ISSN 1359-7345
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Number 44 | 2007
ChemComm
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ges 4549–4696
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Number 44 | 28 November 2007 | Pages 4549–4696
Chemical Communications
FEATURE ARTICLE
Brian M. Zeglis, Valerie C. Pierre and
Jacqueline K. Barton
Metallo-intercalators and metallo-
insertors
FEATURE ARTICLE
Jun Shan and Heikki Tenhu
Recent advances in polymer protected
gold nanoparticles
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Metallo-intercalators and metallo-insertors
Brian M. Zeglis, Valerie C. Pierre
{
and Jacqueline K. Barton
*
Received (in Cambridge, UK) 17th July 2007, Accepted 31st August 2007
First published as an Advance Article on the web 20th September 2007
DOI: 10.1039/b710949k
Since the elucidation of the structure of double helical DNA, the construction of small molecules
that recognize and react at specific DNA sites has been an area of considerable interest. In
particular, the study of transition metal complexes that bind DNA with specificity has been a
burgeoning field. This growth has been due in large part to the useful properties of metal
complexes, which possess a wide array of photophysical attributes and allow for the modular
assembly of an ensemble of recognition elements. Here we review recent experiments in our
laboratory aimed at the design and study of octahedral metal complexes that bind DNA non-
covalently and target reactions to specific sites. Emphasis is placed both on the variety of methods
employed to confer site-specificity and upon the many applications for these complexes.
Particular attention is given to the family of complexes recently designed that target single base
mismatches in duplex DNA through metallo-insertion.
Introduction
DNA is the library of the cell, simultaneously storing and
dispensing the information required for life. Molecules that
can bind and react with specific DNA sites provide a means to
access this cellular information. Over the past few decades,
small molecules that bind to DNA have shown significant
promise as diagnostic probes, reactive agents and therapeutics.
Much attention has focused on the design of organic, DNA-
binding agents.
1
However, over the past twenty five years,
increasing interest has focused on another class of non-
covalent DNA-binding agents: substitutionally inert, octahe-
dral transition metal complexes.
At first glance, transition metal complexes seem an odd
choice for DNA molecular recognition agents. Certainly,
Nature herself offers very little precedent in this regard. With
few exceptions, biological transition metals are confined to
coordination sites in proteins or cofactors, not in discrete, free-
standing coordination complexes.
2
Further, the cell generally
employs organic moieties for the binding and recognition of
DNA. Yet despite the lack of many natural examples,
transition metals complexes offer two singular advantages as
DNA-binding agents. First and foremost, coordination com-
plexes offer a uniquely modular system. The metal center acts
in essence as an anchor, holding in place a rigid, three-
dimensional scaffold of ligands that can, if desired, bear
recognition elements. DNA-binding and recognition proper-
ties can thus be varied relatively easily
via
the facile
interchange of ligands. Second, transition metal centers benefit
from rich photophysical and electrochemical properties, thus
extending their utility far beyond that of mere passive
Division of Chemistry and Chemical Engineering, California Institute of
Technology, Pasadena CA 91125, USA. E-mail: jkbarton@caltech.edu;
Fax: 626-577-4976; Tel: 626-395-6075
{
Present address: Department of Chemistry, University of Minnesota,
Minneapolis, MN, USA.
Brian Zeglis received his BS in
chemistry summa cum laude
from Yale University in 2004.
While at Yale, he studied
iridium N-heterocyclic carbene
complexes in the laboratory of
Robert H. Crabtree. Currently,
Brian is an NSF predoctoral
fellow in Dr. Barton’s labora-
tory working on the develop-
ment of mismatch-specific
metallo-insertors for diagnostic
and therapeutic applications.
Valerie C. Pierre received her
Engineer’s Diploma in
Chemistry and Chemical Engineering from the Ecole
Superieure de Chimie, Lyon, France in 2001. After a year of
internship at BASF-AG in Ludwigshafen am Rhein, Germany,
she pursued her studies at the
University of California,
Berkeley where she received
her PhD in 2005 for her work
with Kenneth N. Raymond on
the development of macromo-
lecular gadolinium complexes
as contrast agents for mag-
netic resonance imaging.
Valerie then moved to the
California Institute of
Technology, Pasadena, CA,
for postdoctoral studies with
J. K. Barton where she struc-
turally determined the binding
mode of metallo-insertors at
mismatched sites. She is currently starting her independent
career as an Assistant Professor at the University of Minnesota,
Twin-Cities.
Brian Zeglis
Valerie C. Pierre
FEATURE ARTICLE
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| ChemComm
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molecular recognition agents. Indeed, these characteristics
have allowed metal complexes to be used in a wide range of
capacities, from fluorescent markers to DNA foot-printing
agents to electrochemical probes.
3
With few exceptions, non-covalent, DNA-binding metal
complexes share a few important characteristics. All are
kinetically inert, a requisite trait due to the paramount
importance of stability. Indeed, most of the complexes are d
6
octahedral or d
8
square-planar. In addition, most exhibit a
rigid or mostly rigid three-dimensional structure, an important
facet considering that in many cases undue fluxionality could
negate recognition. Moreover, the stereochemistry of the
complex, if applicable, can provide specificity, an under-
standable notion given the chirality of the DNA target. Finally
most of the complexes that have been prepared are, by design,
photochemically or photophysically active, properties that
confer tremendous utility in probing or effecting chemistry.
In this review, we do not strive to carry out an exhaustive
survey of the field; instead, we seek to provide a discussion of
more limited scope, highlighting important contributions from
other researchers, yet concentrating principally on the work
from our own laboratory. The early history of non-covalent,
DNA-binding metal complexes is first addressed, followed by
a more comprehensive look at the last two decades of research.
In subsequent sections, complexes that bind DNA in each of
three different non-covalent modes are discussed: groove
binding, intercalation, and insertion (Fig. 1 and 2). Lastly,
recent work on the development of therapeutic and diagnostic
applications for some of these complexes is described. It
should be noted that some of the most well-known research
involving metal complexes and DNA has centered upon
covalent interactions, most remarkably the work on plati-
num-based chemotherapeutics. Given the considerable breadth
of this effort, it is understandably outside the scope of this
review. However, it has been extensively covered elsewhere.
4
Before embarking on our discussion of DNA binding and
recognition, a brief description of the structure of DNA may
be helpful. The most common form of DNA (and the form
addressed almost exclusively in these pages) is the anti-parallel,
right-handed double helix termed B-DNA, though the less
common right-handed A-form and left-handed Z-form
Dr Jacqueline K. Barton is the
Arthur and Marian Hanisch
Memorial Professor of
Chemistry at the California
Institute of Technology.
Barton was awarded the A.B.
summa cum laude at Barnard
College and a PhD in inor-
ganic chemistry at Columbia
University. After a postdoc-
toral fellowship at Bell
Laboratories and Yale
University, she became an
assistant professor at Hunter
College, City University of
New York. In 1983, she
returned to Columbia University, becoming a professor in
1986. In the fall of 1989, she joined the faculty at Caltech.
Professor Barton has pioneered the application of transition
metal complexes to probe recognition and reactions of double
helical DNA. Barton has also carried out seminal studies to
elucidate electron transfer chemistry mediated by the DNA
helix. Barton has received numerous awards, including a
MacArthur Foundation Fellowship and election to the National
Academy of Sciences. This year she is the recipient of the
American Chemical Society’s 2007 Pauling Medal.
Jacqueline K. Barton
Fig. 1
The three binding modes of metal complexes with DNA: (a) groove binding, (b) intercalation, and (c) insertion.
Fig. 2
Geometries of (a) groove binder, (b) metallo-intercalator, (c)
metallo-insertor.
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occasionally enter the discussion.
5
Within the polynucleotide
assembly, the heterocyclic bases – adenine (A), guanine (G),
cytosine (C), and thymine (T) – are bonded to the sugars in an
anti
orientation with a disposition perpendicular to the helical
axis. The base pairs collectively form a central, hydrogen-
bonded
p
-stack that runs parallel to the helical axis between
the two strands of the sugar-phosphate backbone. Each base
forms hydrogen bonds with its complement on the opposite,
anti-parallel strand, A with T and C with G. The rise per base
is 3.4 A
̊
, and there are ten base pairs per helical turn.
Surrounding the central base stack, the polyanionic sugar–
phosphate backbone forms two distinct grooves, a wide major
groove and a narrow minor groove. All of these structural
characteristics can and have been exploited for molecular
recognition.
Early work
The earliest research into the interactions between metals and
DNA focused almost exclusively on the binding strength and
location of metal-aquo ions, both those with and without
biological significance.
6
Perhaps as a result of these studies, the
potential utility of metal–DNA interactions was realized early
on. For example, melting temperature measurements for DNA
in the presence of each of the first row transition metal ions
were obtained to assess which metal ions stabilize or
destabilize the duplex.
7
The use of uranyl-bound nucleosides
was investigated as a possible tool for electron microscopy-
based DNA sequence determination.
8
Further, studies of the
binding of mercury to non-thiolated and thiolated guanosine
residues also portended the growing interest in metals as useful
DNA probes.
9
Importantly, these studies all focused upon the
coordination of metal ions to DNA and as such employed
either aquo ions or complexes with open coordination sites.
Our interest, however, is in the non-covalent binding of
coordinatively saturated metal complexes to DNA. With
respect to this area, clues suggesting the interaction of inert
metal complexes and DNA were evident as early as the 1950s,
most notably in F. P. Dwyer’s work on the biological activity
of metal polypyridyl complexes.
10
Simple tris(chelate) com-
plexes of Ru(
II
) and Ni(
II
) were found to have antiviral and
bacteriostatic activities. Quite remarkably, stereoselective
biological activity was observed in some cases.
It was not until the mid-1970s, however, that a progenitor
non-covalent DNA-binding complex was prepared by S. J.
Lippard and co-workers.
11
During their work on metal-
binding to thiolated bases, it was observed that the planar
complex [Pt(terpy)Cl]
+
(terpy = 2,2
9
:6
9
,2
0
-terpyridine) induced
a spectral shift for 4-thiouridine in the presence of tRNA.
Follow up work, this time using [Pt(terpy)(SCH
2
CH
2
OH)]
+
to
eliminate the labile coordination site, employed a variety of
techniques to establish the intercalative binding mode. X-Ray
fiber diffraction patterns provided further evidence for
intercalation, revealing a periodicity of one platinum unit
every 10
s
(every other base-pair) and a partial un-winding of
the phosphate backbone.
12
Subsequent work expanded the
family of intercalators to include other complexes with planar
heterocyclic ligands, [Pt(bpy)(en)]
2+
and [Pt(phen)(en)]
2+
,
established binding constants in the realm of 10
4
–10
5
M
2
1
for the family with DNA base pairs, and investigated the effects
of sequence context and ionic strength on intercalation.
13
Just as Lippard’s platinum complexes laid the groundwork
for future work on intercalative binding, the study of another
complex, [Cu(phen)
2
]
+
, in the lab of D. S. Sigman during the
late 1970s and early 1980s unearthed the rich chemistry of
groove-binding metal complexes.
14
The complex was serendi-
pitously discovered to degrade DNA during investigations into
the inhibition of
E. coli
DNA polymerase by 1,10-phenanthro-
line, and it was soon learned that the DNA cleavage reaction
was oxygen-dependent.
15
Product isolation and analysis led to
a proposed mechanism that suggested minor-groove binding
by [Cu(phen)
2
]
+
formed
in situ
, a hypothesis later confirmed
through elegant labeling experiments.
16
Additional reactivity
studies have revealed that the complex cleaves not only B-form
duplex DNA but also, though in some cases to a lesser extent,
A-form DNA, RNA, and folded nucleic acid structures.
17
Nature’s example
Before moving on to our main discussion of synthetic
complexes, it is important to address, at least briefly, nature’s
lone example of a non-covalent DNA-binding metal complex:
metallobleomycin. First isolated from
Streptomyces verticillus
in the late 1960s, bleomycins are a widely-studied family of
glycopeptide antibiotics that have been used successfully in the
treatment of some forms of cancer.
18
The structure of
bleomycins can be broken down into three domains: a metal-
binding domain containing a pyrimidine moiety and five
nitrogen atoms for octahedral metal coordination, a peptide
linker region bearing a disaccharide side-chain, and a
bithiazole unit with an appended, positively charged tail.
While the metal-binding region can coordinate a variety of
metals including Zn(
II
), Cu(
II
) and Co(
III
), the majority of
research has focused on understanding the reactivity of Fe-
bleomycin complexes.
19
Significantly, exposure of the Fe-
bleomycin complex to oxygen and a reductant leads to the
formation of activated bleomycin, a species that can, in turn,
affect both single-stranded and double-stranded DNA clea-
vage
via
4
9
-hydrogen atom abstraction by a high valent Fe-oxo
species.
Metallobleomycins bind DNA
via
the minor groove, though
neither affinity nor specificity is particularly high. Over the
past twenty years, extensive synthetic and spectroscopic studies
have helped to elucidate the contribution of each structural
moiety to DNA-binding and reactivity.
20
The bithiazole
subunit and positively-charged tail are considered to play the
most important roles in DNA-binding. The charge of the
cationic tail is generally agreed to provide electrostatic impetus
for binding. The role of the bithiazole, however, is subject to
some debate. While the bulk of the evidence suggests that this
moiety intercalates between base-pairs neighboring the binding
site of the complex,
21
others have suggested that the bithiazole
interacts with the DNA primarily in the minor groove.
22
Hydrogen-bonding of the pyrimidine moiety in the metal-
binding region is thought to help confer 5
9
-G-Py-3
9
cleavage
selectivity.
19
d
,20
b
The definitive roles of the linker region and
disaccharide have proven more subtle and elusive, with the
linker region likely of conformational importance and the
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4567
disaccharide having been given roles ranging from DNA
binding to metal chelation to cellular uptake and localization.
Finally, it is also both interesting and important to note that
metallobleomycins, unlike many of the metal complexes
discussed below, are exquisitely sensitive to structural changes,
for attempts to alter any of the domains have been met with
dramatically reduced cleavage efficiencies.
20
Tris(phenanthroline) complexes
The earliest work on the DNA-binding of octahedral metal
centers focused on tris(phenanthroline) complexes of ruthe-
nium, chromium, zinc, nickel and cobalt (Fig. 3).
23
Extensive
photophysical and NMR experiments suggested that these
complexes bind to DNA
via
two distinct modes: (a) hydro-
phobic interactions in the minor groove and (b) partial
intercalation of a phenanthroline ligand into the helix in the
major groove. Perhaps more important than the discovery of
these dual binding modes, however, was the revelation these
complexes provided regarding the importance of chirality in
DNA-binding.
24
In the case of [Ru(phen)
3
]
2+
, for example, the
D
-enantiomer is preferred in the intercalative binding mode,
while the complementary
L
-enantiomer is favored in the minor
groove binding mode. In subsequent years, it was discovered
that metal centers bearing more sterically demanding phenan-
throline ligand derivatives, such as diphenylphenanthroline
(DIP), display even more dramatic chiral discrimination.
Luminescence and hypochromism assays have revealed enan-
tioselective binding on the part of [Ru(DIP)
3
]
2+
; the
D
-enantiomer
binds
enantiospecifically
to
right-handed
B-DNA and the
L
-enantiomer binds only to left-handed
Z-DNA.
25
This enantiospecificity has been exploited to map
left-handed Z-DNA sites in supercoiled plasmids using
[
L
-Co(phen)
3
]
3+
.
26
Indeed, this trend in enantiomeric selectiv-
ity for octahedral tris(chelate) complexes, matching the
symmetry of the complex to that of the DNA helix, has
repeatedly and consistently been observed for non-covalent
DNA-binding complexes developed in the years since these
initial discoveries.
3,27
These earliest tris(phenanthroline) complexes do not, of
course, represent the only examples of complexes that bind
DNA
via
the minor or major grooves. For instance, the
extensively studied [Cu(phen)
2
]
+
, has been shown to bind
DNA
via
the minor groove. Indeed, these groove-binding
complexes not only bind DNA but also cleave the macro-
molecule in the presence of hydrogen peroxide.
28
Metal
complexes that bind in the groove have come a long way
since these first studies and are now quite sophisticated. Turro
and co-workers, for instance, developed an artificial photo-
nuclease by linking the metallo-groove-binder [Ru(bpy)
3
]
2+
to
an electron-acceptor chain containing two viologen units.
29
Interestingly, the chemistry of metallo-groove-binders also
extends to supramolecular self-assembly. Following the initial
work of Lehn on the interaction and cleavage of DNA with a
cuprous double-helicate,
30
Hannon and co-workers designed a
triple-helicate capable of recognizing three-way junctions in
DNA. This intricate recognition has recently been character-
ized by single crystal X-ray crystallography.
31
Metallo-intercalators
General architecture and binding mode
Intercalators are small organic molecules or metal complexes
that unwind DNA in order to
p
-stack between two base pairs
(Fig. 1). Metallo-intercalators, it then follows, are metal
complexes that bear at least one intercalating ligand (Fig. 2).
As their name suggests, these ligands, oriented parallel to the
base pairs and protruding away from the metal center, can
readily
p
-stack in the DNA duplex. Further, upon binding, the
ligands behave as a stable anchor for the metal complex with
respect to the double helix and direct the orientation of the
ancillary ligands with respect to the DNA duplex. Two well-
known examples of intercalating ligands are phi (9,10-
phenanthrenequinone
diimine)
and
dppz
(dipyrido[3,2-
a
:2
9
,3
9
-
c
]phenazine) (Fig. 4).
3
Ligand intercalation was first demonstrated by photophy-
sical studies.
23,32
However, it was not until extensive NMR
studies
33
and high resolution crystal structures had been
performed that the structural details of this binding mode were
properly illuminated (Fig. 5).
34
Metallo-intercalators enter the
double helix
via
the major groove, with the intercalating ligand
acting in effect as a new base pair. No bases are ejected from
the duplex. Further, intercalation results in a doubling of the
rise and a widening of the major groove at the binding site.
Importantly, this interaction distorts only minimally the
structure of DNA. In the case of B-DNA, for example, the
sugars and bases all maintain their original C
2
9
endo
and
anti
conformations, respectively. Indeed, only the opening of the
phosphate angles, not any base or sugar perturbations, is
necessary for intercalation.
Fig. 3
L
- and
D
-enantiomers of [Rh(phen)
3
]
3+
.
Fig. 4
Chemical structure of two common metallo-intercalators: (a)
D
-[Rh(phen)
2
(phi)]
3+
and
D
-[Ru(bpy)
2
(dppz)]
2+
. The intercalating
ligands are highlighted in blue, the ancillary ligands in yellow.
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