, or (CR.sub.2).sub.n --CR.sub.2 Y; wherein Y is a polyamine group, and wherein Z is C or N; wherein at least one of X', X", or X'" is other than CR.sub.3.

BACKGROUND 
The advancement of molecular biology and genetic engineering has led to a 
greater understanding of the structure and function of nucleic acids on 
the molecular level. This greater understanding has brought about a need 
for reagents that can specifically bind to nucleic acids, particularly 
DNA, without requiring a complementary base sequence for hybridization. 
These reagents can be used to block enzymes that act on nucleic acids as 
well as to detect nucleic acids with high sensitivity. 
Tripyrrole peptides are well known to bind weakly to some nucleic acids. 
Examples of such tripyrrole peptides include distamycin and netropsin and 
analogs thereof. 
Distamycin is an oligopeptide originally isolated from a culture of 
Streptomyces distallicus. Distamycin binds the minor groove of AT-rich 
sequences of B-DNA, directly affects the conformation of bound and 
flanking nucleotides, and exhibits antibiotic activity. The structure of 
Distamycin is as follows. 
##STR4## 
FCE24517 is an analog of distamycin. Unlike distamycin, FCE24517 possesses 
anti-tumor activity. However, both distamycin and FCE24517 are unstable 
molecules. Further, both bind weakly to DNA. The structure of FCE24517 is 
as follows. 
##STR5## 
Netropsin is an oligopeptide that binds the minor groove of DNA without 
intercalating between DNA bases. Typically, netropsin exhibits preference 
for AT stretches and has demonstrated the ability to interfere with the 
actions of DNA topoisomerases I and II. The structure of netropsin is as 
follows. 
##STR6## 
Microgonotropens are another class of tripyrrole peptides which bind to 
the minor groove of double stranded DNA (dsDNA) and extend its binding to 
the major groove. Microgonotropens are analogs of distamycin. 
Microgonotropens are capable of binding the minor groove of DNA sequence 
selectively, reaching up and out of the minor groove with their polyamine 
moieties, and firmly grasping the phosphodiester backbone. In so doing, 
the microgonotropens increase their binding affinities to DNA and alter 
the conformation of DNA. 
Microgonotropens, like the related lexitropsin minor groove binding agents 
distamycin and netropsin, have an affinity for A+T-rich regions. 
The central polyamine groups of the microgonotropens were designed to reach 
the phosphate backbone of the DNA, to point towards the major groove and 
be able to ligate a metal ion, thereby providing putative hydrolytic 
catalysis of the dsDNA or enhanced base pair recognition. 
Dien-microgonotropens are tripyrrole peptides which bind weakly to DNA 
(FIG. 15). They exhibit enhanced minor groove binding due to electrostatic 
interaction of the putative catalytic groups with the phosphodiester 
linkages. The structure of dien-microgonotropens are as follows. 
##STR7## 
Although dien-microgonotropens specifically bind the minor groove of DNA, 
there was a need to develop other reagents with increased binding to DNA 
with increased stability which distamycin and FCE24517 do not provide. 
Stable reagents with higher binding affinities to DNA would provide a more 
advantageous diagnostic or therapeutic agent. For example, a diagnostic 
agent with a high affinity for DNA would provide less false positives when 
a more stringent washing condition is required. Further, a therapeutic 
agent with a high affinity for DNA would remain bound to DNA despite the 
presence of other competing agents having a lower affinity and/or an 
increased concentration. 
SUMMARY OF THE INVENTION 
The present invention provides a triheterocyclic peptide useful for binding 
DNA. The triheterocyclic peptide is one that contains three cyclic 
compounds. Such cyclic compounds may be a pyrrole, furan, thiophene, 
imidazole, oxazole, thiazole, and pyrazole. 
The triheterocyclic peptide has first, second, and third heterocyclic 
rings. One, two or three of these heterocyclic rings have a polyamine 
group attached thereto. The polyamine group extends from a heteroatom of 
the heterocyclic ring towards the phosphate backbone and major groove of 
DNA. 
Additionally, the polyamine groups of the triheterocyclic peptide attach to 
metal ions, phosphate substituents, and/or the floor of the major groove 
of DNA. 
In one embodiment of the invention, the triheterocyclic peptide of the 
invention has the following formula: 
##STR8## 
X' is CR.sub.3, (CR.sub.2).sub.n --NRY, or (CR.sub.2).sub.n --CR.sub.2 Y. 
X" is CR.sub.3, (CR.sub.2).sub.n --NRY, or (CR.sub.2).sub.n --CR.sub.2 Y. 
Additionally, X'" is CR.sub.3, (CR.sub.2).sub.n --NRY, or (CR.sub.2).sub.n 
--CR.sub.2 Y. The R group in the triheterocyclic peptide is a hydrogen (H) 
atom, a lower alkyl group, or halogen atom. 
In another embodiment, the microgonotropen is a tren-microgonotropen having 
the following formula: 
##STR9## 
wherein (CR.sub.2).sub.n represents an alkyl linker of varied chain 
length, n is an integer of between 2-10. R represents H or a lower alkyl 
group. 
Tren-microgonotropens are tripyrrole peptides having first, second, and 
third pyrrole rings. A pyrrole ring is a five atom aromatic ring having 
nitrogen at position 1 of the ring. 
The peptide of the invention is capable of binding DNA. By so doing, the 
peptide is capable of prohibiting the binding of DNA with an enzyme, for 
example, topoisomerase I, which is important in DNA replication and/or 
genetic expression. 
Generally, the peptide of the invention has a polyamine group attached to 
the nitrogen atom of one of the pyrroles of the tripyrrole peptide. In a 
preferred embodiment, the polyamine group is attached to the nitrogen (N) 
atom of the second pyrrole of the tripyrrole peptide. 
Further, the peptide has the following characteristics. The peptide is 
capable of binding the minor groove of DNA with an equilibrium constant of 
.gtoreq.10.sup.9 M.sup.-1. Additionally, the peptide is incapable of 
alkylating the enzyme or DNA. 
In one embodiment of the present invention, the peptide is capable of 
binding the minor and major grooves of DNA so as to alter the conformation 
of DNA. This peptide has a polyamine group attached to the nitrogen atom 
of the second pyrrole of the tripyrrole peptide. The peptide has the 
following formula: 
##STR10## 
wherein n is 3, 4, or 5.

DETAILED DESCRIPTION OF THE INVENTION 
PEPTIDES OF The INVENTION 
The present invention provides five-membered triheterocyclic peptides 
useful for binding DNA. The five-membered triheterocyclic peptides include 
first, second, and third heterocyclic moieties. Each of the first, second, 
and third heterocyclic moieties can be a pyrrole, a furan, a thiophene, an 
imidazole, an oxazole, a thiazole or a pyrazole, (J. Am. Chem. Soc. 1988, 
110, 3641-3649; J. Am. Chem. Soc. 1992, 114, 5911-5919; J. Am. Chem. Soc. 
1933, 115, 7061-7071). 
The heterocyclic moieties of the triheterocyclic peptide may be the same or 
different, i.e., the first heterocyclic compound may be the same or 
different from the second or third heterocyclic moiety. Alternatively, the 
second heterocyclic compound may be the same or different from the first 
or third heterocyclic moiety. Further alternatively, the third 
heterocyclic compound may be the same or different from the first or 
second heterocyclic moiety. 
A polyamine group is attached to the first, second, and/or third 
heterocyclic moieties of the triheterocyclic peptide. The polyamine group 
comprises a methylene linker which extends up from a ring nitrogen, 
towards the phosphate backbone and major groove of DNA and a ligand to 
which the methylene linker is attached. The ligand binds metal ions, 
phosphate substituents, and/or the floor of the major groove of DNA. At 
least one but no more than three polyamine groups are present on the 
triheterocyclic peptides of the invention. 
In one embodiment of the present invention, the triheterocyclic peptide is 
a tripyrrole peptide having first, second, and third pyrrole rings having 
the following formula: 
##STR11## 
The R group in the triheterocyclic peptide is a hydrogen (H) atom, a lower 
alkyl group, or halogen atom. A lower alkyl group includes aliphatic 
hydrocarbons having between one to five carbon atoms. 
Each of X', X", and X'" is CR.sub.3, (CR.sub.2).sub.n -NRY, or 
(CR.sub.2).sub.n -CR.sub.2 Y. At least one of X', X", or X'" is other than 
CR.sub.3. n is an integer from 2 to 10. Y is a polyamine group. 
In accordance with the practice of the invention, when the five membered 
heterocyclic moiety is a pyrrole, imidazole, pyrazole, 3-pyrroline, or 
pyrrolidine, the polyamine linkers extend from the ring nitrogen(s) 
towards the phosphate backbone and major groove. Typically, the peptide of 
the invention is a tripyrrole peptide. 
Preferably, the peptide of the invention is capable of binding DNA. As a 
result of such binding, the peptides of the invention prevent or inhibit 
the binding of DNA with an enzyme important in DNA replication and/or 
genetic expression. 
The peptide of the invention has a polyamine group attached to the nitrogen 
atom of the second pyrrole of the tripyrrole peptide. Further, it has the 
following characteristics. In one embodiment, the peptide of the invention 
is capable of binding the minor groove of DNA with an equilibrium constant 
of .gtoreq.10.sup.9 M.sup.-1. Further, the peptide of the invention is 
incapable of alkylating the enzyme or DNA. 
In one embodiment of the invention, the tripyrrole peptide has a first, 
second, and third pyrrole ring. Moreover, the peptide is capable of 
binding the minor and major grooves of DNA. This binding alters the 
conformation of DNA. In this embodiment, the peptide having a polyamine 
group attached to the nitrogen atom of the second pyrrole of the 
tripyrrole peptide, the peptide having the formula: 
##STR12## 
In accordance with the practice of the invention, (CH.sub.2).sub.n is an 
alkyl linker of varied chain length. Preferably, n is 2, 3, 4, 5, 6, 7, 8, 
9, or 10. R is hydrogen, a lower alkyl, or a halogen such as fluorine, 
chlorine, bromine, and iodine. 
In one embodiment, the tripyrrole peptide is a tren-microgonotropen 
molecule. For example, the tren-microgonotropen molecule comprises a 
polyamine group having the formula --(CH.sub.2).sub.3 NHCH.sub.2 CH.sub.2 
N(CH.sub.2 CH.sub.2 NH.sub.2).sub.2. This polyamine group is attached to 
the molecule at the second pyrrole of the tripyrrole peptide. 
In another embodiment, the polyamine group of tren-microgonotropen has the 
formula --(CH.sub.2).sub.4 NHCH.sub.2 CH.sub.2 N(CH.sub.2 CH.sub.2 
NH.sub.2).sub.2. This polyamine group is attached to the molecule at the 
second pyrrole of the tripyrrole peptide. 
In one embodiment, the polyamine group of the triheterocyclic peptide is a 
molecule which binds the major groove of DNA through the phosphodiester 
linkage and is a lower alkyl group substituted with least one nitrogen 
atom. An example includes cyclen derivative such as 
1,4,7,10-tetraazacyclododecane. 
The structures of two cyclen derivatives are as follows: 
##STR13## 
A is the attachment site to the heterocyclic compound. 
Another suitable polyamine group includes derivatives of 
1,4,7-triazacyclononane having the following structures: 
##STR14## 
A is the attachment site to the heterocyclic compound. 
Additionally, trpn derivatives are suitable polyamine groups of the 
invention. For example, tris(3-aminopropyl)amine is a suitable trpn 
derivative having the following structure: 
##STR15## 
A is the attachment site to the heterocyclic compound. 
Further, suitable examples of polyamine groups include derivatives of 
1,5,9-triazacyclododecane. Some chemical structure of such derivative are 
as follows. 
##STR16## 
A is the attachment site to the heterocyclic compound. 
Additionally, other polyamine groups include the following: 
##STR17## 
A is the attachment site to the heterocyclic compound. 
In the above-described polyamine groups, A indicates the attachment site 
and the wavy line indicates that the substituent could have either a R or 
a S chiral center. Further, m is an integer of 1 to 5. 
In another embodiment of the invention, the peptide of the invention 
exhibits nonintercalative binding to DNA. In this case, the polyamine 
group is capable of forming a complex with a metal ion. Alternatively, or 
additionally, the polyamine group includes four aliphatic amino groups. 
Two of the aliphatic amino groups may be primary amino groups. 
Alternatively, or additionally, one of the aliphatic amino groups may be a 
secondary amino group. Further, in one embodiment of the invention, one of 
the aliphatic amino groups is a tertiary amino group. 
In another embodiment of the invention, the amino terminus of the peptide 
is acetylated. 
In yet another embodiment of the invention, the carboxyl terminus of the 
peptide has an amide linkage to .beta.-(N,N-dimethylamino)propylamine. 
In a further embodiment, the ring nitrogen of the first and third pyrrole 
rings are N-methylated. 
Additionally, in yet a further embodiment, the peptide of the invention 
binds the minor groove of DNA at A+T-rich regions of DNA. 
The present invention further provides a tren-microgonotropen having the 
formula 
##STR18## 
wherein n represents an alkyl linker of varied chain length. Preferably, n 
represents 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the 
tren-microgonotropen is designated 6b as shown in FIG. 15. 6b binds into 
the minor groove at A+T-rich regions of DNA. 
Microgonotropen 6b possesses five aliphatic amino groups: two primary, one 
secondary and one tertiary in the tren substituent (--CH.sub.2 CH.sub.2 
CH.sub.2 CH.sub.2 NHCH.sub.2 CH.sub.2 N(CH.sub.2 CH.sub.2 NH.sub.2).sub.2) 
and one tertiary in the dimethyl propylamino tail (--CH.sub.2 CH.sub.2 
CH.sub.2 N(CH.sub.3).sub.2). The extent of their protonation when 6b is 
lodged in the minor groove is not certain. 
Further, as shown above, when compared with 5c and distamycin, only 6b is 
able to effectively compete with the enzyme topoisomerase I (topoI) once 
the enzyme is bound. Apparently, binding of 6b to DNA alters the 
conformation of DNA. Such an altered DNA conformation could inhibit topoI 
by either preventing enzyme binding to or "tracking" along DNA, or by 
generating conformationally uncleavable sites. 
The three primary amines of 6b's tren amino substituent are located within 
1.75 .ANG. of two phosphodiester oxyanions while the fourth amine 
(tertiary) is 3.0 .ANG. from the same two adjacent phosphodiester 
oxyanions. 
The increased binding affinity of 6b over distamycin is likely due to the 
electrostatic interactions of the polyamino side chain with the 
phosphodiester linkages. The central polyamino groups of 6b are 
significant to its binding affinity. 
6b binds to the A+T-rich region of dsDNA involving one G.C residue flanking 
the A.T binding sites. Unlike 5c, the --CH.sub.2 CH.sub.2 CH.sub.2 
N(CH.sub.3).sub.2 tail of 6b is completely within the minor groove. This 
also contributes to its increased binding affinity to DNA. 
The tren substituent of 6b (--(CH.sub.2).sub.4 NHCH.sub.2 CH.sub.2 
N(CH.sub.2 CH.sub.2 NH.sub.2).sub.2) interacts with two adjacent 
phosphates; this increases 6b's affinity for DNA. The efficiency of 
binding of the tren substituent of 6b (as seen by the embedding of the 
tripyrrole peptide in the minor groove) when compared with the dien 
substituent of 5c can be ascribed to the smaller steric effect around the 
terminal amino groups of the tren allowing a better pairing with the 
phosphate backbone of dsDNA. 
Tren-microgonotropen-b, 6b, (i) penetrates deeper into the minor groove of 
dsDNA than 5c, (ii) exhibits a stronger interaction with the phosphate 
backbone as compared to 5c, and (iii) has a hydrocarbon linker between the 
tripyrrole peptide and the tren substituent that is shorter than the 
linker in 5c. 
METHODS OF MAKING THE PEPTIDES OF THE INVENTION 
FIG. 16 is a schematic diagram showing the organic synthesis of 
tren-microgonotropen as described in Example I. 
In the embodiment when R is a halogen, attachment of the halogen to pyrrole 
carbons is by methods known in the art (J. Org. Chem. 1987 52, 3493-3501). 
Further, attachment of aliphatic groups on the carbons of pyrrole groups 
is by methods known in the art (J. Org. Chem. 1987, 52, 3493-3501). 
Further, attachment of alkyl groups, e.g., a propyl group (Cold Spring 
Harbor Symp. Quant. Biol. 1983, 47, 367-378) or an isoamyl (Bioorg. Khim. 
1982, 8, 1070-1076) group, to the ring nitrogen of a pyrrole compound is 
by methods known to those skilled in the art. Additionally, the methylene 
tail of the dimethylamine carboxy terminus may be of various lengths (J. 
Am. Chem. Soc. 1988, 110, 3641-3649). Moreover, other positively charged 
groups may replace the dimethylamine terminus (i.e., amidine (J. Am. Chem. 
Soc. 1988, 110, 3641-3649)). 
In another embodiment, FIG. 30 is a schematic diagram showing the organic 
synthesis of a microgonotropen molecule having two polyamine groups to 
bind DNA. 
METHODS OF USING THE PEPTIDES OF THE INVENTION 
The invention provides a method for inhibiting the replication of DNA. This 
method comprises contacting the peptide of the invention (for example, 
tren-microgonotropen) with DNA so that the peptide and DNA form a 
peptide-DNA complex. The complex binds tightly and/or induces a 
conformational change in the DNA so complexed. 
The peptide of the invention prevents binding of an enzyme necessary for 
DNA replication with the peptide-DNA complex so as to inhibit the 
replication of DNA so complexed. 
In one embodiment, the enzyme is a topoisomerase. The topoisomerase may be 
mammalian topoisomerase I. 
The present invention provides a composition comprising an effective amount 
of the peptide of the invention and a suitable pharmaceutical carrier. 
Compositions of the invention can be administered using conventional modes 
of administration including, but not limited to, intravenous, 
intraperitoneal, oral, intralymphatic or administration directly into the 
tumor. 
Alternatively, the compositions of the invention may be applied topically, 
like distamycin A, a related compound, in the form of a 1% ointment or 
paste for cutaneous or mucocutaneous infections produced by herpes 
simplex, varicella-zoster, and vaccinia viruses. 
In accordance with the practice of this invention, the composition of the 
invention may be administered to a subject such as human, equine, porcine, 
bovine, murine, canine, feline, and avian subjects. Other warm blooded 
animals may also be administered with the peptide of the invention. 
The most effective mode of administration and dosage regimen for the 
compositions of this invention depends upon the severity and course of the 
disease, the patient's health and response to treatment and the judgment 
of the treating physician. Accordingly, the dosages of the compositions 
should be titrated to the individual patient. 
An effective dose of the compositions of this invention may be in the range 
of from about 1 to about 2000 mg/m.sup.2. Additionally, as a guideline for 
determining dosages of the triheterocyclic peptide of the invention, it is 
important to keep in mind that the LD.sub.50 of distamycin A in mice is 75 
mg/kg i.v. and 500 mg/kg i.p. 
The molecules described herein may be in a variety of dosage forms which 
include, but are not limited to, liquid solutions or suspensions, tablets, 
pills, powders, suppositories, polymeric microcapsules or microvesicles, 
liposomes, and injectable or infusible solutions. The preferred form 
depends upon the mode of administration and the therapeutic application. 
The most effective mode of administration and dosage regimen for the 
molecules of the present invention depends upon the location of the tumor 
being treated, the severity and course of the cancer, the subject's health 
and response to treatment and the judgment of the treating physician. 
Accordingly, the dosages of the molecules should be titrated to the 
individual subject. 
The interrelationship of dosages for animals of various sizes and species 
and humans based on mg/m.sup.2 of surface area is described by Freireich, 
E. J., et al. Cancer Chemother., Rep. 50 (4): 219-244 (1966). Adjustments 
in the dosage regimen may be made to optimize the tumor cell growth 
inhibiting and killing response, e.g., doses may be divided and 
administered on a daily basis or the dose reduced proportionally depending 
upon the situation (e.g., several divided doses may be administered daily 
or proportionally reduced depending on the specific therapeutic situation. 
It would be clear that the dose of the composition of the invention 
required to achieve cures may be further reduced with schedule 
optimization. 
In accordance with the practice of the invention, the pharmaceutical 
carrier may be a lipid carrier. The lipid carrier may be a phospholipid. 
Further, the lipid carrier may be a fatty acid. Also, the lipid carrier 
may be a detergent. As used herein, a detergent is any substance that 
alters the surface tension of a liquid, generally lowering it. 
In one example of the invention, the detergent may be a non-ionic 
detergent. Examples of non-ionic detergents include, but are not limited 
to, polysorbate 80 (also known as Tween 80 or (polyoxyethylenesorbitan 
monooleate), Brij, and Triton (for example Triton WR-1339 and Triton 
A-20). 
Alternatively, the detergent may be an ionic detergent. An example of an 
ionic detergent includes, but is not limited to, alkyltrimethylammonium 
bromide. 
Additionally, in accordance with the invention, the lipid carrier may be a 
liposome. As used in this application, a "liposome" is any membrane bound 
vesicle which contains any molecules of the invention or combinations 
thereof. 
The present invention further provides a method for inhibiting the growth 
of tumor cells. This method comprises contacting tumor cells with an 
effective tumor growth-inhibiting amount of the composition of the 
invention. 
The peptide of the invention has many diagnostic in vitro uses. For 
example, since the peptide binds the major and minor grooves of DNA, the 
peptide may be used to detect the presence of DNA. Frequently, assays 
require DNA free samples. The presence of DNA can often cause false 
positives to increase. Alternatively, the presence of DNA may prohibit the 
proper binding of the reagents. Therefore, no binding occurs. Further, in 
some assays the detection of DNA or RNA, e.g., viral DNA or RNA may be 
useful. 
For example Creuzfeld-Jakob disease is a neurodegenerative disease caused 
by transmissible agents that cause slow, progressive neuronal loss. Even 
after extensive efforts no viral DNA or RNA has been demonstrated as 
infectious material. Moreover, no one has ever determined whether an 
immune response to the virus has been exhibited. 
The neuropathology of Creuzfeld-Jakob is characterized by formation of 
amyloid plaques (insoluble protein deposits), spongiform encephalopathy 
(the appearance of prominent vacuoles in cells), and gliosis (reaction 
proliferation of glia). 
It would be useful to develop a routine screening assay for detecting the 
presence of viral DNA or RNA in infectious material. The DNA or RNA so 
detected may be isolated and identified as a marker for the disease. The 
peptides of the invention would be useful in making this determination. 
Another example of a situation in which the detection of trace amounts of 
nucleic acid is useful arises from the purification of a protein produced 
on a large scale by genetic engineering. Typically, these proteins are 
purified by affinity chromatography or other chromatographic procedures; 
the presence of small quantities of nucleic acid remaining from the 
purification procedure would be unwelcome. It would therefore be highly 
desirable to have a test by which the presence of small quantities of 
nucleic acid in such preparations could be detected, without having to 
rely on hybridization or another sequence-specific assay for nucleic acid. 
Detection of DNA or RNA can be accomplished using various methods by direct 
or indirect labeling methods. For example, using the peptides of the 
invention, a label is attached directly to the peptide by a covalent bond, 
or the label intercalates noncovalently between the double strand of the 
peptide:target complex. The latter method, indirect labeling, employs a 
specific binding partner (e.g., biotin) attached to the nucleic acid 
probe. The hapten is detected using a labeled specific binding protein 
(e.g., antibiotin, avidin, or streptavidin). A slightly more complex 
format uses an intermediate binding protein to bridge between the hapten 
and the labeled binding protein. Alternatively, a binding protein specific 
for double-stranded DNA can be used (e.g., monoclonal anti-dsDNA) and 
complexes are then detected using a labeled antispecies antibody. 
ADVANTAGES OF THE INVENTION 
The present invention has advantages over distamycin and analogs thereof. 
Tren-microgonotropens provide a 2-fold greater binding to DNA than 
generated by the dien-microgonotropens. Further, tren-microgonotropens are 
about twice as effective in inducing structural changes in DNA as are the 
dien-microgonotropens and at least four times as effective in inducing 
structural changes as is Distamycin (Dm). The structure of 
tren-microgonotropen is as follows. 
##STR19## 
Further, the microgonotropens of the present invention are advantageous 
over distamycin and analogs thereof since microgonotropens bind tightly to 
the minor groove of DNA since they are tripeptides of 
3-aminopyrrole-2-carboxylic acid. 
Also, in one embodiment, the ring nitrogens of two of the three pyrrole 
rings are N-methylated. Moreover, the ring nitrogen of the second pyrrole 
carries the ligand such as --(CH.sub.2).sub.3 NH(CH.sub.2).sub.2 
N{(CH.sub.2).sub.2 NH.sub.2 }.sub.2 (6a) and --(CH.sub.2).sub.4 
NH(CH.sub.2).sub.2 N{(CH.sub.2).sub.2 NH.sub.2 }.sub.2 (6b). 
Another advantage is that the electrophoretic mobilities of .phi.X-174-RF 
DNA HaeIII restriction fragments complexed to 6a or 6b revealed a much 
greater conformational change in the DNA fragments when compared to 
distamycin (Dm) bound to the same fragments. Further, the result of this 
greater conformational change is about a 2-fold greater binding to DNA 
than generated by the dien-microgonotropens. 
For example, complete inhibition of mammalian topoisomerase I with 30 .mu.M 
6b was observed while dien-microgonotropen-b and Dm only partially 
inhibited topoisomerase I at 150 .mu.M. 
Evidence from equilibrium constants for complexation, electrophoretic 
mobilities, and topoisomerase I assays suggests that 6b alters the 
conformation of DNA in a manner that is not directly related to the 
affinity of complexation. Further, the ability to alter the conformation 
of DNA with small organic molecules at selected sites may have profound 
consequences on influencing DNA modifying enzymes and on controlling 
regulation of genetic expression. 
In order that the invention described herein may be more fully understood, 
the following examples are set forth. It should be understood that these 
examples are for illustrative purposes only and are not to be construed as 
limiting the scope of this invention in any manner. 
EXAMPLE I 
Experimental Section 
Organic Synthesis Materials 
Reagent grade chemicals were used without purification unless otherwise 
stated. Methanol was refluxed and distilled from CaH.sub.2. 
Dimethylformamide (DMF) was dried by CaH.sub.2 overnight and distilled 
under reduced pressure. Triethylamine was dried by KOH and distilled. 
Methanol, DMF, and trimethylamine were stored over 4A molecular sieves. 
Tetrahydrofuran (THF) was refluxed with sodium (Na) metal and distilled 
before use. Tris(2-aminoethyl)amine, diisopropylethylamine, and diethyl 
cyanophosphonate (DECP) were purchased from Aldrich (Milwaukee, Wis.). 
2-(Trimethylsilyl)ethyl p-nitrophenyl carbonate and t-butyl 
S-4,6-dimethyl-pyrimid-2-yl thiocarbonate were purchased from Fluka 
(Ronkonkoma, N.Y.). After treatment with 1M NaOH, ion-exchange resin 
(Aldrich) was washed with distilled water and methanol before using. 
Ethyl 1-(3-propal)-4-nitro-2-pyrrole-carboxylate (7a), ethyl 
1-(4-butal)-4-nitro-2-pyrrolecarboxylate (7b), and dimethyl 
3-(1-methyl-4-nitro-2-pyrrolecarboxamido)-propionamine (11) were 
synthesized (He, G.-X.; Browne, K. A.; Groppe, J. C.; Blasko, A.; Mei, 
H.-Y.; Bruice, T. C. J. Am. Chem. Soc. 1993, 115, 7061). 
1-Methyl-4-nitro-2-pyrrolecarbonyl chloride was synthesized from reaction 
of 1-methyl-4-nitro-2-pyrrolecarboxylic acid, prepared by nitration of 
1-methyl-2-pyrrolecarboxylic acid (Aldrich) ((a) Bialer, M.; Yagen, B.; 
Mechoulam, R. Tetrahedron 1978, 34, 2389; (b) Lown, J. W.; Krowicki, K. J. 
Org. Chem. 1985, 50, 3774; (c) Youngquist, R. S. Ph. D. Dissertation, 
California Institute of Technology, 1988), and thionyl chloride according 
to the published methods (Lown, J. W., et al., supra; Rao, K. E.; Bathini, 
Y.; Lown, J. W. J. Org. Chem. 1990, 55, 728). 
General organic synthesis methods 
Infrared (IR) spectra were obtained in KBr or neat on a Perkin-Elmer 
monochromator grating spectrometer (Model 1330). Low-resolution mass 
spectra (LRMS) were recorded on a VG Analytical spectrometer (Model 
VGII-250) by fast atom bombardment (FAB) using m-nitrobenzyl alcohol (NBA) 
as a matrix. High-resolution mass spectrometry (HRMS) was performed at the 
Midwest Center for Mass Spectrometry Laboratory at the University of 
Nebraska (Lincoln, Nebr.) using FAB technique and NBA matrix. .sup.1 H NMR 
spectra were obtained in CDCl.sub.3 or in DMSO-d.sub.6 with a General 
Electric GN-500 spectrometer (Blasko et al., 1993, supra). Chemical shifts 
are reported in sigma (ppm) (sigma means units of chemical shifts) 
relative to CHCl.sub.3 (7.24 ppm) or to DMSO (2.49 ppm) with s, d, t, q, 
and m signifying singlet, doublet, triplet, quartet, and multiplet; 
coupling constants (J) are reported in hertz (Hz). 
Chromatographic Silica Gel (Fisher Chemical, 200-425 mesh) was used for 
flash chromatography and glass-backed plates of 0.25-mm SiO.sub.2 
60-F.sub.254 (Merck, Darmstadt, Germany) were used for thin-layer 
chromatography (TLC). All nonaqueous reactions were run under argon with 
rigorous exclusion of water unless otherwise noted. 
The methods described hereinafter are shown in the schematic diagram of 
FIG. 16. 
(8a) To a solution of tris(2-aminoethyl)amine (5.0 g, 34 mmol) and acetic 
acid (5.0 g, 83 mmol) in 200 mL MeOH, ethyl 
1-(3-propal)-4-nitro-2-pyrrolecarboxylate (7a) (1.3 g, 5.4 mmol) in 100 mL 
MeOH was added dropwise over 30 min at 0.degree. C. 
After addition the solution was stirred at room temperature for 72 h. The 
reaction was followed by TLC (SiO.sub.2, hexane:EtOAc=3:1). After complete 
disappearance of 7a, the solution was concentrated and the residue 
obtained was dissolved in 300 mL CH.sub.2 Cl.sub.2. The CH.sub.2 Cl.sub.2 
solution was washed with 50 mL 1N aqueous NaOH and dried over K.sub.2 
CO.sub.3. 
Removal of the solvent gave crude 8a which contained &lt;5% impurity as shown 
by .sup.1 H NMR, and 8a thus obtained was used in the next reaction 
without further purification. 
8a: 1.7 g, 85%; pale yellow oil; .sup.1 H NMR(CDCl.sub.3): .delta.1.34(t, 
J=7, --COO--C--CH.sub.3, 3H), 1.87(bs, --NH+H.sub.2 O), 1.95(m, 
--C--CH.sub.2 --C--, 2H), 2.49-2.76(m, --C--CH.sub.2 --N--, 14H), 4.28(q, 
J=7, --COOCH.sub.2 --C, 2H), 4.44(t, J=7, pyrrole N--CH.sub.2 --C--, 2H), 
7.41(d, J=2, pyrrole Ar--H, 1H), 7.72(d, J=2, pyrrole Ar--H, 1H); 
LRMS(FAB): 371 (M+H.sup.+). 
(8b) The procedure used for the synthesis of 8b was much the same as 
employed for 8a. 
8b: 3.1 g, 97%; pale yellow oil; .sup.1 H NMR(DMSO-d.sub.6): .delta.1.28(t, 
J=7, --COO--C--CH.sub.3, 3H), 1.32-1.60(m, --C--CH.sub.2 --C--, 2H), 
1.65-1.78(m, --C--CH.sub.2 --C--, 2H), 2.38-2.60(m, --C--CH.sub.2 --N--, 
14H), 3.04(bs, --NH+H.sub.2 O), 4.25(q, J=7, --COOCH.sub.2 --C, 2H), 
4.34(t, J=7, pyrrole N--CH.sub.2 --C--, 2H), 7.33(d, J=2, pyrrole Ar--H, 
1H), 8.32(d, J=2, pyrrole Ar--H, 1H); LRMS(FAB): 385 (M+H.sup.+). 
(9a) A solution of 8a (1.7 g, 5 mmol), diisopropylethylamine (5 mL), and 
2-(trimethylsilyl)ethyl p-nitrophenyl carbonate in 100 mL MeOH was stirred 
at 60.degree. C. for 10 h. TLC (SiO.sub.2, hexane:EtOAc=1:1) showed 
complete disappearance of the reactant. After cooling, the solution was 
concentrated and the residue obtained was dissolved in 300 mL CH.sub.2 
Cl.sub.2. 
The organic solution was washed with 100 mL 5% Na.sub.2 CO.sub.3 aq. and 
100 mL sat. aqueous NaCl, and dried over Na.sub.2 SO.sub.4. Removal of the 
solvent gave a yellow-oil product mixture which was loaded on SiO.sub.2 
column, and elution with a solvent mixture of hexane:EtOAc:Et.sub.3 
N=20:10:3 gave pure 9a as a pale yellow viscous oil. 
9a: 2.1 g, 52%; TLC (SiO.sub.2, hexane:EtOAc:Et.sub.3 N=30:10:3): R.sub.f 
=0.37; .sup.1 H NMR(DMSO-d.sub.6): .delta.-0.10(s, --SiCH.sub.3, 27H), 
0.82-0.90(m, --CH.sub.2 Si--, 6H), 1.25(t, J=7, --COO--C--CH.sub.3, 3H), 
1.85-1.95(m, --C--CH.sub.2 --C--, 2H), 2.39-2.51(m, --C--CH.sub.2 --N--, 
6H), 2.94-3.18(m, --OCON--CH.sub.2 --C--, 8H), 3.93-4.00(m, 
--NCOO--CH.sub.2 --C--, 6H), 4.22(q, J=7, --COOCH.sub.2 --C, 2H), 4.30(bs, 
pyrrole N--CH.sub.2 --C--, 2H), 6.77(bs, --OCONH--, 2H), 7.28, 8.29(2s, 
pyrrole Ar--H, 2H); LRMS(FAB): 803 (M+H.sup.+). 
(9b) The procedure used for the synthesis of 9b was much the same as 
employed for 9a. 
9b: 4 g, 61%; TLC (SiO.sub.2, hexane:EtOAc:Et.sub.3 N=30:10:3): R.sub.f 
=0.37; IR(Neat): .nu..sub.N--H =3300-3500 cm.sup.-1, .nu..sub.C.dbd.O 
=1680-1720 cm.sup.-1, .nu..sub.N--O =1320, 1510 cm.sup.-1 ; .sup.1 H 
NMR(DMSO-d.sub.6): .delta.-0.11(s, --SiCH.sub.3, 27H), 0.85-0.92(m, 
--CH.sub.2 Si--, 6H), 1.26(t, J=7, --COO--C--CH.sub.3, 3H), 1.39-1.43(m, 
--C--CH.sub.2 --C--, 2H), 1.64-1.67(m, --C--CH.sub.2 --C--, 2H), 
2.41-2.51(m, --C--CH.sub.2 --N--, 6H), 2.94-3.17(m, --OCON--CH.sub.2 
--C--, 8H), 3.97-4.02(m, --NCOO--CH.sub.2 --C--, 6H), 4.25(q, J=7, 
--COOCH.sub.2 --C, 2H), 4.36(t, J=7, pyrrole N--CH.sub.2 --C--, 2H), 
6.79(bs, --OCONH--, 2H), 7.31, 8.30(2s, pyrrole Ar--H, 2H); LRMS(FAB): 817 
(M+H.sup.+). 
(10a) NaOH (0.32 g, 8 mmol) in 20 mL H.sub.2 O was added to a solution of 
9a (2.0 g, 2.5 mmol) in 20 mL EtOH. The resulting solution was stirred at 
room temperature for 10 h. Et.sub.3 N.HCl (2.2 g, 16 mmol) was added to 
the solution when TLC (SiO.sub.2, hexane:EtOAc:Et.sub.3 N=20:10:3) showed 
disappearance of the reactant. The color of the solution turned from 
orange to pale yellow. 
The solution was concentrated to dryness under reduced pressure and the 
residue was dissolved in 200 mL CH.sub.2 Cl.sub.2. The pale yellow organic 
phase was washed with 30 mL H.sub.2 O and dried over Na.sub.2 SO.sub.4. 
Removal of the solvent gave the product as a yellow viscous oil. 
10a: 1.6 g, 73%; .sup.1 H NMR(DMSO-d6): .delta.-0.10(s, --SiCH.sub.3, 27H), 
0.86-0.92(m, --CH.sub.2 Si--, 6H), 1.13(t, J=7, --N--C--CH.sub.3, 9H), 
1.89-1.95(m, --C--CH.sub.2 --C--, 2H), 2.40-2.48(m, --C--CH.sub.2 --N--, 
6H), 2.94-3.18(m, --OCON--CH.sub.2 --C--+--CH.sub.2 N.sup.+ --, 14H), 
3.33(bs, --NH.sup.+ --+H.sub.2 O), 3.97-4.03(m, --NCOO--CH.sub.2 --, 6H), 
4.38(bs, pyrrole N--CH.sub.2 --C--, 2H), 6.86(bs, --OCONH--, 2H), 7.03, 
8.06(2s, pyrrole Ar--H, 2H); LRMS(FAB): 775 (M-Et.sub.3 N+H.sup.+). 
(10b) The procedure used for the synthesis of 10b was much the same as 
employed for 10a. 
10b: 4 g, 92%; IR(KBr): .nu..sub.N--H =3400-3700 cm.sup.-1, .nu..sub.N 
+.sub.-H =2600-2900 cm.sup.-1, .nu..sub.C.dbd.O =1680-1720 cm.sup.-1, 
.nu..sub.N--O =1260, 1520 cm.sup.-1 ; .sup.1 H NMR(DMSO-d.sub.6): 
.delta.-0.10(s, --SiCH.sub.3, 27H), 0.84-0.92(m, --CH.sub.2 Si--, 6H), 
1.14(t, J=7, --N--C--CH.sub.3, 9H), 1.35-1.45(m, --C--CH.sub.2 --C--, 2H), 
1.60-1.70(m, --C--CH.sub.2 --C--, 2H), 2.40-2.48(m, --C--CH.sub.2 --N--, 
6H), 2.95-3.15(m, --OCON--CH.sub.2 --C--+--CH.sub.2 N.sup.+ --, 14H), 
3.38(bs, --NH.sup.+ --+H.sub.2 O), 3.97-4.03(m, --NCOO--CH.sub.2 --C--, 
6H), 4.43(t, J=7, pyrrole N--CH.sub.2 --C--, 2H), 6.86(bs, --OCONH--, 2H), 
7.02, 8.03(2s, pyrrole Ar--H, 2H); LRMS(FAB): 789 (M-Et.sub.3 N+H.sup.30 
). 
(12a) A solution of 11 (0.46 g, 1.8 mmol) in 100 mL MeOH was hydrogenated 
at atmospheric pressure over 10% palladium on charcoal (0.5 g) at room 
temperature. The catalyst was removed by filtration and the filtrate was 
concentrated. To the residue, 10a (1.6 g, 1.8 mmol) in dry DMF (100 mL) 
was added. After cooling to 0.degree. C., DECP (0.33 g, 2.0 mmol) and 
Et.sub.3 N (1.0 g, 10 mmol) were added dropwise to the solution. 
The solution was stirred at 0.degree. C. for 2 h and at room temperature 
for another 10 h. Solvent was evaporated to dryness in vacuo, and the 
resulting residue was dissolved in 400 mL CH.sub.2 Cl.sub.2. The organic 
phase was washed with 80mL 5% Na.sub.2 CO.sub.3 aq. and dried over K.sub.2 
CO.sub.3. The crude product was purified with a flash column (SiO.sub.2, 
EtOAc:MeOH:Et.sub.3 N=50:10:3) to give 12a as a yellow glassy solid. 
12a: 1 g, 64%; TLC (SiO.sub.2, EtOAc:MeOH:Et.sub.3 N=50:10:3): R.sub.f 
=0.59; .sup.1 H NMR (DMSO-d.sub.6): .delta.-0.17(s, --SiCH.sub.3, 27H), 
0.84-0.92(m, --CH.sub.2 Si--, 6H), 1.55-1.62(m, --CON--C--CH.sub.2 --C--, 
2H), 1.92-2.00(m, --C--CH.sub.2 --C--, 2H), 2.12(s, --NCH.sub.3, 6H), 
2.23(t, J=7, --CH.sub.2 NMe, 2H), 2.43-2.48(m, --C--CH.sub.2 --N--, 6H), 
2.95-3.02(m, Ar--CON--CH.sub.2 --C--, 2H), 3.12-3.23(m, --OCON--CH.sub.2 
--C--, 8H), 3.79(s, pyrrole N--CH.sub.3, 3H), 3.97-4.03(m, 
--NCOO--CH.sub.2 --C--, 6H), 4.40(bs, pyrrole N--CH.sub.2 --C--, 2H), 
6.78(bs, --OCONH--, 2H), 6.79, 7.19, 7.59, 8.22(4s, pyrrole Ar--H, 4H); 
8.04, 10.21(2bs, --CO--NH--, 2H); LRMS(FAB): 981 (M+H.sup.+). 
(12b) The procedure used for the synthesis of 12b was much the same as 
employed for 12a. 
12b: 2.7 g, 60%; TLC (SiO.sub.2, EtOAc:MeOH:Et.sub.3 N=50:10:3): R.sub.f 
=0.59; IR(KBr): .nu..sub.N--H =3200-3600 cm.sup.-1, .nu..sub.C.dbd.O 
=1650-1720 cm.sup.-1, .nu..sub.N--O =1310, 1520 cm.sup.-1 ; .sup.1 H NMR 
(DMSO-d.sub.6): .delta.-0.17(s, --SiCH.sub.3, 27H), 0.84-0.92(m, 
--CH.sub.2 Si--, 6H), 1.38-1.45(m, --C--CH.sub.2 --C--, 2H), 1.57-1.64(m, 
--CON--C--CH.sub.2 --C--, 2H), 1.65-1.73(m, --C--CH.sub.2 --C--, 2H), 
2.14(s, --NCH.sub.3, 6H), 2.24(t, J=7, --CH.sub.2 NMe, 2H), 2.43-2.47(m, 
--C--CH.sub.2 --N--, 6H), 2.96-3.00(m, Ar--CON--CH.sub.2 --C--, 2H), 
3.10-3.20(m, --OCON--CH.sub.2 --C--, 8H), 3.79(s, pyrrole N--CH.sub.3, 
3H), 3.97-4.04(m, --NCOO--CH.sub.2 --C--, 6H), 4.42(t, J=7, pyrrole 
N--CH.sub.2 --C--, 2H), 6.79(bs, --OCONH--, 2H), 6.80, 7.19, 7.58, 
8.19(4s, pyrrole Ar--H, 4H); 8.09, 10.20(2bs, --CO--NH--, 2H); LRMS(FAB): 
995 (M+H.sup.+). 
(13a) A solution of 12a (1.0 g, 1.0 mmol) in 100 mL DMF was hydrogenated at 
atmospheric pressure over 10% palladium on charcoal (0.5 g) at 50.degree. 
C. The catalyst was removed by filtration, the filtrate was concentrated, 
and the resulting residue was dissolved in dry DMF (100 mL). After cooling 
down to 0.degree. C., 1-methyl-4-nitro-2-pyrrolecarbonyl chloride (0.2 g, 
1.1 mmol) and Et.sub.3 N (0.3 g, 3 mmol) were added. The solution was 
stirred at 0.degree. C. for 2 h and at room temperature for another 10 h. 
The solution was concentrated to dryness in vacuo, and the resulting 
residue was dissolved in 300 mL CH.sub.2 Cl.sub.2. 
The organic phase was washed with 50 mL aqueous 5% Na.sub.2 CO.sub.3 and 
dried over K.sub.2 CO.sub.3. The crude product was purified with a flash 
column (SiO.sub.2, EtOAc:MeOH:Et.sub.3 N=50:10:5) to give 13a as a pale 
yellow glassy solid. 
13a: 0.6 g, 54%; TLC (SiO.sub.2, EtOAc:MeOH:Et.sub.3 N=50:5:5): R.sub.f 
=0.33; .sup.1 H NMR (DMSO-d.sub.6): .delta.-0.18(s, --SiCH.sub.3, 27H), 
0.84-0.91(m, --CH.sub.2 Si--, 6H), 1.56-1.63(m, --CON--C--CH.sub.2 --C--, 
2H), 1.86-1.92(m, --C--CH.sub.2 --C--, 2H), 2.14(s, --NCH.sub.3, 6H), 
2.24(t, J=7, --CH.sub.2 NMe, 2H), 2.43-2.48(m, --C--CH.sub.2 --N--, 6H), 
2.94-3.00(m, Ar--CON--CH.sub.2 --C--, 2H), 3.12-3.20(m, --OCON--CH.sub.2 
--C--, 8H), 3.79, 3.95(2s, pyrrole N--CH.sub.3, 6H), 3.97-4.04(m, 
--NCOO--CH.sub.2 --C--, 6H), 4.29(t, J=7, pyrrole N--CH.sub.2 --C--, 2H), 
6.78(bs, --OCONH--, 2H), 6.80, 7.01, 7.19, 7.33, 7.59, 8.18(6s, pyrrole 
Ar--H, 6H); 8.05, 9.93, 10.28(3bs, --CO--NH--, 3H); LRMS(FAB): 1103 
(M+H.sup.+). 
(13b) The procedure used for the synthesis of 13b was much the same as 
employed for 13a. 
13b: 2 g, 66%; TLC (SiO.sub.2, EtOAc:MeOH:Et.sub.3 N=50:5:5): R.sub.f 
=0.33; IR(KBr): .nu..sub.N--H =3100-3500 cm.sup.-1, .nu..sub.C.dbd.O 
=1650-1720 cm.sup.-1, .nu..sub.N--O =1310, 1520 cm.sup.-1 ; .sup.1 H NMR 
(DMSO-d.sub.6): .delta.-0.16(s, --SiCH.sub.3, 27H), 0.85-0.94(m, 
--CH.sub.2 Si--, 6H), 1.38-1.43(m, --C--CH.sub.2 --C--, 2H), 1.58-1.63(m, 
--CON--C--CH.sub.2 --C--, 2H), 1.60-1.65(m, --C--CH.sub.2 --C--, 2H), 
2.13(s, --NCH.sub.3, 6H), 2.24(t, J=7, --CH.sub.2 NMe, 2H), 2.43-2.48(m, 
--C--CH.sub.2 --N--, 6H), 2.95-3.02(m, Ar--CON--CH.sub.2 --C--, 2H), 
3.12-3.20(m, --OCON--CH.sub.2 --C--, 8H), 3.79, 3.95(2s, pyrrole 
N--CH.sub.3, 6H), 3.97-4.05(m, --NCOO--CH.sub.2 --C--, 6H), 4.31(t, J=7, 
pyrrole N--CH.sub.2 --C--, 2H), 6.78(bs, --OCONH--, 2H), 6.81, 7.00, 7.18, 
7.32, 7.58, 8.18(6s, pyrrole Ar--H, 6H); 8.06, 9.92, 10.28(3bs, 
--CO--NH--, 3H); LRMS(FAB): 1117 (M+H.sup.+). 
(14a) A solution of 13a (0.6 g, 0.6 mmol) in 100 mL DMF was hydrogenated at 
atmospheric pressure over 10% palladium on charcoal (0.5 g) at 60.degree. 
C. The catalyst was removed by filtration, the filtrate was concentrated, 
and the resulting residue were dissolved in dry DMF (100 mL). After 
cooling down to 0.degree. C., CH.sub.3 COCl (0.08 g, 1.0 mmol) and 
Et.sub.3 N (0.3 g, 3.0 mmol) were added dropwise. 
The solution was stirred at 0.degree. C. for 2 h and at room temperature 
for another 10 h. The solution was concentrated to dryness in vacuo, and 
the resulting residue was dissolved in 300 mL CH.sub.2 Cl.sub.2. The 
organic phase was washed with 50 mL aqueous 5% Na.sub.2 CO.sub.3 and dried 
over K.sub.2 CO.sub.3. The crude product was purified with a flash column 
(SiO.sub.2, EtOAc:MeOH:Et.sub.3 N=50:20:5) to give 14a as a pale yellow 
glassy solid. 
14a: 0.3 g, 45%; TLC (SiO.sub.2, EtOAc:MeOH:Et.sub.3 N=50:20:5): R.sub.f 
=0.27; .sup.1 H NMR (DMSO-d.sub.6): .delta.-0.15(s, --SiCH.sub.3, 27H), 
0.86-0.90(m, --CH.sub.2 Si--, 6H), 1.58-1.63(m, --CON--C--CH.sub.2 --C--, 
2H), 1.86-1.92(m, --C--CH.sub.2 --C--, 2H), 1.96(s, CH.sub.3 CON--, 3H), 
2.17(s, --NCH.sub.3, 6H), 2.28(t, J=7, --CH.sub.2 NMe, 2H), 2.43-2.48(m, 
--C--CH.sub.2 --N--, 6H), 2.94-3.00(m, Ar--CON--CH.sub.2 --C--, 2H), 
3.15-3.22(m, --OCON--CH.sub.2 --C--, 8H), 3.79, 3.82(2s, pyrrole 
N--CH.sub.3, 6H), 3.98-4.06(m, --NCOO--CH.sub.2 --C--, 6H), 4.27(t, J=7, 
pyrrole N--CH.sub.2 --C--, 2H), 6.79(bs, --OCONH--, 2H), 6.81, 6.86, 7.02, 
7.14, 7.18, 7.31(6s, pyrrole Ar--H, 6H); 8.06, 9.82, 9.88, 9.90(4bs, 
--CO--NH--, 4H); LRMS(FAB): 1115 (M+H.sup.+). 
(14b) The procedure used for the synthesis of 14b was much the same as 
employed for 14a. 
14b: 1.5 g, 74%; TLC (SiO.sub.2, EtOAc:MeOH:Et.sub.3 N=50:20:5): R.sub.f 
=0.27; .sup.1 H NMR (DMSO-d.sub.6): .delta.-0.13(s, --SiCH.sub.3, 27H), 
0.86-0.90(m, --CH.sub.2 Si--, 6H), 1.40-1.48(m, --C--CH.sub.2 --C--, 2H), 
1.58-1.65(m, --C--CH.sub.2 --C--+--CON--C--CH.sub.2 --C--, 4H), 1.96(s, 
CH.sub.3 CON--, 3H), 2.16(s, --NCH.sub.3, 6H), 2.27(t, J=7, --CH.sub.2 
NMe, 2H), 2.43-2.48(m, --C--CH.sub.2 --N--, 6H), 2.96-3.00(m, 
Ar--CON--CH.sub.2 --C--, 2H), 3.11-3.22(m, --OCON--CH.sub.2 --C--, 8H), 
3.79, 3.82(2s, pyrrole N--CH.sub.3, 6H), 3.98-4.06(m, --NCOO--CH.sub.2 
--C--, 6H), 4.29(t, J=7, pyrrole N--CH.sub.2 --C--, 2H), 6.79(bs, 
--OCONH--, 2H), 6.81, 6.85, 7.00, 7.13, 7.17, 7.28(6s, pyrrole Ar--H, 6H); 
8.05, 9.79, 9.86, 9.88(4bs, --CO--NH--, 4H); LRMS(FAB): 1129 (M+H.sup.+). 
(6a) Ten mL of CF.sub.3 COOH was cooled in an ice-bath before slowly being 
added to a solution of 14a (0.18 g, 0.15 mmol) in 10 mL CH.sub.2 Cl.sub.2 
with stirring at 0.degree. C. The solution was stirred at 0.degree. C. for 
2 h and at room temperature for another 2 h. CF.sub.3 COOH and CH.sub.2 
Cl.sub.2 was removed by evaporation and the resulting residue was 
dissolved in 50 mL MeOH. After addition of 20 g ion-exchange resin 
(HO.sup.- form) the mixture was stirred for 30 min at room temperature. 
The resin was removed by filtration and the filtrate was concentrated 
under vacuum to give pure 6a as a pale yellow glassy solid. 
6a: 0.1 g, 98%; .sup.1 H NMR (DMSO-d.sub.6): .delta.1.58-1.63(m, 
--CON--C--CH.sub.2 --C--, 2H), 1.78-1.82(m, --C--CH.sub.2 --C--, 2H), 
1.96(s, CH.sub.3 CON--, 3H), 2.12(s, --NCH.sub.3, 6H), 2.23(t, J=7, 
--CH.sub.2 NMe, 2H), 2.34(t, J=7, --C--CH.sub.2 --N, 4H), 2.42-2.46(m, 
--C--CH.sub.2 --N--, 6H), 2.54-2.64(m, --C--CH.sub.2 --N--, 6H), 
3.16-3.20(m, Ar--CON--CH.sub.2 --C--, 2H), 3.25(bs, --C--NH.sub.2 +H.sub.2 
O), 3.79, 3.82(2s, pyrrole N--CH.sub.3, 6H), 4.32(t, J=7, pyrrole 
N--CH.sub.2 --C--, 2H), 6.82, 6.85, 7.00, 7.14, 7.17, 7.27(6s, pyrrole 
Ar--H, 6H); 8.11(t, J=5.5, Ar--CO--NH--C--, 1H), 9.80(s, --CO--NH--, 1H), 
9.90(bs, --CO--NH--, 2H); LRMS(FAB): 683 (M+H.sup.+); HRMS(FAB): 683.4467 
(calculated for C.sub.33 H.sub.55 N.sub.12 O.sub.4 (M+H.sup.+) 683.4469). 
(6b) The procedure used for the synthesis of 6b was much the same as 
employed for 6a. 
6b: 0.65 g, 96%; .sup.1 H NMR (DMSO-d.sub.6): .delta.1.33-1.40(m, 
--C--CH.sub.2 --C--, 2H), 1.56-1.64(m, --CON--C--CH.sub.2 --C--, 2H), 
1.64-1.71(m, --C--CH.sub.2 --C--, 2H), 1.96(s, CH.sub.3 CON--, 3H), 
2.13(s, --NCH.sub.3, 6H), 2.23(t, J=7, --CH.sub.2 NMe, 2H), 2.38(t, J=7, 
--C--CH.sub.2 --N, 4H), 2.42-2.46(m, --C--CH.sub.2 --N--, 6H), 
2.54-2.64(m, --C--CH.sub.2 --N--, 6H), 3.15-3.20(m, Ar--CON--CH.sub.2 
--C--, 2H), 3.22(bs, --C--NH.sub.2 +H.sub.2 O), 3.79, 3.82(2s, pyrrole 
N--CH.sub.3, 6H), 4.28(t, J=7, pyrrole N--CH.sub.2 --C--, 2H), 6.81, 6.86, 
7.00, 7.14, 7.17, 7.28(6s, pyrrole Ar--H, 6H); 8.08(t, J=5.5, 
Ar--CO--NH--C--, 1H), 9.82, 9.89, 9.92(3s, --CO--NH--, 3H); LRMS(FAB): 697 
(M+H.sup.+); HRMS(FAB): 697.4631 (calcd for C.sub.34 H.sub.57 N.sub.12 
O.sub.4 (M+H.sup.+) 697.4625). 
Discussion 
Synthesis 
Our synthesis of 6a,b (FIG. 15) began with the preparation of the central 
pyrrole units (8a,b) (FIG. 16) in which the tren group was attached to the 
pyrrole through the desired linker arms. Attempts at purification of 8a,b 
through column chromatography failed. 
Very poor separation was obtained over an Al.sub.2 O.sub.3 column. In 
addition, use of a SiO.sub.2 column led to the hydrolysis of the ester 
group in 8. 
Compound 8 (5%) was obtained only as a mixture of the methyl ester (5%) and 
the carboxylate (20%) with SiO.sub.2 column chromatography by elution with 
MeOH:conc. NH.sub.3 (aq.)=80:20. It seems that the polyamino group can 
complex trace amounts of metal ion from the SiO.sub.2 which consequently 
catalyzes the hydrolysis of the ester group. Due to these complexities in 
attempted purification, crude 8, which by .sup.1 H NMR showed only 
.sup..about. 5% impurity, was used in the next reaction without 
purification. 
Attempts to employ t-butyl S-4,6-dimethylpyrimid-2-yl thiocarbonate as an 
agent to deliver the t-butyl carbamate (Nagasawa, T.; Kuroiwa, K.; Narita, 
K.; Isowa, Y. Bull. Chem. Soc. Jpn. 1973, 46, 1269) (Boc) as a protecting 
group for the primary and secondary amines of 8 provided but .sup..about. 
30% yields of product. Changing the synthetic methodology by using 
2-trimethylsilylethyl carbamate ((a) Carpino, L. A.; Tsao, J.-H. J. Chem. 
Soc., Chem. Commun. 1978, 358; (b) Rosowsky, A.; Wright, J. E. J. Org. 
Chem. 1989, 54, 5551) (Teoc) for the protection of the tren polyamino 
group on 8 provided 9 in a 52-61% yield after separation by SiO.sub.2 
column chromatography (FIG. 16). 
Additional factors in favor of the choice of this protecting group included 
its stability toward the conditions of hydrogenation over Pd/C and other 
harsh conditions employed in the synthetic steps, including the last step 
of the synthesis. 
Compound 6, like 8, was difficult to purify since it did not migrate on 
SiO.sub.2 TLC even with the elution solvent mixture of MeOH:conc. NH.sub.3 
(aq.)=60:40. Fortunately, the deprotection reaction of 14 (acid catalyzed 
removal of the Teoc group with CF.sub.3 COOH) produces only the desired 
product (6) and volatile compounds ((a) Carpino, et al., 1978, supra). 
Subsequent treatment of the crude 14 product with HO.sup.- exchange resin 
gave very pure 6 as shown by .sup.1 H NMR. 
DNase I footprint analysis of 6a and 6b. 
DNase I was employed as the DNA cleaving agent for footprint generation 
(Galas, D. J.; Schmitz, A. Nucleic Acids Res. 1978, 5, 3157) in the 
comparative analysis of the interactions of 6a, 6b, and distamycin with 
the 167 bp EcoRI/Rsa I pBR322 restriction fragment. 
Four A+T-rich distamycin (Harshman, K. D.; Dervan, P. B. Nucleic Acids Res. 
1985, 13, 4825), bromoacetyldistamycin (Baker, B. F.; Dervan, P. B. J. Am. 
Chem. Soc. 1989, 111, 2700), and dien-microgonotropen (He, G.-X., 1993, 
supra) binding sites have been previously identified (bold typeface, FIG. 
1), making this an ideal DNA fragment for the comparative study of the 
tren-microgonotropens with distamycin. 
DNase I has an advantage over Tullius' HO. (Burkhoff, A. M., Tullius, T. D. 
Cell, 1987, 48, 935) and Dervan's MPE.Fe(II) (Van Dyke, M. W.; Hertzberg, 
R. P.; Dervan, P. B. Proc. Natl. Acad. Sci. USA 1982, 79, 5470) in that it 
cleaves precisely at the 5' edge of an agent's minor groove binding site, 
producing a protected region with a sharp, well-defined 5' border ((a) 
Dabrowiak, J. C.; Goodisman, J. In Chemistry & Physics of DNA-Ligand 
Interactions; Kallenbach, N. R., Ed.; Adenine Press: New York, 1989; pp 
143-174; (b) Goodisman, J.; Dabrowiak, J. C. Biochemistry 1992, 31, 1058). 
Thus, even though DNase I cleavage at the 3' edge of the binding site is 
not precisely defined, complementary strand analysis provides sharply 
defined 5' borders on both DNA strands (FIG. 1), and, hence, precisely 
defined binding sites that correspond closely to those previously defined 
(He, et al., 1993, supra). 
DNase I footprinting analysis of the 3'-.sup.32 P! labeled 167 bp 
EcoRI/RsaI restriction fragment with 6a and 6b (FIG. 2b), when coupled 
with results from the 5'-labeled material (FIG. 2a), defined binding sites 
similar to those for distamycin. Pre-incubation of the 167 bp 3'- and 
5'-.sup.32 P! labeled restriction fragments with 5 .mu.M 6a, 6b, or 
distamycin (0.05 ligand/bp DNA) did not produce detectable inhibition of 
DNase I cleavage at any of the four A+T-rich binding sites (FIGS. 2a/b). 
In contrast, specific inhibition of cleavage was observed at three of the 
four sites (FIG. 1 at sites II, III, IV) after pre-incubation with 25 and 
50 .mu.M 6a, 6b, or distamycin (0.25 and 0.5 ligand/bp DNA). Site I could 
only be distinguished at 100 .mu.M, and even then, site definition was 
vague. 
Pre-incubation of the restriction fragment with 100 .mu.M ligand (1.0 
ligand/bp DNA) resulted in additional protection from DNase I cleavage 
within the spacer regions which flank the A+T-rich binding sites. Dervan 
and co-workers have observed a similar binding isotherm for distamycin on 
the 516 bp RsaI/EcoRI restriction fragment of pBR332 (Van Dyke et al., 
1982, supra). At higher concentrations of distamycin (3.1 ligand/bp DNA), 
spacer regions which flanked A+T-rich binding sites coalesced into a 
single, broad, protected zone (Van Dyke, et al., 1982, supra). 
Analysis of the 5' footprint edges of the binding sites of 50 .mu.M 6a, 6b, 
and distamycin shows cleavage patterns that are very similar to those seen 
previously for the dien-microgonotropens (He, et al., 1993, supra). Closer 
scrutiny reveals small changes for sites II and III while site IV is 
unchanged. Site III is one base smaller on the 3' strand and site II is 
two bases smaller on the 3' strand than was found for the 
dien-microgonotropens (He, et al., 1993, supra) (FIG. 1). 
Even at the highest concentrations of 6a and 6b, enhancements in or 
increased rates of DNase I cleavage were not observed at specific 
sequences for the 5'- and the 3'-.sup.32 P! labeled restriction fragments 
(Dm showed enhancements similar to those found previously) (He, et al., 
1993, supra). 
Equilibrium constants for the association of 6a and 6b with oligomeric DNA 
were assessed by the complexing of tren-microgonotropen-a and -b to the 
hexadecamer d(GGCGCAAATTTGGCGG)(SEQ ID NO:1)/d(CCGCCAAATTTGCGCC)(SEQ ID 
NO:2) in aqueous solutions at 35.degree. C. (2.8 mL solutions containing 
0.01M phosphate buffer, pH 7.0, and 0.01M NaCl). 
These reactions were followed by the competition of the dye Hoechst 33258 
(Ht) with 6a and 6b for the A.sub.3 T.sub.3 minor groove binding site (an 
extension of Ht alone binding to dsDNA). The concentrations of 6a and 6b 
were confirmed by .sup.1 H NMR peak integration of resonances with those 
of an equivalent concentration of mesitoate. 
As shown previously (Browne, K. A.; He, G.-X.; Bruice, T. C. J. Am. Chem. 
Soc. 1993, 115, 7072), monitoring the increase in fluorescence intensity 
as the association of Ht with the hexadecamer displaces prebound 
nonfluorescent ligands is an excellent method for determining equilibrium 
binding constants. FIG. 17 relates the equilibrium constants for the 
complexing of one and two Ht species to the hexadecamer with one and two L 
(where L=6a or 6b) binding to the hexadecamer, plus equilibrium constants 
for the simultaneous binding of one Ht and one L at the same site. 
##EQU1## 
Eq 1, derived from FIG. 17, relates each of the equilibrium binding 
constants, the total fluorescence (.SIGMA..theta.), and L! in terms of 
fluorescence (F) and Ht!. The rationale behind FIG. 17 and the subsequent 
derivation of eq 1 have been described in considerable detail (Browne, et 
al., 1993, supra). The values of log K.sub.Ht1 =7.6 and log K.sub.Ht2 =9.1 
used were determined from a reevaluation of data previously collected 
(Browne, et al., 1993, supra) and are very close to the previously 
determined values. A concentration independent static quenching term, Q', 
is included in eq 1 to account for the lessened fluorescent emission of 
the DNA:Ht:L complex compared to the DNA:Ht and DNA:Ht.sub.2 complexes. 
The equilibrium association constants calculated as best fits to the 
experimental data points for 6a and 6b with eq 1 are presented in Table I. 
Plots of F vs. Ht! using these constants at 8.0.times.10.sup.-9, 
1.0.times.10.sup.-8, and 1.2.times.10.sup.-8 M ligand and 
5.0.times.10.sup.-9 M in hexadecamer duplex are shown in FIGS. 3a and 3b. 
Inspection of Table I shows that the values of K.sub.L1 (1.6.times.10.sup.9 
and 7.9.times.10.sup.8 M.sup.-1 for 6a and 6b, respectively) and K.sub.L2 
(1.6.times.10.sup.9 and 1.0.times.10.sup.9 M.sup.-1 for 6a and 6b, 
respectively) have only a small, if any, cooperative effect for the 
binding of the tren-microgonotropens to d(GGCGCAAATTTGGCGG)(SEQ ID 
NO:1)/d(CCGCCAAATTTGCGCC)(SEQ ID NO:2). 
A reevaluation of the previously studied dien-microgonotropens (5a,b,c) 
(Browne, et al., 1993, supra) indicates that the second association 
constants are more than 3-fold greater than the first (Table I). The 
complex association constants (K.sub.L1 K.sub.L2) are greater for 6a,b 
than for 5a,b,c since both K.sub.L1 and K.sub.L2 are slightly greater for 
the tren- than for the dien-microgonotropens. This is as expected since 
there are 4 amines (including 2 primary amines) in the tren moiety verses 
3 tertiary amines in the dien group. Primary amines have a higher pK.sub.a 
than tertiary amines (Perrin, D. D. "Dissociation Constants of Organic 
Bases in Aqueous Solution"; Butterworths: London, 1965) and are more prone 
to hydrogen bond to phosphate linkages. 
In addition, there is little difference in the association constants of the 
different microgonotropens within a given series (tren- or dien-) even 
though the chain lengths of the linkers differ. This is likely due to the 
fact that binding ability is a function of both the minor groove binder 
and the polyamine, with all chain lengths being long enough to permit 
efficient electrostatic grasping of the phosphodiester backbone. 
The degree of fluorescence quenching of Ht in the DNA:Ht:L complexes when 
L=6a and 6b was also found to be different than for the 
dien-microgonotropens while the mode of quenching (intracomplex) was the 
same for both sets of microgonotropens. From values of Q'=0.41 and 0.64 
for 6a and 6b, respectively, quenching of Ht fluorescence was determined 
to be 59% and 36%. 
In contrast, all three of the dien-microgonotropens quenched fluorescence 
to a constant degree (.sup..about. 45%). A small difference in the 
quenching terms within a given series (tren- or dien-) would be expected 
since a given series has a common polyamine. But, in fact, the 
fluorescence quenching that 6a causes is considerably more efficient than 
that due to any of the other microgonotropens. This difference in 
quenching is likely because of a special position that the 3 methylene 
linker of 6a confers upon its tren moiety such that the quenching amino 
groups are in greater intimate contact with the Ht fluorochrome than is 
the case with 5a,b,c or 6b. 
The fluorescence of solutions containing (i) the hexadecameric DNA duplex 
plus Ht in the ratio of 1:2 or (ii) the hexadecameric DNA duplex, Ht, and 
6b in the ratio of 1:1:1 did not change on titration with a solution of 
tris(2-aminoethyl)amine. Thus, as for the dien-microgonotropens (Browne, 
et al., 1993, supra) amine quenching is not bimolecular but, rather, to 
intracomplex quenching within the DNA:Ht:L complex. 
TABLE I 
__________________________________________________________________________ 
Mean values of the association and quenching 
constants for Ht, Dm, 2, 5a, 5c, and the new ligands 
6a and 6b to d(GGCGCAAATTTGGCGG)(SEQ ID NO: 1) /d(CCGCCAAATTTGCGCC) 
(SEQ ID NO: 2) 
{in H.sub.2 O, 10 mM phosphate buffer, pH 7.0; and 10 mM NaCl at 
35.degree. C}. 
Ligand 
log K.sub.L1 
log K.sub.L2 
log K.sub.L1 K.sub.L2 
log K.sub.HtL 
log K.sub.LHt 
Q' 
__________________________________________________________________________ 
Ht.sup.a,b 
7.6 .+-. 0.1 
9.1 .+-. 0.2 
Dm.sup.a,c 
7.6 .+-. 0.09 
8.4 .+-. 0.08 
16.0 8.8 .+-. 0.09 
8.8 
2.sup.a,d 
6.8 .+-. 0.1 
6.2 .+-. 0.5 
13.0 -1.2 .+-. 0.1 
-1.3 
5a.sup..sup.a,c 
8.5 .+-. 0.3 
8.9 .+-. 0.02 
17.4 10.0 .+-. 0.07 
8.9 0.53 .+-. 0.2 
5b.sup.a,c 
8.3 .+-. 0.2 
8.8 .+-. 0.2 
17.1 10.0 .+-. 0.06 
9.2 0.57 .+-. 0.064 
5c.sup.a,c 
8.2 .+-. 0.2 
8.8 .+-. 0.05 
17.0 9.9 .+-. 0.02 
9.2 0.55 .+-. 0.081 
6a.sup.e 
9.2 .+-. 0.1 
9.2 .+-. 0.1 
18.4 10.7 .+-. 0.01 
8.8 0.41 .+-. 0.11 
6b.sup.e 
8.9 .+-. 0.08 
9.0 .+-. 0.2 
17.9 10.3 .+-. 0.1 
8.8 0.64 .+-. 0.046 
__________________________________________________________________________ 
.sup.a A recalculation of previously determined association constants wit 
the curve fitting program SigmaPlote .RTM. (Jandel Scientific). .sup.b Th 
constants were calculated from the mean values of 3 titration experiments 
of the hexadecamer with Ht. .sup.c The standard deviations are 
.sigma..sub.n, are from the mean values of the constants calculated at 8. 
.times. 10.sup.-9, 1.0 .times. 10.sup.-8, 1.2 .times. 10.sup.-8, and 1.4 
.times.10.sup.-8 M ligand. .sup.d The standard deviations, .sigma..sub.n, 
are from the mean values of the constants calculated at 5.0 .times. 
10.sup.-8 and 1.0 .times. 10.sup.-7 M 2. .sup.e The standard deviations, 
.sigma..sub.n, are from the mean values of the constants from 2 
experiments calculated at 8.0 .times. 10.sup.-9, 1.0 .times. 10.sup.-8, 
and 1.2 .times. 10.sup.-8 M in 6a or 6b. 
Electrophoretic mobility shift assay for 6a and 6b. The effect of the 
binding of 6a and 6b to DNA on the electrophoretic migration has been 
investigated with .phi.X-174-RF DNA HaeIII restriction digest fragments 
(FIG. 4). Our use of .phi.X-174-RF DNA restriction digests in 
electrophoretic mobility shift assays (He, et al., 1993, supra) is 
predicated on the common use of this material as molecular weight size 
standards. 
Applicants have calculated (He, et al., 1993, supra) 246 A-tracts (AAAA, 
AAAT, or TAAA; independent or overlapping) approximately evenly spaced 
throughout the restriction digest fragments. These are the most preferred 
binding sites for 6a and 6b (loc. cit.). 
When increasing the concentrations of 6a and 6b from 20 to 40, 60, and 80 
.mu.M (0.088 to 0.176, 0.264, and 0.352 ligand/bp, respectively), the 
mobility of DNA restriction fragments decreases. Moreover, the decreases 
in the otherwise approximately logarithmic mobility of the DNA fragments 
are proportional to their lengths (largest effect seen with the largest 
fragments). This suggests that the conformation of the DNA is altered 
significantly by the binding of the tren-microgonotropens, especially in 
the largest fragments (1358, 1078, and 872 bp). 
An alternative explanation of the decreased mobility that must be 
considered is a change in the charge to mass ratio of the DNA:ligand 
complex. This is unlikely, however, since the shortest fragments do not 
show the greatest change in mobility as dictated by the logarithmic nature 
of DNA fragments in an electric field on an agarose gel. 
Meanwhile, a "smearing" of the bands is evident in the intermediate 
fragments (603, 310, 281/271, 234, and 194 bp), especially at 60 and 80 
.mu.M tren-microgonotropen. This indicates not simply a conformational 
change but a population of differing conformations of 
DNA:tren-microgonotropen complexes leading to a distribution of apparent 
electrophoretic molecular weights. 
Distamycin brings about smaller changes at 150 .mu.M (0.66 ligand/bp) than 
6a or 6b at 40 .mu.M. Tris(2-amino-ethyl)amine, the tren moiety of the 
tren-microgonotropens, produces no apparent change in electrophoretic 
behavior at 150 .mu.M compared with the control lanes. 
To gain a more quantitative appreciation for the magnitude of the DNA 
structural changes occurring with the association of the 
tren-microgonotropens, the migration data has been reduced to a graphical 
form. 
The electrophoretic mobilities of the .phi.X-174-RF DNA HaeIII restriction 
digest fragments have been calculated as the R.sub.L values when 
coelectrophoresed with 6a, 6b, Dm, and tris(2-aminoethyl)amine. 
R.sub.L is the ratio of the apparent length to real length where apparent 
length is the length of uncomplexed dsDNA (interpolated or slightly 
extrapolated from the standards) with same mobility (Wu, H.-M.; Crothers, 
D. M. Nature 1984, 308, 509). 
The representative plot of R.sub.L vs. bp at 80 .mu.M 6a and 6b, or 150 
.mu.M distamycin (FIG. 5a) shows that as the size of the fragment 
increases, the effect of these agents is to increase the apparent size of 
DNA fragments (decrease the mobility) relative to the control 
(.phi.X-174-RF DNA with no added agent). 
The order of effectiveness in increasing the apparent length of 
.phi.X-174-RF DNA HaeIII restriction digest fragments is 6a.sup..about. 
6b&gt;&gt;distamycin&gt;tren. The R.sub.L value does not vary as a simple function 
with increasing DNA fragment size. Instead, variation in migration 
patterns is probably contingent on the number of A+T-rich sequences in 
each fragment, the relative positions of the A+T-rich sequences within a 
given fragment (Levene, S. D.; Wu, H.-W.; Crothers, D. M. Biochemistry 
1986, 25, 3988), and the porosity of the gel (Thompson, J. F.; Landy, A. 
Nucleic Acids Res. 1988, 16, 9687). 
In addition, the plot of R.sub.L vs. agent for the 1078 bp fragment in FIG. 
5b shows that tren-microgonotropen-a and -b's influence on the DNA 
conformation is quite concentration dependent and sigmoidal in response. 
Distamycin does not demonstrate very marked changes even at the highest 
concentrations examined. In fact, the effect of distamycin on these 
fragments is nearly concentration independent over the concentration 
ranges examined. 
As is evident from the above discussion and previous work from this 
laboratory (He, et al., 1993, supra), the tren-microgonotropens are about 
twice as effective in inducing structural changes in DNA as are the 
dien-microgonotropens (nearly the same decrease in electrophoretic 
mobility is seen for 6a,b at ca. half of the concentration that was used 
for 5a,b,c) and at least four times as effective in inducing structural 
changes as is Dm. 
The fact that the tren-microgonotropens are only approximately twice as 
effective as the dien-microgonotropens in retarding gel electrophoretic 
migration of DNA fragments is somewhat surprising considering the fact 
that the complex equilibrium association constants (K.sub.L1 K.sub.L2, 
Table I) for 6a (i.e., 2.times.10.sup.18 M.sup.-2)) and 6b (i.e., 
8.times.10.sup.17 M.sup.-2) are considerably higher than those of the 
dien-microgonotropens (i.e., 1.times.10.sup.17 to 2.times.10.sup.17 
M.sup.-2). 
This suggests that the mode of inhibition of DNA mobility in an 
electrophoretic field is not simply a function of how tight the ligand 
binds to the DNA. Instead, the degree of inhibition is likely due to a 
less well understood quality of the microgonotropen which induces a DNA 
conformational change upon association. 
Topoisomerase I inhibition by 6b 
Mammalian topoisomerase I (topoI) is an enzyme that relaxes both positive 
and negative superhelical turns in covalently closed circular DNA. It 
performs this ATP-independent reaction by transiently breaking the 
phosphodiester linkage of one strand of DNA, passing the intact strand 
through the break, and then religating the gap. In this manner, the enzyme 
effectively decreases the superhelical density by changing the linking 
number of the closed circular DNA by integral values (Lewin, B. Genes, 2nd 
Edition, John Wiley & Sons, New York, 1985). 
Inhibition of topoI's action on supercoiled pBR322 by 6b was compared to 
inhibition by Dm and 5b (dien-microgonotropen-b). In the first set of 
experiments, each agent was allowed to incubate with the DNA for 1 hr 
prior to the 18 hr topoI reaction period. The topoI (+) control (Enz, 18h) 
shows the extent of superhelical relaxation found in the absence of added 
5b, 6b, or Dm while the (-) control (.phi.) shows the spontaneous 
background relaxation. 
The amount of relaxation seen in the (+) control is roughly the same amount 
seen in the presence of 150 .mu.M 5b (4.95 molecules of 5b/bp). With 150 
.mu.M Dm, a continuous family of topological isomers separated by single 
linking numbers is generated from supercoiled to completely relaxed 
circular pBR322 indicating partial inhibition (the number of topological 
isomers is somewhere between the (+) and the (-) controls). At 10 .mu.M 6b 
(0.33 molecules of 6b/bp) a considerable number of the same topological 
isomers as for Dm at 150 .mu.M can be seen even though the predominant 
isomer is the fully supercoiled species. By 30 .mu.M 6b (0.99 molecules of 
6b/bp), complete inhibition of topoI takes place. 
In closely related experiments, the mode of inhibition of topoI was 
examined. This was accomplished by allowing the supercoiled DNA to be 
partially relaxed for 0.5 hr with topoI before any other DNA ligands were 
added (FIG. 6). The 0.5 hr control shows the state of unwinding at the 
time 5b, 6b, and Dm were added. While 150 .mu.M 5b and Dm demonstrated no 
effect (compare with the topoI (+) control), 30 .mu.M 6b inhibited topoI 
even after pBR322 was considerably unwound. 
This indicates that of the three compounds surveyed, only 6b is able to 
effectively compete with topoI once the enzyme is bound. Extrapolating 
from experiments with the hexadecamer d(GGCGCAAATTTGGCGG)(SEQ ID 
NO:1)/d(CCGCCAAATTTGCGCC)(SEQ ID NO:2) (Table I), one might anticipate the 
binding affinity of 6b to pBR322 to be in the range of 2- to 4-fold 
greater than the binding affinity of 5b to the same hexadecamer. The 2- to 
4-fold difference in binding does not explain the inhibition data (FIG. 
6). 
This suggests that, as with the electrophoretic mobility shift assay, 
binding of 6b to DNA alters the conformation of DNA. Such an altered DNA 
conformation could inhibit topoI by either preventing enzyme binding to or 
"tracking" along DNA, or by generating conformationally uncleavable sites. 
Reagents and methods for DNA binding studies were exactly the same as used 
previously (He, et al., 1993, supra; Browne, et al., 1993, supra) unless 
stated otherwise. The values for the equilibrium constants for 2, 5a,b,c, 
Dm, and Ht were recalculated from previously collected data (Browne, et 
al., 1993, supra) using the curve fitting program SigmaPlot.RTM. 4.1.4 
(Jandel Scientific, San Rafael, Calif.). The equilibrium constants for 6a 
and 6b were calculated with SigmaPlot.RTM. 4.1.4 using the reevaluated 
constants for Ht (K.sub.Ht1 and K.sub.Ht2). 
Topoisomerase I inhibition assays 
The buffer for all of the 50 .mu.L reactions was composed of 50 mM 
Tris-HCl, pH 7.5, 50 mM KCl, 10 mM MgCl.sub.2, and 1 mM EDTA that was 
filtered through a sterile 0.45.mu. Gelman Sciences Acrodisc. Every 
reaction mixture contained 1 .mu.g of supercoiled pBR322 plasmid 
(Pharmacia) and, except for the supercoiled control, every reaction 
mixture included 10 units of calf thymus topoisomerase I (Bethesda 
Research Laboratories). 
For the supercoil relaxation assays, no added agent, 10 .mu.M or 30 .mu.M 
6b, or 150 .mu.M distamycin (Dm) or dien-microgonotropen-b (5b) (He, et 
al., 1993, supra) were preincubated with the supercoiled DNA for 60 min 
before topoisomerase I was added. These reactions were allowed to run for 
18 h at 37.degree. C. at which time the reactions were stopped with the 
addition of 2 .mu.L of 250 mM EDTA, pH 7.5. 
For the supercoil partial relaxation assays, topoisomerase I was incubated 
with the supercoiled DNA for 30 min at 37.degree. C. before the addition 
of 30 .mu.M 6b, or 150 .mu.M Dm or 5b. These reactions were allowed to run 
for an additional 18 h at 37.degree. C. after which time they were stopped 
as described above. 
The supercoil partial relaxation control was stopped after the initial 30 
min at 37.degree. C. All reactions were extracted twice with 
water-saturated phenol, extracted once with chloroform, and precipitated 
with ammonium acetate and ethanol. 
After the DNA pellets were dissolved in 9 .mu.L of 10 mM Tris-HCl, pH 8.0, 
and 1 mM EDTA, 1.0 .mu.L of loading buffer (Sambrook, J.; Fritsch, E. F.; 
Maniatis, T. Molecular Cloning, A Laboratory Manual; 2nd Edition, Cold 
Spring Harbor, N.Y., 1989) (10% (w/v) glycerol, 0.1% (w/v) sodium dodecyl 
sulfate, and 0.1% (w/v) bromophenol blue) was added to each sample. 
The different helical forms of pBR322 created by the relaxation assays were 
electrophoretically separated through a 4% NuSieve 3:1 (hydroxyethylated) 
agarose gel (vertical, 0.8 mm) in 40 mM Tris-acetate, pH 8.0 and 1 mM EDTA 
for 8 hr at 2 V/cm. The gel was stained with a 0.5 .mu.g/mL solution of 
ethidium bromide in deionized water for 30 min, destained for 15 min in 
deionized water, and photographed on a UV (302 nm) transilluminator with 
Polaroid type 667 film. 
EXAMPLE II 
MATERIALS AND METHODS 
The synthesis of 6b was described in Example I. The self complementary 
d(CGCAAATTTGCG (SEQ ID NO:3)).sub.2 was obtained by annealing (Browne et 
al., 1993, supra) the single stranded DNA oligomer prepared and purified 
at the Biomolecular Resource Center, University of California, San 
Francisco. 
The NMR samples contained either 0.38 or 2.5 mM (.mu.=0.079 and 1.2, 
respectively) d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 in 10 mM 
potassium phosphate buffer and 10 mM NaCl at pH 7.0 with 0.1% DSS in 0.4 
mL D.sub.2 O. Concentrations of ssDNA were determined from the absorbance 
at 260 nm (.epsilon..sub.260,single-stranded=1.36.times.10.sup.5 M.sup.-1 
cm.sup.-1, 60.degree. C.) 
One equivalent of 6b was added to 0.4 mL of 2.5 mM oligomer and this sample 
was lyophilized twice from 99.9% D.sub.2 O, once from 99.96% D.sub.2 O, 
and finally dissolved in 0.4 mL of 99.96% D.sub.2 O (Aldrich) under a 
nitrogen atmosphere. (The titration sample was dried in an analogous 
manner in the absence of 6b.) The solution was kept refrigerated at 
4.degree. C. between uses. All NMR spectra were recorded at 500 MHz on a 
GN-500 (General Electric) spectrometer at 10.degree. C., unless otherwise 
specified. Chemical shifts were referenced to the signal of DSS (0 ppm). 
1D NMR. 
The titration experiment was performed in D.sub.2 O at 21.degree. C. in 
0.25 mole equiv. steps of 6b/d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 
at 3.8.times.10.sup.-4 M of d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2. 
Mesitoate (2,4,6-trimethylbenzoate) was present at 3.8.times.10.sup.-4 M 
as an internal standard. The melting study of dsDNA was performed at 
3.8.times.10.sup.-4 M of d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 
between 20.degree. and 60.degree. C. with DSS 
(2,2-dimethyl-2-silapentane-3,3,4,4,5,5-d.sub.6 -5-sulfonate) as an 
internal standard. 
2D NMR. 
NOESY experiments were recorded in the phase sensitive mode using the 
hypercomplex NOE pulse sequence (States, D. J.; Haberkorn, R. A.; Ruben, 
D. J. J. Magn. Reson., 1982, 48,286) with mixing times of 50, 100 and 180 
ms for the d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 : 6b complex. 
Spectra were collected into 4K complex points for 512 t.sub.1 increments 
with a spectral width of 5681 Hz in both dimensions. 
The data matrix was zero filled to 2K and appodized with a gaussian 
function to give a line broadening of 1 Hz in both frequency domains. The 
ROESY experiment was recorded at 10.degree. C. using the Kessler pulse 
sequence (Kessler, H.; Griesinger, C.; Kerssebaum, R.; Wagner, E.: Ernst, 
R. J Am. Chem. Soc. 1987, 109, 607) with a mixing time of 50 ms and a 
locking field strength of 2.5 kHz. 
Notations 
Here, as elsewhere (Blasko et al., 1993, supra; (a) Patel, D. J.; Shapiro, 
L. Biochimie 1985, 67, 887, (b) Patel, D. J.; Shapiro, L. J. Biol. Chem. 
1986, 261, 1230, (c) Patel, D. J.; Shapiro, L.; Hare, D. Q. Rev. Biophys. 
1987, 20, 35, (d) Gao, X.; Patel, D. J. Q. Rev. Biophys. 1989, 22, 93), 
the numbering of DNA protons follows the rule that the sugar protons will 
be denoted by prime and double prime superscripts and preceded by the name 
of the residue to which they belong. 
When reference is made to the same proton of more than one residue, all 
residues are listed followed by the proton type (e.g. A.sub.6 T.sub.7 
T.sub.8 H2" means the H2" (sugar) protons which belong to the A.sub.6, 
T.sub.7, and T.sub.8 residues; G.sub.2 G.sub.10 G.sub.12 H8 means the H8 
(base) protons of the G.sub.2, G.sub.10 and G.sub.12 residues). When both 
H2' and H2" protons are involved in discussion, we used the H2'2" 
abbreviation. 
Distance calculations were made by measuring the volume integrals of the 
NOE enhancements from the 180 ms NOESY spectrum which were then related to 
interproton distances by eq 2 where r.sub.a and 
EQU r.sub.a =r.sub.b (NOE.sub.b /NOE.sub.a).sup.1/6, .ANG. eq (2) 
r.sub.b are the distances corresponding to the unknown and known (C.sub.1 
H5-C.sub.1 H6, 2.45 .ANG.) interactions of a pair of protons with their 
corresponding NOE.sub.a and NOE.sub.b (Zhang, X.; Patel, D. J. 
Biochemistry 1990, 29, 9451). 
The linearity of the NOE build-up with t.sub.m was checked for most of the 
dsDNA proton interactions between 50 and 180 ms and a 5-20 fold increase 
was found in the NOE volume integrals from the 50 to 180 ms mixing times. 
The exchange rate (k.sub.ex) was calculated from eq 3 as follows: 
EQU k.sub.ex =ln((1+R)/2.tau..sub.m (1-R)), s.sup.-1 eq(3) 
using the ratio of peak intensities (R), expressed in number of contour 
levels (off diagonal/diagonal) from a short mixing time (.tau..sub.m) 
ROESY spectrum (Ernst, R. R.; Bodenhausen, G.; Wokaun, A. "Principles of 
Nuclear Magnetic Resonances in One and Two Dimensions", Clarendon Press, 
Oxford, 1987). 
The free energy of activation, .DELTA.G*, for this exchange process at a 
certain temperature, T (K), was calculated from eq 4 (Gunther, H. "NMR 
Spectroscopy: An Introduction", John Wiley, New York, 1980, p. 241). 
EQU .DELTA.G*=19.14T(10.32-log(k.sub.ex /T)), J/mol eq (4) 
Computational Analysis and Restrained Molecular Modeling were performed on 
a Silicon Graphics (Mountain View, Calif,) Iris 4D/340GTX workstation 
using CHARMm (Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. 
J.; Swaminathan, S.; Karplus, M. J. Comp. Chem. 1983, 4, 187) (version 
21.3) and QUANTA (version 3.3.1) programs (Molecular Simulations, Waltham, 
Mass.). 
The solution structure of 5c in a complex with d(CGCA.sub.3 T.sub.3 
GCG).sub.2 was used as initial coordinates for 6b (Blasko, et al., 1993, 
supra). The aliphatic chain and dien polyamino group on the central 
pyrrole nitrogen of 5c was replaced with a (CH.sub.2).sub.4 methylene 
chain and a tren moiety {--NHCH.sub.2 CH.sub.2 N(CH.sub.2 CH.sub.2 
NH.sub.2).sub.2 } using 3D Molecular Editor (QUANTA). 
Atomic partial charges of the atoms in 6b and d(CGCA.sub.3 T.sub.3 GCG (SEQ 
ID NO:3)).sub.2 were generated from CHARMm's force field's parameter 
files. Primary, secondary, and tertiary amines were modeled as fully 
protonated with a total charge of +5 for 6b (partial charge of +0.35 for 
each protonated amine of 6b). 
To the solution structure of the dodecamer (Blasko, et al., 1993, supra) 6b 
was docked into the minor groove to initiate structural refinement of the 
1:1 complex of d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 : 6b. CHARMm 
minimization was subsequently conducted exactly as previously described 
for 5c (Blasko, et al., 1993, supra) with the following exception: only 2 
Na.sup.+ gegenions were removed from vicinity of the phosphates nearest to 
where the protonated polyamine sidechain and dimethylamine tail of 6b were 
initially located. 
Molecular and helical parameters were also measured exactly as before 
(Blasko, et al., 1993, supra; NEWHEL93 was generously provided by R. E. 
Dickerson. The program was run on a VAXstation 3100 with coordinates in 
Brookhaven's Protein Data Bank format. The best helicies were generated 
from the sugars' C1', the pyrimidine's N1, and the purine's N9 atoms. For 
more information on an earlier version of this program, see Prive, G. G.; 
Yanagi, K.; Dickerson, R. E. J. Mol. Biol. 1991, 217, 177). Dihedral angle 
constraints were not included in the simulations. 
The distances of 6b to the DNA (-) and (+) strands were measured from the 
pyrrolic nitrogens to P.sub.-4 P.sub.-5 P.sub.-6 and P.sub.8 P.sub.9 
P.sub.10, respectively. The depth of 6b binding was defined by measuring 
the distances from the amide nitrogens N1, N2, and N3 to the lines 
connecting T.sub.-6 O2 and A.sub.6 H2, A.sub.-7 H2 and T.sub.7 O2, and 
A.sub.-8 H2 and T.sub.8 O2 atoms, respectively. 
Results 
Titration of d(CGCAAATTTGCG (SEQ ID NO:3)).sub.2 with 6b 
All changes in the imino proton region (12-15 ppm) occur prior to reaching 
a 1:1 ratio of d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 and 6b when 
recording the .sup.1 H NMR spectra in 9:1 H.sub.2 O:D.sub.2 O solvent. The 
titration of d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 
(3.8.times.10.sup.-4 M) with 6b was carried out in 0.25 mole equiv. steps 
in D.sub.2 O at 21.degree. C. (FIG. 7). 
In contrast with the H.sub.2 O experiment, the nonexchangeable proton 
signals continue to change after reaching a mole ratio of 1:1 in 
6b/d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 when titrating in D.sub.2 
O (vide infra). In these titrations we employed mesitoate 
(2,4,6-trimethylbenzoate), at a 1:1 mole ratio with respect to dsDNA, as 
an internal standard. The mesitoate CH.sub.3 protons resonate at 2.22 ppm 
(2,6-position) and 2.24 ppm (4-position) while the aromatic protons 
(3,5-position) are at 6.90 ppm. The titration was followed up to a 2.5 
mole ratio of 6b to d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2. 
The affected dsDNA resonances double at the 1:1 mole ratio and give line 
broadenings. At the 2:1 mole ratio, the resonances corresponding to the 
1:1 ratio have collapsed and one observes only one set of equivalent 
resonances when monitoring the thymidine methyl signals (1.2-1.7 ppm). 
There is a downfield shift of the aromatic adenosine signals of 
d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 and an upfield shift of the 
pyrrole aromatic signals of 6b (FIG. 7a-c). 
The assignment of the resonances of 6b in H.sub.2 O (DQF-COSY, FIG. 21) is 
shown in Table II. These assignments were used as a lead for the 
assignment of the resonances of 6b in the dsDNA: 6b 1:1 complex. The 
Double Quantum Filtered Homonuclear J-Correlated Spectroscopy (DQF-COSY) 
spectrum of the 1:1 complex (FIGS. 22-25) shows the connectivities in the 
R2 and R3 propylamine and tren-polyamine groups; their chemical shifts are 
summarized in Table II. 
The H2, H4, and H6 pyrrole resonances of 6b (FIG. 19a) are found in the 
6.5-6.8 ppm region. They give NOEs with the aromatic adenosine A.sub.-7 
A.sub.-8 H2 protons of the (-) strand and with the sugar A.sub.5 H1' and 
A.sub.-8 H1' protons, respectively. The H1, H3, and H5 resonances of 6b 
were assigned using their intramolecular interactions with the 
CH.sub.2.sup.n (i) methylenes of the central hydrocarbon linker and with 
the CH.sub.3.sup.R1 group of the acetamide substituent (FIGS. 8 and 9). 
The assignment of the 6b resonances were confirmed by the NOE enhancements 
in the NOESY spectrum (FIGS. 8, 9, 10, 26 and 27). 
Assignment of .sup.1 H chemical shifts of d(CGCAAATTTGCG (SEQ ID 
NO:3)).sub.2 in the 1:1 complex with 6b 
The finding of two sets of Watson-Crick G.tbd.C and A=T resonances and two 
sets of thymidine CH.sub.3 resonances at the 1:1 mole ratio of 
6b/d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 is indicative of an 
asymmetric, monomeric binding of the ligand to the DNA molecule, as was 
found in the case of the d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 : 
5c complex (Blasko, A., et al., 1993, supra). 
Expansion of the NOESY spectrum in the (1.1-3.0).times.(6.7-8.5) ppm region 
(FIG. 9) shows the general pattern of NOESY interactions of H6/8-H2'2", 
H6/8-T.sub.i CH.sub.3, and T.sub.i CH.sub.3 -T.sub.i+1 CH.sub.3 used for 
the assignment of sugar H2'2" resonances (Table III). 
A good point to initiate assignments of the dsDNA resonances is at the 
signals of T.sub.7 T.sub.-6 CH.sub.3. This procedure was used in the case 
of free d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 and the d(CGCA.sub.3 
T.sub.3 GCG (SEQ ID NO:3)).sub.2 : 5c complex (Blasko, A., et al., 1993, 
supra). The T.sub.7 T.sub.-6 CH.sub.3 signals were used for the assignment 
of A.sub.6 A.sub.-7 H8, T.sub.7 T.sub.8 T.sub.9 H6 and T.sub.-4 T.sub.-5 
T.sub.-6 H6 proton resonances (FIG. 9). Here and elsewhere (Blasko, A., et 
al., 1993, supra), we use the convention that the (+) strand is the 
binding site side and the (-) strand is the complementary DNA strand. 
The remaining aromatic resonances were assigned using the known resonances 
of cytidine H6/5 (DQF-COSY, FIG. 22) which give strong intraresidual NOEs 
(FIG. 8) and using the interactions between two adjacent A.sub.n-1 A.sub.n 
H8 protons (8.05 and 8.25 ppm). We also used the proven fact that 6b binds 
into the minor groove at A+T-rich regions (He, G.-X., et al., 1993, 
supra). We saw NOE enhancements between A.sub.-8 H8 and A.sub.-9 H8 and 
also weak enhancements between A.sub.5 H8 and A.sub.6 H8. Both 
enhancements were used for the dsDNA sequential assignment. 
The guanosine H8 resonances (7.8-8.0 ppm) were used to define the C.sub.1 
G.sub.2 G.sub.12 H1' and T.sub.-4 H1' resonances (FIG. 8). We did not see 
NOE build-ups between G.sub.10 H8 and any of the H3' or H5'5" protons and 
no NOEs between adenosine H8 and H5'5" protons. Defining the position of 
A.sub.6 H1' is important in the intracomplex interactions (vide infra). We 
found weak NOEs between A.sub.6 H8 and A.sub.6 H1' (FIG. 8). The crowded 
region of H4' and H5'5" was resolved (where possible) using their NOEs 
with H1' protons (FIG. 26 and Table III). 
Intracomplex interactions of d(CGCAAATTTGCG (SEQ ID NO:3)).sub.2 and 6b 
Tren-microgonotropen-b (6b) binds into the A+T-rich region of the minor 
groove of d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 in 1:1 and 
noncooperative 2:1 mole ratios. These complexations also involve one G.C 
bp (vide supra). Expansion of the NOESY spectrum in the 
(5.3-8.3).times.(5.3-8.3) ppm region reveals strong NOE interactions 
between the H2, H4 and H6 pyrrole protons and the A.sub.-8 H2 and A.sub.-7 
H2 protons as well as a small NOE for H4 with the sugar A.sub.-8 H1' 
proton (FIGS. 8 and 26). The acetamido CH.sub.3.sup.R1 methyl protons of 
6b give NOEs with T.sub.-4 H6 and A.sub.6 H1' (FIGS. 9 and 10) defining 
the orientation of the 6b molecule in the minor groove. The 
dimethylpropylamino substituent, R3, approaches the G.sub.10 residue, 
defined by the NOE build-up between the CH.sub.3.sup.R3 and G.sub.10 H1' 
(FIG. 10) 
The tren polyamino substituent of the central pyrrole ring of 6b strongly 
interacts with the sugar protons of T.sub.8 and T.sub.9. We saw NOEs 
between CH.sub.2.sup.n (2) and T.sub.8 T.sub.9 H3', between CH.sub.2.sup.n 
(3) and T.sub.9 H3' (FIG. 10) and between CH.sub.2.sup.n (4) and T.sub.8 
H5" (FIG. 27). Other intracomplex interactions were seen between 
CH.sub.3.sup.R5 and T.sub.9 H4' (FIG. 26) and between H5 and A.sub.-8 H2" 
(FIG. 9). An inter-residual NOE was also seen between T.sub.7 H5" and 
A.sub.6 H2 (FIG. 28). 
TABLE II 
__________________________________________________________________________ 
.sup.1 H Chemical Shifts for 6b, Free and in the 1:1 Complex with 
d(CGCA.sub.3 T.sub.3 GCG(SEQ ID NO: 3)).sub.2 in D.sub.2 O. 
Residue 
Proton 
d(CGCA.sub.3 T.sub.3 GCG(SEQ ID NO: 3)).sub.2 :6b 
d(CGCA.sub.3 T.sub.3 GCG(SEQ ID NO: 
.DELTA..delta..sup.b 
__________________________________________________________________________ 
pyrrole 
H1 7.07 7.18 -0.11 
pyrrole 
H3 7.24 7.01 0.23 
pyrrole 
H5 7.26 7.06 0.20 
pyrrole 
H2 6.63 6.70 -0.07 
pyrrole 
H4 6.74 6.74 0.00 
pyrrole 
H6 6.57 6.71 -0.14 
R1 methyl 
2.08 1.98 0.10 
R3 methyl 
2.87 2.63 0.24 
R4 methyl 
3.97 3.76 0.21 
R5 methyl 
3.97 3.76 0.21 
CH.sub.2.sup.R2 
(1) 2.36 2.70 -0.34 
CH.sub.2.sup.R2 
(2) 2.62 2.92 -0.30 
CH.sub.2.sup.R2 
(1') 
3.01 2.94 0.07 
CH.sub.2.sup.R2 
(2') 
2.77 2.68 0.09 
CH.sub.2.sup.R3 
(1) 3.12 3.33 -0.21 
CH.sub.2.sup.R3 
(2) 1.87 1.90 -0.03 
CH.sub.2.sup.R3 
(3) 2.03 2.88 -0.85 
CH.sub.2.sup.n 
(1) 5.41 4.18 1.23 
CH.sub.2.sup.n 
(2) 1.76 1.73 0.03 
CH.sub.2.sup.n 
(3) 2.16 1.60 0.56 
CH.sub.2.sup.n 
(4) 3.14 2.85 0.29 
__________________________________________________________________________ 
.sup.a .delta. in ppm relative to TSP at 10.degree. C.; dsDNA! = 2.5 
.times. 10.sup.-3 M (10 mM phosphate buffer, pH 7.0, 10 mM NaCl). .sup.b 
.delta..sub.complex - .delta..sub.free dsDNA. 
TABLE III 
______________________________________ 
.sup.1 H Chemical Shifts for d(CGCA.sub.3 T.sub.3 GCG).sub.2 in the 1:1 
Complex with 6b in D.sub.2 O..sup.a 
H2/5/ 
Base H1' H2' H2" H3' H4' H5' H5" H6/8 CH.sub.3 
______________________________________ 
(+) 
strand 
5'-C.sub.1 
5.71 1.95 2.37 4.68 4.04 4.03 3.70 7.60 5.82 
G.sub.2 
5.84 2.64 2.68 4.94 4.33 4.40 4.35 7.94 
C.sub.3 
5.75 1.90 2.33 4.82 4.18 4.18 4.12 7.39 5.42 
A.sub.4 
5.81 2.74 2.80 5.06 4.38 4.47 4.22 8.20 7.18 
A.sub.5 
5.55 2.72 2.78 5.03 nd.sup.b 
4.46 4.36 8.25 6.98 
A.sub.6 
5.84 2.68 2.77 5.06 4.22 4.40 4.22 8.13 7.46 
T.sub.7 
5.36 1.97 2.41 4.62 nd 4.02 3.88 6.93 1.23 
T.sub.8 
5.64 2.00 2.31 4.63 3.70 3.88 3.35 7.18 1.46 
T.sub.9 
5.42 1.98 2.30 4.78 4.10 4.20 4.10 7.11 1.56 
G.sub.10 
5.84 2.55 2.66 4.98 4.01 4.35 4.12 7.80 
C.sub.11 
5.70 1.93 2.33 4.83 4.03 4.18 4.13 7.36 5.41 
G.sub.12 
6.15 2.36 2.62 4.67 4.18 4.17 4.06 7.95 
(-) 
strand 
C.sub.-12 
5.71 1.95 2.37 4.68 4.04 3.98 3.70 7.58 5.82 
G.sub.-11 
5.84 2.64 2.68 4.94 4.33 4.40 4.35 7.94 
C.sub.-10 
5.75 1.90 2.33 4.82 4.18 4.18 4.12 7.39 5.42 
A.sub.-9 
5.81 2.79 2.89 5.06 nd 4.47 4.22 8.15 7.53 
A.sub.-8 
5.52 2.78 2.89 5.08 4.22 4.46 4.36 8.23 8.08 
A.sub.-7 
5.86 2.71 2.81 5.05 nd 4.40 4.22 8.08 8.12 
T.sub.-6 
5.70 1.97 2.41 4.64 nd 4.15 3.92 6.87 1.21 
T.sub.-5 
6.17 2.00 2.40 4.62 3.70 4.00 3.85 7.22 1.48 
T.sub.-4 
5.75 1.98 2.42 4.78 4.10 4.15 4.10 7.28 1.62 
G.sub.-3 
5.78 2.36 2.66 4.97 4.01 4.10 3.98 7.92 
C.sub.-2 
5.70 1.93 2.32 4.83 4.03 4.18 4.13 7.36 5.35 
G.sub.-1 
6.15 2.36 2.62 4.67 4.18 4.17 4.06 7.95 
______________________________________ 
.sup.a .delta. in ppm relative to TSP at 10.degree. C.; dsDNA! = 2.5 
.times. 10.sup.-3 M (10 mM phosphate buffer pH 7.0, 10 mM NaCl). The 
WatsonCrick imino protons (recorded in H.sub.2 O) are in the range: A = T 
13.5-14.2 and G .ident. C 12.5-13.1 ppm (Blasko, A., Bruice, T.C. PNAS 
(USA) 1993 90: 10018). 
.sup.b not determined. 
No NOEs were detected between the R2 polyamino substituent of 6b and 
d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2. However, the NOE build-up 
between CH.sub.2.sup.n (4) and CH.sub.2.sup.R2 (2') protons (FIGS. 11 and 
27) will define the position of this part of the R2 polyamine group of 6b 
with regard to the dsDNA molecule (note that the position of the 
CH.sub.2.sup.n chain was already defined from their NOEs with d(CGCA.sub.3 
T.sub.3 GCG (SEQ ID NO:3)).sub.2 ; vide supra). A survey of the sequential 
NOEs for the DNA selected protons in the ligated dsDNA is shown in Table 
IV. 
Induced chemical shift differences (.DELTA..delta.) are observed in certain 
proton resonances (FIG. 12) due to the minor groove binding. This is 
primarily due to the ring current effect from both the dsDNA and the 
tripyrrole peptide. The .DELTA..delta. extends beyond the binding site due 
to distortion of the dsDNA upon binding. 
With the exception of the T.sub.8 H5" (.DELTA..delta.=-0.8 ppm (FIG. 12), 
the differences are greater for the H1' protons (minor groove pointers) 
than for any other selected protons. The increase in .DELTA..delta. 
follows the order H2'&lt;H6/8&lt;H3'&lt;H2".apprxeq.H5'&lt;H1'. The aromatic pyrrole 
protons, H3 and H5, give upfield shifts upon binding (.DELTA..delta.=0.2 
ppm) while H1, H2 and H6 give downfield shifts (.DELTA..delta.=-0.1 ppm) 
(Table II). 
All the CH.sub.3 groups of R1, R3, R4 and R5 give upfield shifts 
(.DELTA..delta.=0-0.2 ppm). Small downfield shifts were seen in the case 
of CH.sub.2.sup.R2 (1'), CH.sub.2.sup.R3 (1), CH.sub.2.sup.R3 (2) 
(.DELTA..delta.&lt;-0.1 ppm) and small upfield shifts in the case of 
CH.sub.2.sup.R2 (2') and CH.sub.2.sup.n (2') (.DELTA..delta.=0.2 ppm). 
Large upfield shifts are exhibited by the hydrocarbon linker methylene 
resonances (.DELTA..delta.=0.3-1.2 ppm), the highest (.DELTA..delta.=1.2 
ppm) being at CH.sub.2.sup.n (1). Large downfield shifts were seen in the 
case of CH.sub.2.sup.R3 (3) (.DELTA..delta.=-0.8 ppm) and in the case of 
CH.sub.2.sup.R2 (1) and CH.sub.2.sup.R2 (2) (.DELTA..delta.=-0.3 ppm). 
These are due to their adjacent protonated amines which are involved in 
hydrogen bonding to phosphates. 
Sugar puckerings of d(CGCAAATTTGCG (SEQ ID NO:3)).sub.2 
From the DQF-COSY spectrum of the d(CGCA.sub.3 T.sub.3 GCG (SEQ ID 
NO:3)).sub.2 : 6b complex (FIGS. 22-25), coupling constants can be 
estimated and, therefore, some sugar residues can be characterized in 
terms of their vicinal proton dihedral angles. 
TABLE IV 
__________________________________________________________________________ 
Comparison of the Sequential NOEs for: (a) d(CGC.sub.3 T.sub.3 GCGA(SEQ 
ID NO: 3)).sub.2 and (b) the 1:1 
Complex of d(CGCA.sub.3 T.sub.3 GCG(SEQ ID NO: 3)).sub.2 with 
__________________________________________________________________________ 
6b. 
a. (.+-.) strand 
C.sub.1 
G.sub.2 
C.sub.3 
A.sub.4 
A.sub.5 
A.sub.6 
T.sub.7 
T.sub.8 
T.sub.9 
G.sub.10 
C.sub.11 
G.sub.12 
(SEQ ID NO: 3) 
H6/8--CH.sub.3 o---o----o----o 
H6/8--H1' 
o----o o----o----o 
H6/8--H2" 
o----o o----o----o----o----o----o----o----o----o 
CH.sub.3 --CH.sub.3 o----o 
H6--H6 o---o---o---o 
b. (+) strand: 
C.sub.1 
G.sub.2 
C.sub.3 
A.sub.4 
A.sub.5 
A.sub.6 
T.sub.7 
T.sub.8 
T.sub.9 
G.sub.10 
C.sub.11 
G.sub.12 
(SEQ ID NO: 3) 
H6/8--CH.sub.3 /H5/6/8 o----o----o----o o----o 
H6/8/5--H1' 
o----o o----o 
H6/8/CH.sub.3 --H2" 
o---o----o o----o o----o o----o----o 
H6/8--H3' o----o 
H2/CH.sub.3 --H/CH.sub.3 
o----o o----o 
b. (-) strand: 
C.sub.-12 
G.sub.-11 
C.sub.-10 
A.sub.-9 
A.sub.-8 
A.sub.-7 
T.sub.-6 
T.sub.-5 
T.sub.-4 
G.sub.-3 
C.sub.-2 
G.sub.-1 
(SEQ ID NO: 3) 
H6/8--CH.sub.3 /H5/6/8 
o----o 
o----o----o----o 
o----o 
H6/8--H1' o----o o----o 
H6/8/CH.sub.3 --H2" 
o----o o----o----o o----o 
H6/8--H3' o----o 
H2/CH.sub.3 --H2/CH.sub.3 
o----o----o----o----o 
__________________________________________________________________________ 
In terms of sugar puckering, the DNA's backbone conformation is dictated by 
the glycosidic torsion angle defined by C5'-C4'-C3'-O3'. The exact .sup.3 
J coupling constants involving H3' are hard to determine due to their 
passive coupling including phosphorus (Kim, S.-G.; Lin, L.-J.; Reid, B. R. 
Biochemistry 1992, 31, 3564). However, they can be qualitatively 
constrained into restricted ranges from the corresponding cross-peaks 
intensities (Kim, S.-G., et al., 1992, supra). 
Cross-peaks between H3'-H2" and H3'-H4' were weak or nonexistent in the 
DQF-COSY spectrum of the 1:1 complex (FIG. 22), except for some terminal 
base pairs. These very small coupling constants are indicative of the 
presence of the B-form of dsDNA (Kim, S.-G., et al., 1992, supra). Since 
the sugar conformation can be determined from the NOESY-derived distance 
data, the coupling constants estimated from the DQF-COSY complements the 
NOESY/RM characterization of the complexed dsDNA. 
In the cases of the well resolved H1'-H2" and H1'-H2' cross-peaks, sugar 
coupling constants were estimated for G.sub.10, G.sub.12, C.sub.1, 
C.sub.3, and C.sub.11 to be 3-5 Hz and 1.5 Hz for A.sub.4 and A.sub.6 
(FIGS. 23-25). In all cases .sup.3 J.sub.H1'-H2' &gt;.sup.3 J.sub.H1'-H2". 
This limits the deoxyribose pseudorotational phase angles (P) to 
90.degree.-190.degree. (Kim, S.-G., et al., 1992, supra). 
In the case of the terminal base pairs C.sub.1, C.sub.3, and G.sub.12, the 
coupling constants for H3'-H4' were 3-5 Hz, while for the binding site 
residue T.sub.9 /T.sub.-4, 2.5 Hz, placing them close to P=126.degree., 
H1'-exo and P=140.degree.-162.degree., H2'-endo, respectively. No other 
cross-peaks could be seen and/or resolved. 
Distance calculations and restrained molecular modeling refinements 
For the 1:1 complex of the dodecamer d(CGCA.sub.3 T.sub.3 GCG (SEQ ID 
NO:3)).sub.2 and 6b, 155 intramolecular interactions were found for both 
NMR-nonequivalent strands. Of these, 17 were used in refining the DNA 
distances of the previously determined solution structure of d(CGCA.sub.3 
T.sub.3 GCG (SEQ ID NO:3)).sub.2 (Blasko, A., et al., 1993, supra) (Table 
V). 
These intramolecular interactions represent the only well separated cross 
peaks (Table IV). In addition, 17 interactions between 6b and the dsDNA 
and intramolecular 6b interactions were used for docking (FIG. 11; Table 
V). The same minimization procedure used previously (Blasko, A., et al., 
1993, supra) was employed to obtain the most probable solution structure 
of the 1:1 complex of 6b with d(CGCA.sub.3 T.sub.3 GCG (SEQ ID 
NO:3)).sub.2 (FIGS. 13A-C). 
All deviations in the refined structure from the calculated NOE distances 
were less than 0.6 .ANG.. The ROESY spectrum (FIG. 29) confirms most of 
the NOESY enhancements. 
TABLE V 
______________________________________ 
Experimental (NOESY) and Refined (Molecular Modeling) Distances 
for the 1:1 Complex of d(CGCA.sub.3 T.sub.3 GCG(SEQ ID NO: 3)).sub.2 with 
6b. in 
D.sub.2 O..sup.a (Refined distances are in parentheses.) 
______________________________________ 
a. Distances involving only d(CGCA.sub.3 T.sub.3 GCG)SEQ ID NO: 3)).sub.2 
protons: 
CH.sub.3 / 
H1' H2' H5' H6/8 H5/H2* 
______________________________________ 
G.sub.2 
H8 3.9.sup.b (4.0) 
C.sub.3 
H6 4.1 (4.0) 
A.sub.4 
H8 4.9 (4.3) 
A.sub.5 
H8 3.9 (3.9) 
A.sub.6 
H8 4.1.sup.b (4.1) 
A.sub.6 
H2 3.4.sup.c (3.9) 
T.sub.8 
H6 4.3.sup.b (4.2) 3.8.sup.c (3.8) 
T.sub.8 
CH.sub.3 4.3.sup.b (4.4) 
G.sub.12 
H8 3.7 (3.8) 
4.9.sup.b (4.8) 4.0.sup.b (4.2) 
T.sub.-5 
H6 3.8.sup.c (3.8) 
T.sub.-6 
H6 4.4.sup.c (4.4) 
A.sub.-7 
H8 4.3.sup.c (4.3) 
A.sub.-8 
H2 4.7*.sup.c (4.6) 
A.sub.-9 
H8 4.2 (4.1) 
b. Distances involving d(CGCA.sub.3 T.sub.3 GCG(SEQ ID NO: 3)).sub.2 and 
6b protons: 
H2-A.sub.-7 H2 3.4(3.4); H4-A.sub.-7 H2 3.7(3.7); H4-A.sub.-8 H1' 
4.0(4.3); 
H4-A.sub.-8 H2 3.6(3.6); H6-A.sub.-8 H1' 4.0(3.8); H6-A.sub.-8 H2 
3.8(3.8); 
CH.sub.3.sup.R1--A.sub.6 H1' 4.2(4.1); CH.sub.2.sup.R1 -T.sub.-4 H6 
4.0(4.5); CH.sub.2.sup.n (3)--T.sub.9 H3' 3.6 (4.0); 
CH.sub.2.sup.n (2)--T.sub.9 H3' 3.8(3.8); CH.sub.3.sup.R3 --G.sub.10 H1' 
4.8(4.8); 
H1--CH.sub.3.sup.R1 3.8(4.2); H3--CH.sub.2.sup.n (1) 3.0(3.0); 
H3--CH.sub.2.sup.n (3) 3.8(4.4); 
H5--CH.sub.2.sup.n (2) 3.3(3.8); CH.sub.2.sup.n (4)--CH.sub.2.sup.R2 (2) 
4.0(4.2); 
CH.sub.2.sup.n (4)--CH.sub.2.sup.R2 (2') 4.0(4.2) 
______________________________________ 
.sup.a In .ANG., with the same residue. .sup.b Distances with the (n - 1) 
residue. .sup.c Distances with the (n + 1) residue. Distances marked with 
asterisks (*) belong to the protons marked with asterisks. 
Comparison of solution structures of d(CGCA.sub.3 T.sub.3 GCG (SEQ ID 
NO:3)).sub.2 (Blasko, A., et al., 1993, supra) and the d(CGCA.sub.3 
T.sub.3 GCG (SEQ ID NO:3)).sub.2 : 6b complex shows that the minor groove 
widens considerably between the T.sub.-5 to T.sub.9 and T.sub.-4 to 
T.sub.8 phosphates (4-3 .ANG., respectively) upon complexation of 6b. The 
ligand binds 7.3-9.0 and 5.5-6.4 .ANG. from the (-) and (+) strands, 
respectively, when examining the regions from T.sub.-4 to T.sub.-6 and 
G.sub.10 to T.sub.8 (distances from the pyrrole nitrogens to P.sub.-4 
P.sub.-5 P.sub.-6 and P.sub.8 P.sub.9 P.sub.10 respectively; Experimental 
section). 
In addition, the 6b complexed dodecamer lengthens 1 .ANG. relative to the 
solution structure of the dodecamer (Blasko, A., et al., 1993, supra) as 
is evidenced by the unit height (34.93 .ANG./repeat). This is due to a 
combination of a relatively unwound helix (turn angle=35.90.degree./bp), a 
large axial rise (3.50 .ANG./bp), and a fairly large helical rise (10.03 
bp/repeat). The angle of the bend (.alpha.; FIG. 14) in the helical axis 
of the solution structure of the d(CGCA.sub.3 T.sub.3 GCG (SEQ ID 
NO:3)).sub.2 complexed with 6b (22.2.degree.) is more than twice the same 
angle for the crystal (10.8.degree.) and only 0.8.degree. greater than the 
solution (21.4.degree.) structure of the d(CGCA.sub.3 T.sub.3 GCG (SEQ ID 
NO:3)).sub.2 alone (Blasko, A., et al., 1993, supra). In the solution 
structure, the molecular contact surface area between d(CGCA.sub.3 T.sub.3 
GCG (SEQ ID NO:3)).sub.2 and 6b is 518 .ANG..sup.2. 
Dynamics of ligand exchange 
The signals of the H2, H4, and H6 resonances of 6b exhibit different line 
broadenings (.DELTA.v.sub.1/2 =14, 15, and 10 Hz, respectively) when in 
the 1:1 complex with d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 (FIG. 
8). This is in accord with minor groove binding (Umemoto, K.; Sarma, M. 
H.; Gupta, G.; Luo, J.; Sarma, R. H. J. Am. Chem. Soc. 1990, 112, 4539). 
As previously discussed (Blasko, A., et al., 1993, supra) the broadening 
could be due to the relatively slow exchange of 6b between two equivalent 
binding sites and/or to a fast sliding motion in the minor groove. 
Exchanges between two equivalent binding sites have been proposed for dsDNA 
complexes of distamycin (Pelton et al., 1990, supra) and netropsin (Patel 
et al., 1985, supra). If we consider that the exchange is governed by a 
"flip-flop" mechanism (Pelton et al., 1990, supra) (FIG. 18), not 
excluding the possible existence of a fast sliding motion of 6b in the 
minor groove, the rate of exchange can be calculated (Experimental 
Section). In studying the identical line shapes of the diagonal and cross 
peaks, the rate of exchange for this process was found to be 1.3.+-.0.2 
s.sup.-1 (10.degree. C., Experimental Section) corresponding to an 
activation energy (.DELTA.G*) of .sup..about. 17 kcal/mol. 
The association constant of 6b with A.sub.3 T.sub.3 sites (e.g. 
d(GGCGCA.sub.3 T.sub.3 GGCGG)(SEQ ID NO:1)/d(CCGCCA.sub.3 T.sub.3 
GCGCC)(SEQ ID NO:2)} has been determined (Example I) to be 
8.times.10.sup.8 M.sup.-1. From this information, dissociation of 6b from 
the hexadecamer is much slower than association and, therefore, one can 
consider the rate of exchange equal to the off-rate (k.sub.ex 
.apprxeq.k.sub.off). 
Here, and elsewhere (Blasko, A., et al., 1993, supra), we consider these 
values as estimates and their determination does not include studies 
beyond our goal of cross relaxations contributing to the peak intensities 
and the mixing time profile (Klevit, R. E.; Wemmer, D. E.; Reid, B. R. 
Biochemistry 1986, 25, 3296). 
Discussion 
Both 1:1 and 2:1 complexes of d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 
with 6b have been observed. The solution structure of the 1:1 complex of 
d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 with 6b has been determined 
by 2D NMR spectroscopy and restrained molecular modeling. Due to the 
complexity of ligation and the dynamics of 6b in the complex with dsDNA, 
small populations of the free dodecamer or of dodecamer:ligand complexes 
of structures other than reported here may exist in solution. 
The titration of d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 with 6b in 
H.sub.2 O/D.sub.2 O 9:1 (at 1.8.times.10.sup.-4 M of dsDNA) (Blasko, A., 
et al., 1993, supra) was carried out to a ratio of 2:1 of 6b to dsDNA. No 
detectable spectral changes in the imino protons' resonances were observed 
above a 1:1 mole ratio of 6b to d(CGCA.sub.3 T.sub.3 GCG (SEQ ID 
NO:3)).sub.2 (in D.sub.2 O we could detect a 2:1 complex, vide infra). 
The spectral changes in the imino proton region when titrating with 6b show 
that 6b targets the A+T-rich region involving one G.C residue. The 
titration in D.sub.2 O (dsDNA!=3.8.times.10.sup.-4 M) was carried out to 
a ratio of 2.5:1 of 6b to dsDNA. In this experiment spectral changes in 
the nonexchangeable protons extended from below a 1:1 ratio to a 2:1 ratio 
of 6b/d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 (FIG. 7). 
The doubling of the dsDNA resonances (in the D.sub.2 O experiment) below a 
1:1 mole ratio is indicative of an asymmetrical type of binding (see 
thymidine CH.sub.3 's (1.2-1.6 ppm)) of 6b to d(CGCA.sub.3 T.sub.3 GCG 
(SEQ ID NO:3)).sub.2. The collapse of these resonances to only one set at 
a 2:1 mole ratio is indicative of a symmetrical binding mode for two 6b 
per one d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2. 
In a study based on fluorescence measurements, it was found that the 
equilibrium constants for binding of the first and second molecule of 6b 
to d(GGCGCA.sub.3 T.sub.3 GGCGG)(SEQ ID NO:1)/d(CCGCCA.sub.3 T.sub.3 
GCGCC)(SEQ ID NO:2) shows slight cooperativity (Example I). Using 
d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 with 6b, our .sup.1 H NMR 
examination shows no (or undetectable) cooperativity in binding. 
The inability to observe .sup.1 H NMR spectral changes in the imino region 
above a 1:1 ratio suggests that at any given time only one of two 6b 
molecules resides inside the groove (FIG. 19). In the 2:1 complex there 
should be a fast exchange between the two molecules of 6b when binding to 
d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 such that the minor groove 
widens (and remains wide during the exchange of two 6b molecules) and, as 
a result, spectral changes occur. On decreasing the temperature to 
-5.degree. C., the internal motions of the 2:1 complex of 6b/d(CGCA.sub.3 
T.sub.3 GCG (SEQ ID NO:3)).sub.2 (dsDNA!=4.times.10.sup.-4 M) decrease. 
At -5.degree. C. broadening of the A.T resonances occur while the G.C 
signals remain sharp. It was previously shown that the 4:1 
distamycin/d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 complex maintains 
its A.T and G.C resonance line widths when going to -10.degree. C. The 
broadening of the A.T resonances of the 2:1 complex of 6b/d(CGCA.sub.3 
T.sub.3 GCG (SEQ ID NO:3)).sub.2 at -5.degree. C. could be due to (a) an 
asymmetric 2:1 rigid binding mode in which 6b exchanges between two 
equivalent sites of the dsDNA or (b) a symmetrical 2:1 binding mode in 
which two molecules of 6b exchange as shown in FIG. 19. The possibility of 
an asymmetric, rigid 2:1 binding can be ruled out due to the existence of 
only one set of 6b resonances. 
The binding of the flat tripyrrole peptide portion of 6b in the A+T-rich 
region of the 1:1 complex results in broadening and downfield shifting of 
the involved resonances (Leupin, W.; Chazin, W. J.; Hyberts, S.; Denny, W. 
A.; Wuthrich, K. Biochemistry 1986, 25, 5902). Assignment of the 
nonexchangeable protons (Table III) revealed two sets of DNA resonances, 
but only one set of 6b resonances (Table II). This indicates that the 
predominant structure involves a single type of monomeric binding. 
Induced chemical shift differences reveal that the most affected protons 
involved in the dsDNA to 6b interactions are H1' and H2" (FIG. 12). These 
chemical shift differences also show the changes which occur at the 
binding site by the perturbation of the involved protons. 
The large chemical shift difference, .DELTA..delta., for T.sub.8 H5" 
indicates strong interactions between this proton and the CH.sub.2.sup.n 
hydrocarbon linker of the central pyrrole ring of 6b, consistent with the 
large .DELTA..delta. found for the CH.sub.2.sup.n protons (Table II). This 
observation is in agreement with the refined solution structure of the 
d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 : 6b complex (FIGS. 13A-C). 
The increase in the number of NOEs observed for H6/8 with CH.sub.3 /H5/6/8 
protons (not involved in the exchange phenomena) as compared to the free 
DNA (Blasko et al., 1993, supra) can be ascribed to the stiffening of the 
DNA molecule at the binding site (Table IV, see H6/8 interactions with 
CH.sub.3 /H5/6/8) and/or to the dynamic motion of the dodecamer around a 
position which would bring the aromatic units of the binding site closer 
together as seen in the case of 5c (Blasko et al., 1993, supra). 
By convention, we assigned this sequence to the (+) strand. The 
characteristics of the reduced electrophoretic mobilities on agarose gels 
of DNA restriction digest fragments after preincubation with 6b suggest a 
distortion of DNA (He et al., 1993, supra). 
Although the differences in the induced chemical shifts beyond the binding 
site are generally small, even in the case of the terminal base pairs 
(C.sub.1, G.sub.-1 and G.sub.12, C.sub.-12) structural distortions occur 
upon binding as is evidenced by .DELTA..delta..noteq.0 (FIG. 12). The 
significant .DELTA..delta. for G.sub.10 H2' enforces our observation that 
this proton is involved in an interaction with 6b. 
A small effect on the proton resonances of the aromatic bases suggests that 
the binding of 6b does not significantly affect the positions of those 
protons that are major groove pointers. The upfield shift of the H5' and 
H5" resonances suggests high electron density around these protons. These 
electron densities derive from the central tren polyamino substituent of 
6b. 
The acetamido function of 6b affects the position of A.sub.6 H5" to a small 
extent while perturbation of G.sub.10 H5' is by the dimethylpropylamino 
substituent R3. These chemical shift differences suggest that, aside from 
the minor groove protons which experience disruption of the DNA ring 
currents due to 6b binding, all other affected protons are influenced by 
the conformational changes of the DNA which occurs upon complex formation. 
There are changes in base pairing and stacking as well as sugar puckering 
of d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 upon formation of the 
d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)) .sub.2 : 6b complex. From the 
derived dihedral angles of the ribose moieties, we can state that the A.T 
regions of the complexed dsDNA maintains its B-conformation (Kim et al., 
1992, supra) and the terminal G.C ends do not. 
Instead, the G.C ends appear to exist in an intermediate B- to A-DNA form 
when monitored by the H3'-H4' dihedral angles. Since the conformation of 
the terminal base pairs is not strictly maintained due to the dynamic 
"fraying" of the ends, it is not surprising that those dihedral angles do 
not correspond to B-DNA. 
The --CH.sub.2 CH.sub.2 CH.sub.2 N(CH.sub.3).sub.2 tail at the carboxyl 
terminus of 6b is completely within the minor groove. This observation is 
consistent with the induced chemical shift differences for the R3 protons 
of 6b in the complex (Table II). The CH.sub.3 protons of the acetamido 
moiety R1 are slightly deshielded while R3, R4, and R5 methyl protons are 
strongly deshielded due to their proximities with the phosphate backbone. 
A strong deshielding is observed on the first, third and fourth methylene 
groups of the CH.sub.2.sup.n chain attached to the nitrogen of the central 
pyrrole ring. This suggests that these three methylenes have proximities 
with the dsDNA phosphates as shown by the structure of the 1:1 
d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 /6b complex (FIGS. 13A-C). 
The deshielding of H3 and H5 was ascribed to the pyrrole ring interactions 
with the phosphate ridge on the minor groove side. 
Microgonotropen 6b possesses five aliphatic amino groups: two primary, one 
secondary and one tertiary in the tren substituent (--CH.sub.2 CH.sub.2 
CH.sub.2 CH.sub.2 NHCH.sub.2 CH.sub.2 N(CH.sub.2 CH.sub.2 NH.sub.2).sub.2) 
and one tertiary in the dimethyl propylamino tail (--CH.sub.2 CH.sub.2 
CH.sub.2 N(CH.sub.3).sub.2). The extent of their protonation when 6b is 
lodged in the minor groove is not certain. In solution at pH 7.0, 6b would 
be expected to have at least four of its five amino groups protonated 
(Bruice, T. C.; Mei, H.-Y.; He, G.-X.; Lopez, V. Proc. Natl. Acad. Sci. 
(USA) 1992, 89, 1700; Lowry, T. H.; Richardson, K. S. "Mechanism and 
Theory in Organic Chemistry", 3rd Edition, Harper & Row, New York, 1987, 
p. 311). 
The upfield shift of the CH.sub.2.sup.R3 (3) resonance suggests protonation 
of the --CH.sub.2 CH.sub.2 CH.sub.2 N(CH.sub.3).sub.2 nitrogen. The latter 
is involved in hydrogen bonding with C.sub.11 O4'. The deshielding of the 
tren polyamino end methylenes, CH.sub.2.sup.R2 (1')/(2'), by &lt;0.1 ppm is 
also suggestive of protonation of the corresponding terminal tren 
nitrogens involved in hydrogen bondings (the dominant effect on 
.DELTA..delta.) with the phosphate oxygens of T.sub.9 P and G.sub.10 P as 
shown by the molecular modeling results (FIG. 13A-C). 
We have assumed (vide infra), in our restrained molecular modeling, that 
all five amino functions are fully protonated. This is in agreement with 
the induced chemical shift differences for the methylene protons flanking 
the involved amino groups (Table II). When complexed to dsDNA, the four 
tren amino groups are intimately associated with two negatively charged 
phosphates, T.sub.9 P and G.sub.10 P. 
Examination of the X-ray structure of the d(CGCA.sub.3 T.sub.3 GCG (SEQ ID 
NO:3)).sub.2 : distamycin complex (Coll, M.; Frederick, C. A.; Wang, A. 
H.-J.; Rich, A. Proc. Natl. Acad. Sci. (USA) 1987, 84, 8385) and the 
d(CGCGAATT.sup.Br CGCG (SEQ ID NO:3)).sub.2 : netropsin complex (Kopka, M. 
L.; Yoon, C.; Goodsell, D.; Pjura, P.; Dickerson, R. E. Proc. Natl. Acad. 
Sci. (USA) 1985, 82 1376) leads to the conclusion that the minor groove 
can increase its width upon binding to lexitropsins. Using X-ray 
structures, comparison of the width (phosphate to phosphate at the A.T 
binding site) of the minor grooves of d(CGCA.sub.3 T.sub.3 GCG (SEQ ID 
NO:3)).sub.2 (9.4-9.9 .ANG.) and d(CGCA.sub.3 T.sub.3 GCG (SEQ ID 
NO:3)).sub.2 : distamycin complex (9.4-10.8 .ANG.) shows an increase of 
0-0.9 .ANG. (Coll et al., 1987, supra). 
Using the NMR solution structures, comparison of the width of the minor 
grooves of d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 (6.5-10 .ANG.) 
(Blasko, A., et al., 1993, supra) and d(CGCA.sub.3 T.sub.3 GCG (SEQ ID 
NO:3)).sub.2 : 6b (9.2-9.6 .ANG.) shows an increase of 0.4-3.1 .ANG.. 
There is some variability in the positioning of ligands within the minor 
groove of B-DNA even when there is a common motif such as the "flat 
sickle-shape" of 6b, 5c, and distamycin. Thus, the amide nitrogens of 6b 
are embedded to a distance of 3.1-4.5 .ANG. from the floor of the groove. 
The crystal structure of the d(CGCA.sub.3 T.sub.3 GCG (SEQ ID NO:3)).sub.2 
: distamycin complex (Coll et al., 1987, supra) shows distamycin 
penetrating to within 4.2-4.5 .ANG. from the bottom of the minor groove. 
Examination of FIGS. 13A-C shows how the positively charged 
dimethylpropylamino tail (R.sub.3) of 6b resides at a position which is 
adjacent to the C.sub.11 O2 and O4' in the minor groove while the 
protonated tren moiety is paired with the phosphates of T.sub.9 and 
G.sub.10. The three primary amines of 6b's tren amino substituent are 
located within 1.75 .ANG. of two phosphodiester oxyanions while the fourth 
amine (tertiary) is 3.0 .ANG. from the same two adjacent phosphodiester 
oxyanions. 
The binding of distamycin in the minor groove is enhanced by its amidine 
tail forming bifurcated hydrogen bonds to the bottom of the minor groove 
(Coll et al., 1987, supra). 
Changing the amidine tail (--CH.sub.2 CH.sub.2 C(.dbd.NH)NH.sub.2) } of the 
carboxyl terminus of distamycin to a (--CH.sub.2 CH.sub.2 CH.sub.2 
N(CH.sub.3).sub.2 group and the formyl substituent at the amino terminus 
to acetamide causes a decrease in the equilibrium constant for 1:1 complex 
formation with d(GGCGCA.sub.3 T.sub.3 GGCGG)(SEQ ID NO:1)/d(CCGCCA.sub.3 
T.sub.3 GCGCC)(SEQ ID NO:2) from 4.times.10.sup.7 for distamycin to 
6.times.10.sup.6 M.sup.-1 (Browne et al., 1993, supra). 
However, further change of the N-methyl group on its central pyrrole to 
include a four methylene linker and a tren polyamino side chain (6b) leads 
to a binding constant of 8.times.10.sup.8 M.sup.-1 to the same oligomer 
(He et al., 1993, supra). This increase from 6.times.10.sup.6 to 
8.times.10.sup.8 M.sup.-1 in the binding constant must be due to the 
electrostatic interactions of the polyamino side chain with the 
phosphodiester linkages (He, G.-X., supra, 1993). 
The significance of the central polyamino groups of 6b can be seen when 
comparing the bending angle of the d(CGCA.sub.3 T.sub.3 GCG (SEQ ID 
NO:3)).sub.2 : 6b complex (22.2.degree.) with the angles found in 
distamycin complexed (1:1 and 2:1) to d(CGCA.sub.3 T.sub.3 GCG (SEQ ID 
NO:3)).sub.2 (13.9.degree. and 11.3.degree., respectively; FIG. 14). 
The molecular contact surface area between d(CGCA.sub.3 T.sub.3 GCG (SEQ ID 
NO:3)).sub.2 and 6b is 518 .ANG..sup.2 
In the 1:1 complexes of the dodecamer d(CGCA.sub.3 T.sub.3 GCG (SEQ ID 
NO:3)).sub.2 with 6b, 5c (Blasko et al., 1993, supra), or distamycin 
(Pelton et al., 1990, supra), exchange is between two equivalent (A.sub.3 
T.sub.3) binding sites via the "flip-flop" mechanism. The rate constant 
for exchange (which equals the off-rate) for 6b (10.degree. C.) is ca. 1.3 
s.sup.-1. This may be compared to 0.2 s.sup.-1 for distamycin at 
30.degree. C. (Pelton et al., 1990, supra). Thus, the exchange rate with 
6b at identical A.sub.3 T.sub.3 sites appreciably exceeds that for 
distamycin. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 6 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GGCGCAAATTTGGCGG16 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
CCGCCAAATTTGCGCC16 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
CGCAAATTTGCG12 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 105 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
AATTCTCATGTTTGACAGCTTATCATCGATAAGCTTTAATGCGGTAGTTTATCACAGTTA60 
AATTGCTAACGCAGTCAGGCACCGTGTATGAAATCTAACAATGCG105 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 105 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
TTAAGAGTACAAACTGTCGAATAGTAGCTATTCGAAATTACGCCATCAAATAGTGTCAAT60 
TTAACGATTGCGTCAGTCCGTGGCACATACTTTAGATTGTTACGC105 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GCGTTTAAACGC12 
__________________________________________________________________________