Chem. Rev.

2009, 109, 4921–4960

4921

Application of Metal Coordination Chemistry To Explore and Manipulate Cell Biology
Kathryn L. Haas and Katherine J. Franz*
Department of Chemistry, Duke University, 124 Science Drive, Durham, North Carolina 27708-0346 Received April 3, 2009

Contents
1. Introduction 2. Principles of Metal-Ligand Coordination Chemistry 2.1. Donor Atom Preference 2.2. Chelate Rings, Steric Strain, and Preorganization 2.3. Complex Geometry 3. Commonly used Chelators To Alter Bioavailability of Metal Ions in Cell Biology 3.1. Iron Chelators 3.2. Copper Chelators 3.3. Zinc Chelators 4. Selectivity by Reactivity or Localization 5. Complexes That Release Metals 6. Metal Complexes that Bind/Release Small Molecules 6.1. NO and CO 6.2. Metal-Based NO Sensors 6.3. Metal Complexes as NO-Releasing Compounds 6.3.1. NO Donors Sensitive to Visible and Near-IR Light 6.3.2. Trackable NO Donors 6.3.3. NO Donors Sensitive to Two-Photon Excitation 6.3.4. Other NO Donors 6.4. Metal Complexes as CO-Releasing Compounds 6.5. Metal Complexes That Bind Phospho Anions 6.6. Metal Complexes for Phosphoprotein Detection 6.6.1. Phosphoprotein Detection by Zinc Complexes 6.6.2. Phosphoprotein Detection by Calcium and Magnesium Complexes 6.6.3. Phosphoprotein Detection by Lanthanide Complexes 6.7. Metal Complexes That Release Bioactive Small Molecules 6.7.1. Photorelease of Bioactive Molecules 6.7.2. Reductive Activation and Ligand Exchange 6.7.3. Catalytic Release of Bioactive Molecules 7. Metal Complexes as Enzyme Inhibitors 7.1. Inhibitors with Metal-Binding Headgroups for Targeting Metalloproteins 7.2. Inhibitors That Recruit Endogenous Metals To Increase Potency 7.3. Inert Metal Complexes as Enzyme Inhibitors 4921 4924 4924 4924 4924 4925 4925 4927 4927 4927 4928 4929 4929 4930 4930 4931 4932 4932 4933 4933 4934 4936 4936 4938 4938 4938 4938 4939 4940 4940 4940 4941 4942

7.4. Metal Complexes as Catalytic Protein Inactivators 8. Metal Complexes for Probing DNA 8.1. Sequence-Specific DNA Probes 8.2. Metal Complexes That Recognize Mismatched DNA 8.3. Metal Complexes That Recognize G-Quadruplex DNA 8.4. Metal Complexes That Recognize Single-Double Strand Junctions 9. Metal-Responsive MRI Agents 9.1. MRI Agents for Calcium 9.2. MRI Agents for Zinc and Copper 10. Luminescent Metal Complexes for Cellular Imaging 10.1. Luminescent Transition Metal Complexes 10.2. Luminescent Lanthanide Complexes 11. Using Coordination Chemistry To Label Proteins 11.1. Polycysteine Tags 11.2. Polyhistidine Tags 11.3. Polyaspartate Tags 11.4. Covalent Protein Labeling Facilitated by Metal Chelation 11.5. Lanthanide Binding Tags 11.6. Unnatural Metal-Binding Amino Acids 12. Conclusions 13. Abbreviations 14. Acknowledgments 15. References

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1. Introduction
Our desire to understand how the individual molecules that make up cells organize, interact, and communicate to form living systems has led to the burgeoning field of chemical biology, an interfacial area of science that combines aspects of chemistry (the study of matter and its transformations) and biology (the study of living things and their interactions with the environment). The defining feature of chemical biology is the use of chemical approaches and small molecules to interrogate or manipulate biology.1,2 These small molecules are synthetic or naturally occurring ones that, for example, bind to DNA to affect protein expression levels, bind to proteins to inhibit their function, interact with lipids to alter membrane integrity, or become fluorescent in response to a metabolic event. Because small molecules can affect biochemical function, there is a clear link between chemical biology and pharmacology and medicine.3 While small molecules are usually implied as being organic compounds,4 inorganic small molecules also have a long

10.1021/cr900134a CCC: $71.50 © 2009 American Chemical Society Published on Web 08/28/2009

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Haas and Franz

Kathryn L. Haas was born in Harrisburg, PA, and studied chemistry at Gettysburg College. As an undergraduate, she conducted research on electronic tuning effects in titanium-catalyzed Diels-Alder transformations using disubstituted binapthol ligands. After receiving her B.S. in chemistry in 2004, she joined the lab of Prof. Katherine Franz in the Department of Chemistry at Duke University and received an NSF Integrative Graduate Education and Research Traineeship in the Center for Biologically Inspired Materials and Materials Systems. Her graduate work currently focuses on the manipulation of redox-active metals to solve biological problems and on elucidating mechanisms of copper transport via the human copper transporter, hCtr1.

Katherine J. Franz grew up in Williamsport, PA, and received her B.A. from Wellesley College in 1995. As an undergraduate, she conducted research with Prof. James Loehlin at Wellesley and with Dr. Richard H. Fish at the Lawrence Berkeley National Laboratory as a Department of Energy Research Assistant. She received her Ph.D. in 2000 from MIT, where part of her thesis work with Prof. Stephen J. Lippard focused on creating metal complexes as fluorescent sensors of nitric oxide. As an NIH postdoctoral fellow with Prof. Barbara Imperiali at MIT, Kathy developed lanthanide-binding peptides as natively expressible protein fusion tags for a variety of biochemical applications. She began her independent career in 2003 in the Department of Chemistry at Duke University, where her research group is working on developing new compounds to manipulate and probe the concentration, reactivity, and distribution of metal ions inside cells.

history in both biology and medicine. Ancient civilizations used gold and copper for healing purposes, and the modern era of drug discovery was ushered in when arsenic-containing salvarsan was discovered as an antisyphilis agent to become the world’s first blockbuster drug.5 Inorganic compounds should therefore not be overlooked in the realm of chemical biology, since their distinctive electronic, chemical, and photophysical properties render them particularly useful for a variety of applications.6-8 What are the properties of metal ions that impart utility to biology? Because inorganic elements comprise the bulk of the periodic table, the diversity of these properties is

likewise broad and has been thoroughly covered by several books in the field of bioinorganic chemistry.9-11 A brief summary of the general chemical properties of metals is given below. 1. Charge. Metal ions are positively charged in aqueous solution, but that charge can be manipulated depending on the coordination environment so that a metal complexed by ligands can be cationic, anionic, or neutral. 2. Interactions with ligands. Metal ions bind to ligands via interactions that are often strong and selective. The ligands impart their own functionality and can tune properties of the overall complex that are unique from those of the individual ligand or metal. The thermodynamic and kinetic properties of metal-ligand interactions influence ligand exchange reactions. 3. Structure and bonding. Metal-ligand complexes span a range of coordination geometries that give them unique shapes compared with organic molecules. The bond lengths, bond angles, and number of coordination sites can vary depending on the metal and its oxidation state. 4. Lewis acid character. Metal ions with high electron affinity can significantly polarize groups that are coordinated to them, facilitating hydrolysis reactions. 5. Partially filled d-shell. For the transition metals, the variable number of electrons in the d-shell orbitals (or f-shell for lanthanides) imparts interesting electronic and magnetic properties to transition metal complexes. 6. Redox activity. Coupled with the variability of electrons in the d-shell is the ability for many transition metals to undergo one-electron oxidation and reduction reactions. Biology has taken advantage of these chemical properties of metals to perform several functional roles, which are summarized in Table 1. This is by no means an exhaustive list but rather a primer to highlight important themes. Some metal ions, particularly the alkali and alkaline earth metals, are stable in aqueous solution as cations, making Na+, K+, and Ca2+ ideal for maintaining charge balance and electrical conductivity.10 On the other hand, the distinct architectures accessible via metal-ligand bonding interactions impart important structural roles to metal ions that encompass both macroscopic structural stabilization, as in biomineralized tissues,12 and molecular structural stabilization, as in proteins and nucleic acids that are stabilized in a preferred fold by metal ions.13-16 Metal-ligand bonding is also significant in its reversibility. For example, Nature takes advantage of reversible binding of metal ions like Ca2+ and Zn2+ to proteins or other storage repositories in order to propagate various biochemical signals.13,17 Metal ions themselves can be their own signal to adjust DNA transcription, as in the case of metalloregulatory proteins.18,19 Reversible metal-ligand coordination is also exploited to bind and release molecules to and from a metal center, a prime example being O2 binding and release from hemoglobin. The reactivity of metallic centers in biology rests mostly in their Lewis acid or redox-active characters. Metal centers that are strong Lewis acids can activate coordinated ligands for reactivity, so for example, a water molecule coordinated to a Zn(II) center becomes a potent nucleophile for amide bond hydrolysis of a protein substrate.20 In terms of redox activity, a wide variety of transition metals that can access variable oxidation states are found incorporated as enzyme

Application of Metal Coordination Chemistry

Chemical Reviews, 2009, Vol. 109, No. 10 4923

Table 1. Functional Roles of Inorganic Elements Found in Biology with Selected Representative Examples function charge balance structure (macroscopic) structure (protein structure) structure (nucleic acid structure) signaling signaling to DNA acid-base catalysis atom or group transfer electron transfer redox catalysis inorganic element Na, K, Ca Ca, Si Zn, Ca Mg, Mn Ca, NO, Zn Hg, Cu, Zn, Pb, As, Sb, Cd, Ni, Fe, Mn, Co, Ag Zn, Fe, Ni, Mn, Mg V, Fe, Co, Ni, Cu, Mo, W Fe, Cu, Mo V, Mn, Fe, Co, Ni, Cu, W representative examples K+ channels responsible for electrical conduction in nervous systems biominerals in bone, teeth, and shell Zn finger proteins, extracellular Ca proteins Mg2+ stabilization of tRNA and the hammerhead ribozyme release of Ca2+, Zn2+, and NO instigates diverse biochemical signaling pathways metal-responsive transcriptional regulators (metalloregulatory proteins) hydrolysis reactions carried out by carboxypeptidase, purple acid phosphatase, urease, arginase, etc. dioxygen transport (hemoglobin), alkyl group transfer (cobalamin) iron-sulfur proteins, cytochromes, blue copper proteins enzymes involved in oxygen metabolism, nitrogen fixation, radical formation ref 10 12 13, 16 14, 15 13, 17, 21 18, 19 20 11 22 23

Table 2. Functional Roles of Inorganic Elements Applied to Biology with selected Representative Examples and Reference to the Relevant Section of This Review function structure signaling acid-base catalysis visualization: luminescence magnetic resonance radioactive X-ray alteration of metal bioavailability bonding ligand exchange electron transfer redox inorganic element Ru, Rh, Pt Ca, NO Co, Zr, Pt, Ln Tb, Eu, other Ln Gd, Mn 99m Tc, 64Cu, others Os, Ln Fe, Cu, Zn, Ca, other Zn, Ni, others Gd, Co, others Rh, Ru Fe, Cu, others representative examples kinetically inert complexes serve as structural scaffolds that interact in novel ways with biomolecules compounds that release Ca, NO, or other signaling molecules complexes that induce hydrolytic cleavage of biomolecules subcellular imaging agents metal-responsive MRI imaging SPECT, PET imaging heavy atom derivatives for biomolecular X-ray structure determination chelating agents that alter normal metal homeostasis metal-ligand bond formation for analyte sensing, metalloprotein inhibition, or protein labeling change in coordination sphere to increase MRI signal or release bioactive molecule photoinduced electron transfer to produce potent photooxidants oxidative degradation of proteins, DNA; chelation to manipulate redox activity 7.3, 8 5, 6 7.4 10 9 (see refs 30, 31) 11.5 3, 4, 5 6.5, 6.6, 7.1, 11 6.7, 9 8 3.1, 3.2, 4, 7.4, 8.4, 11.6 sections

cofactors to carry out oxidation/reduction chemistry. Electron transfer units like cytochromes, iron-sulfur clusters, and blue copper proteins shuttle electrons to other proteins that require redox chemistry for their function, while other redox proteins catalyze multielectron oxidation/reduction reactions directly on a substrate. Examples here involve oxygen metabolism, including the reduction of dioxygen to water by cytochrome c oxidase and hydrocarbon oxidation catalyzed by cytochrome P-450 enzymes, to name just a few. When it comes to applying inorganic compounds to biology, chemists are not restricted to the naturally bioavailable set of metals and can take advantage of the properties of biologically exotic elements, including second and third row transition elements and the lanthanide (Ln) elements. This expansion leads to the list of functional roles of inorganic elements applied to biology shown in Table 2. Many of the functions listed in Table 2 mirror those of Table 1, but applied in novel ways. For example, the structures of kinetically inert metal complexes are found to interact with proteins and nucleic acids in unique ways, and the acid-base and redox activity of native and non-native metals can be harnessed for artificial reactivity. Metal complexes can also impart additional functionality not found naturally. The most

striking addition to the list is in visualization, where the photophysical, magnetic, and radioactive properties of metals make possible studies based on luminescence, magnetic resonance, PET, and SPECT imaging modalities. This review will explore how the properties of inorganic coordination complexes are applied in the context of inorganic chemical biology, with a particular focus on applications related to cellular trafficking and regulation. We will delve into the structure, bonding, spectroscopy, and reactivity of transition metal coordination compounds and explore how their unique properties can be used as probes and tools to understand or control biological processes. Our discussion will expand on the functions and examples listed in Table 2, which is really only a partial list, because the examples of inorganic and organometallic complexes applied to biology continue to grow. Our focus is on compounds used in cells to understand the trafficking or regulation of something, be it the metal itself or some other molecule or process that is enabled or visualized by a metal complex. Many of the compounds that are discussed have potential applications in medicine, but the reader is referred to other excellent sources for implicit coverage of medicinal and diagnostic uses of metal complexes.24-33 There have been

4924 Chemical Reviews, 2009, Vol. 109, No. 10
Table 3. Classification of Select Metal Ions and Donor Atoms According to Pearson’s HSAB Principle hard Lewis acids H+, Li+, Na+, K+, Be2+, Mg2+, Ca2+, Sr2+, Sc3+, Ti4+, Zr4+, Cr3+, Al3+, Ga3+, La3+, Gd3+, Co3+, Fe3+ hard Lewis bases F-, OH-, H2O, ROH, Cl-, RO-, R2O, CH3CO2-, NH3, RNH2, NH2NH2, CO32-, NO3-, O2-, SO42-, PO43-, ClO4borderline acids Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Pb2+, Bi3+, Rh3+, Ir3+ borderline bases NO2-, Br-, N3-, N2, C6H5NH2, pyridine, imidazole soft acids

Haas and Franz

Cu+, Au+, Ag+, Tl+, Hg+, Pd2+, Cd2+, Pt2+, Hg2+ soft bases RSH, RS-, R2S, S2-, CN-, RNC, CO, I-, R3As, R3P, C6H5, C2H4, H2S, HS-, H-, R-

significant advances in the development of fluorescent probes for monitoring cellular metals. Such molecules are clearly important tools in inorganic chemical biology but will not be covered here, because there are several excellent reviews available.34-39 We define a metal complex that is a “probe for biological systems” as one that can be used in ways to teach us about the chemical biology of living cells. It may be used in vivo or in vitro with the aim of understanding how cells operate. With this definition in mind, we will discuss metal chelators and metal complexes that are being used or have potential to be used to this end, with an emphasis on those that are applied in cellular studies.

an adjustment might actually decrease the effective metal binding at a desired pH. Because of proton competition, overall stability constants (β) do not reflect the actual affinity of a ligand for a metal under biologically relevant solution conditions. A pH-dependent conditional binding constant can be calculated from known β and pKa values.47-49 Alternatively, an apparent binding constant (Kapp, also called Keff for effective binding constant) can be measured directly as the equilibrium constant under the specified solution conditions of pH and buffer components. For convenience, binding constants are often inverted and discussed as dissociation constants (Kd).

2. Principles of Metal-Ligand Coordination Chemistry
The principles governing metal-ligand complex stability and specificity depend on the properties of both the metal ion and the chelating agent, as summarized briefly in the following sections. More comprehensive reviews on ligand design for selective complexation of metal ions in aqueous solution are available.40-45 This discussion sets the stage for understanding the properties of the compounds presented throughout this review.

2.2. Chelate Rings, Steric Strain, and Preorganization
Polydentate ligands that present multiple donor atoms for metal binding provide greater complex stability compared with monodentate analogs due to the chelate effect. This effect can be maximized if the number and size of the chelate rings are optimized for the size of the cation in a way that minimizes steric strain upon metal binding.41,42 The chelate rings formed when two donor groups from the same ligand bind a metal center are most favorable for five- and sixmembered rings. Adjacent six-membered rings formed from polydentate ligands, however, can induce unfavorable steric strain that is relieved in ligands containing adjacent five- and six-membered rings.41,42 In general, ligands that minimize steric strain in the complex on coordination of the ligand to the metal ion or that preorganize their donor atoms spatially as required for complexation or both are preferred for highaffinity binding.43 Macrocycles that incur minimal strain upon metal complexation can therefore retain their metal ion by tight complexation.

2.1. Donor Atom Preference
The principle of hard and soft acids and bases (HSAB) was developed in 1965 by R.G. Pearson following criteria introduced by Irving, Williams, Arhland, Chatt, and Davies.46 The classification is based on an atom’s polarizability, where nonpolarizable acids or bases are small with high charge density and are classified as “hard”. Polarizable acids and bases are usually large with low charge density and are classified as “soft”. Acids and bases that have intermediate hard/soft character are classified as “borderline”. The HSAB principle predicts that hard acids prefer hard bases, soft acids prefer soft bases, and borderline acids prefer borderline bases. Pearson’s classifications of metal ions (Lewis acids) and their ligands (Lewis bases) are shown in Table 3, which serves as a useful starting point for predicting the preference of metal ions for ligands with various donor groups. For example, soft donor groups such as thioethers (R2S) and thiolates (RS-) prefer soft metal ions, like Cu+, whereas hard oxygen donors like carboxylates and phenolates are appropriate for hard metal ions, like Fe3+.46 In fitting with the Lewis acid-Lewis base description of metal-ligand coordination, it would seem apparent that increasing the Lewis basicity of the donor would enhance metal-ligand bonding. While this principle can be used to tune metal-ligand affinity, other factors must also be considered. For example, increasing the basicity of a phenolate also increases its pKa. Since metal ions compete with protons for ligand binding in aqueous solution, such

2.3. Complex Geometry
On the basis of its number of valence d electrons, a metal ion may prefer certain binding geometries over others. This preference is based on ligand field stabilization energy (LFSE), a full description of which can be found in standard inorganic chemistry text books.50,51 Comparing the geometric preferences of iron, copper, and zinc illustrates the point. The common biologically relevant oxidation states of iron are Fe2+ and Fe3+, which prefer octahedral and distorted octahedral geometries. Copper, on the other hand, exists primarily as Cu+ and Cu2+, with Cu2+ favoring square planar, square pyramidal, or axially distorted octahedral geometries due to Jahn-Teller distortions of its d9 electron configuration. Its reduced Cu+ form has a filled d10 configuration with no preference for geometry based on LFSE and can therefore be found in a range of coordination geometries including two-, three-, and four-coordinate sites. The geometry of the ligand field can influence the redox state of copper. For

Commonly used Chelators To Alter Bioavailability of Metal Ions in Cell Biology Altering the bioavailability of metal ions by using metal chelating agents is an important strategy for studying cellular processes related to metal ion transport. LFSE is also a factor in the trend observed in the Irving-Williams series of relative complex stabilities of firstrow divalent metal ions: Mn2+ < Fe2+ < Co2+ < Ni2+ < Cu2+ > Zn2+. that a high affinity for a particular metal ion does not imply that other metal ions do not also bind with considerable affinity. cell-permeable chelator that is quenched upon iron binding. There is a very large body of literature on the design of iron chelators for medical applications (see. however.71 . The structures are shown in Chart 1. Most Fe3+ chelators also have reasonable affinity for Cu2+ and Zn2+. DFO is one of the first chelators used to treat iron overload disorders. DFO is also used in the laboratory for cell studies to control iron bioavailability. The extent of restored fluorescence gives an estimate of the chelator-accessible. Vol. especially transcription. The ligand-induced change in reduction potential can be used to purposefully select a desired oxidation state.56 The iron siderophore literature is also helpful for understanding selective iron sequestration. however. 3.57 It is important to repeat. A common iron(III) chelator used both in the laboratory and in the clinic is desferrioxamine (DFO. Geometric preferences imposed by the ligand are also important for differentiating Cu2+ and Zn2+. it has a short half-life in cell culture media and plasma due to hydrolysis of the hydrazone linkage. and it is still widely used today. and trafficking.52-54 The catalytic capacity of iron in Fenton reactions requires it to cycle between Fe2+ and Fe3+. a naturally occurring siderophore that binds Fe3+ with three hydroxamate groups to give a six-coordinate complex. it also complexes Cu2+ and Zn2+. Chelating agents for both oxidation states are discussed below.55 Chelating agents that shift the reduction potential to favor either Fe3+ Iron(III) is unique to biological transition metal ions in that it has a high charge-to-radius ratio and as a hard acid forms high-affinity complexes with hard oxygen ligands. In order to affect the availability of only one metal ion at a time. The ability to limit the bioavailability of only one target metal is crucial to the integrity of the experiment and interpretation of results. use.62-65 It forms a 2:1 ligand/Fe(III) complex that inhibits iron-dependent ROS formation. another application of SIH has been in the determination of labile iron pools in cells by fluorescent chelators.58 DFO is a potent chelator for Fe3+ that inhibits iron-dependent ROS formation and is itself an antioxidant. For a comprehensive discussion on designing ligands for iron chelation.59. or labile. 2) and DTPA (diethylenetriamine pentaacetic acid. Rarely. The greater stability of copper(II) complexes compared with zinc(II) complexes revealed by this series indicates an inherent challenge in designing chelating agents that are selective for Zn2+ over Cu2+. iron pool. although sites that impose tetrahedral geometry will prefer Zn2+ to Cu2+.61). which means that the reduction potential of the iron center is a key determinant in its ROS production. and Zn2+ shows a diminished stability due to a lack of LFSE for its d10 configuration. Fe2+ + H2O2 f Fe3+ + OH. Up-regulation or downregulation of specific genes can indicate the proteins either directly or indirectly related to metal ion handling.Application of Metal Coordination Chemistry Chemical Reviews. their geometric preference. the complex equilibria that exist in the compartmentalized environment of a cell (even more so in a whole organism) make metal selection a tricky proposition.60 The difficulty of DFO in crossing biological membranes and its subsequent slow rate of intracellular iron chelation has spurred the search for other Fe3+ chelating agents. “selective” metal chelators are often employed. 4) has emerged as a useful lipophilic chelator that readily crosses biological membranes. 10 4925 example. does selectivity imply exclusivity. which can lead to depletion of these metals. complex stability increases as the ionic radius decreases across the series. The following sections highlight some of the most widely used chelating agents in cell biology and point out potential pitfalls.1. especially with prolonged exposure to the chelating agent. In this context. 109. General chelators like EDTA (ethylenediamine tetraacetic acid. however. The general protocol for these measurements has been to use a fluorescently labeled. a typical approach is to change the availability of one metal and observe changes in cell processes.67 In addition to protecting cells against iron-induced oxidative stress.68. see the review by Liu and Hider. and other properties to make it difficult to select for one specific metal in the presence of others. Iron Chelators The well-known redox activity of iron makes it a useful metal cofactor for biology but also poses a potential risk because it can participate in redox cycles with oxygen to produce damaging reactive oxygen species (ROS) through the Fenton reaction (eq 1). ligands that impose a tetrahedral arrangement that is unfavorable for Cu2+ but reasonable for Cu+ will destabilize the Cu2+ form. the tridentate chelator salicylaldehyde isonicotinoyl hydrazone (SIH. storage. the review by Orvig in this issue32 as well as others56. it does not mean that it binds that metal to the exclusion of all others. 3) have high affinity for Fe3+ but also bind several metal ions and are not considered selective. No. shifting the reduction potential more positive in favor of Cu+. So while an agent may have a thermodynamic preference for a particular metal ion. or Fe2+ without redox cycling can in principle keep iron from producing ROS.70 In addition. 1). When using live cells in probing for biomolecules related to metal homeostasis. Many metals are close enough in their HSAB character.+ OH• (1) 3. Ligands can therefore be designed to have a thermodynamic preference for Fe3+ over dicationic metal ions like Cu2+ and Zn2+. In general. where the selectivity is a measure of its affinity for a particular metal ion over others. then use SIH to complex iron and compete it away from the probe to restore fluorescence. so the focus here is rather to highlight select examples with utility for manipulating iron concentrations in cell culture. tetrahedral Zn complexes are common. but Cu2+ shows a sharp spike in stability that can be attributed to LFSE obtained through Jahn-Teller distortion. Indeed. for example. It is charged and hydrophilic and does not diffuse easily across the cell membrane.58 It is believed to enter the cell only by endocytosis to diminish labile iron pools in the cytoplasm and most drastically inside of endosomes.66. While its affinity for Fe3+ is high.58 It is slow to chelate intracellular iron at room temperature but increases efficiency at 37 °C due to increased endocytosis. Because Zn2+ is d10 and has no geometric preference based on LFSE. 2009.69 SIH is not without its drawbacks.

ferrozine is known to cause nonspecific reduction of Fe(III) chelates.4926 Chemical Reviews. Ferrozine (7) is another commonly used chelator for Fe2+ in cell culture to create a “defined Fe medium” or “Fedepleted medium”. Because of its lower charge density and larger size. which is not surprising considering the variable chelate stoichiometry possible with BPS and related phenanthroline derivatives. especially Cu2+ and Zn2+.75 There is evidence that ferrozine forms ternary complexes with Fe and amino acids. amino acids or other components in the media likely . One chelator that fits these criteria is bathophenanthroline disulfonic acid (BPS. which act as potential antioxidants. Vol. No. The oxidation states Fe2+ and Fe3+ differ in terms of their ligand preferences and the ease with which they can be selectively chelated over other metals based on donor atom identity. 109.79 In addition to using chelating agents to limit iron bioavailability. Unchelated Fe2+ ion is more soluble and more bioavailable than unchelated Fe3+.72. so it must be kept in mind that Fe2+ salts added to the culture medium or aqueous buffers are likely to oxidize readily to Fe3+. Fe2+ is more similar in HSAB character to other biologically relevant transition metal ions. 5). which may not be soluble.48 BPS can easily form a bis complex with square planar geometry preferred by Cu2+. it does complex other metals. Fe2+ has a preferred ligand field geometry that is different from Cu2+ and Zn2+. a sulfonated watersoluble derivative of phenanthroline (6) that is frequently used in cell culture as an iron-limiting reagent. Chelators with borderline donor groups that can also adopt an octahedral geometry can potentially select Fe2+ over other biometals. Like BPS.74 Because of its large positive reduction potential. An understanding of iron coordination chemistry is also helpful to avoid potential problems. both because Fe2+ is a substrate for divalent metal ion transporters in mammalian cells and because Fe3+ rapidly hydrolyzes in water to give insoluble iron hydroxides. Commonly Used Chelators for Cell Biology Haas and Franz Chelators for Fe2+ are also used to control iron bioavailability. especially histidine.76-78 It selects for Fe2+ by forming a tris complex of borderline donor atoms in an octahedral binding geometry with a very large binding constant. 2009. In cell culture media. Although it may be difficult to select Fe2+ over other borderline metals. Fe2+ is a d6 metal that has a favorable LFSE in a low-spin octahedral complex. Typical cell culture conditions exist in an oxygen-rich environment. especially Cu2+. BPS is known to cause nonspecific reduction of Fe(III) chelates. 10 Chart 1.73 BPS forms a tris-chelate iron(II) complex with octahedral geometry and is membrane impermeable due to its charged sulfate groups. irons salts are also used to supplement cell culture media.75 Although it has good affinity for Fe2+.

the affinity of TPEN for Fe2+ (log Ka 14.5 V compared with uncomplexed Cu2+. However. As described in section 3. TPEN (N.91. In fact. while its affinity for Cu2+ (log Ka 20. especially phosphate buffers. a metal-binding ligand is masked with a H2O2-sensitive protecting group to prevent metal coordination under normal conditions. Copper Chelators Like iron.85. Instead. forcing it into a tetrahedral geometry and inducing its reduction to Cu+. In our prochelator strategy. No.105 TPEN has six possible donor groups.74 Because BCS is charged and not membrane permeable.105 It is necessary to consider the Cu. This tetrahedral geometry combined with the large bite size of the five-membered chelate ring effectively binds Cu+ over other metals. which highlights the fact that these “Cu(I)-selective” chelators are not innocent chelators of Cu+.102. but in the reducing conditions inside the cell.Application of Metal Coordination Chemistry Chemical Reviews.2. the actual nature of its Cu complex has long been under debate. Neocuproine (8).94 4. bathocuproine (BC. There is strong evidence that cuprizone stabilizes a Cu3+ oxidation state in a square planar d8 complex. Soft character makes Cu+ unique among the biological metal ions. 10 4927 keep iron in solution. will result in insoluble and bio-unavailable iron.4. This means Cu+ can adopt tetrahedral.106 BSIH itself has only weak interactions with metal ions.N′-tetrakis-(2-pyridylmethyl)ethylenediamine.106-108 The concept is based on the hypothesis that oxidative stress is exacerbated by Fenton reactions wherein labile metal ions. is chelated by only five of the six possible donor groups and forms a distorted square pyramid with high stability. so a sulfonated version (BCS) was developed to give a more soluble derivative. Reaction with H2O2 converts the prochelator to the chelator. it should be considered that BCS is a potential promoter of Cu2+ reduction.N. Examples are shown in Chart 2. Zn2+ is very close to Cu2+ in size and charge density and can be difficult to select for over Cu2+. Zn2+ is also a smaller metal ion.3. Vol.90 In fact BCS has been shown to inhibit Cudependent redox cycles. BSIH (13. when binding to a metal in a bis complex.107 Significantly.87-89 Although they may be able to promote reduction of Cu2+ to Cu+.9-Dimethyl-substituted phenanthroline ligands are wellknown to select for Cu+. 109. once the reduced state is reached.0). Neocuproine (8) is more hydrophobic and is used for intracellular and extracellular Cu chelation since it can diffuse over the cell membrane.6) is higher than that for Zn2+ (log Ka 18. Because Cu2+ is the dominant extracellular form of copper. 2. Typically under the oxidizing extracellular environment.82. Our own lab has introduced a prochelator strategy that uses the reactivity of oxidative stress to generate metal-binding agents to inhibit further oxidative damage.N′. Fe. the redox chemistry of Cu2+/+ makes it essential to biological processes but also potentially dangerous if it is not handled properly by the cell and becomes available for Fenton-like chemistry to produce ROS. cells that were not stressed with H2O2 remained viable even after repetitive.91-93 The cytotoxic and neurotoxic effects of cuprizone may be related to Cu2+-Cu3+ redox cycling induced by this chelator under biological conditions. prolonged . the metal is forced into a tetrahedral binding geometry with the two chelate ligands nearly perpendicular to each other. Zn2+ is also a d10 ion but is harder in character than Cu+. and Zn binding properties of TPEN before interpreting the results of an experiment where TPEN is used as a “selective” chelator. particularly iron and copper. The BSIH to SIH conversion also inhibits copper-catalyzed Fenton reactions.9-dimethyl-substituted phenanthroline chelates used in cell culture. 12) is the most common zinc chelator used in the literature. react with H2O2 to produce damaging hydroxyl radicals (see eq 1).109 demonstrating that the strategy is not metal specific per se. it is stabilized and does not participate in redox cycling. and for Zn and Fe. and it is d10 with no real preference for ligand field geometry.6) is relatively significant. It has borderline HSAB character. so it has potential to be selected based on ligand donor groups. healthy metal ion distribution. Cu+ is a d10 ion. or even linear geometries that are disfavored by other metals. trigonal. 10) are the three commonly used 2.108 BSIH was shown to protect cultured retinal pigment epithelial cells against cell death induced by hydrogen peroxide. In addition. but it is difficult to design ligands that can select for the metal ion causing the damage without altering normal. Selectivity by Reactivity or Localization The challenges associated with selective metal chelation have led to the development of new generation chelating agents that achieve selectivity based on reactivity or localization.95-101 however there is an apparent misconception that TPEN is Zn2+-specific. A boronic ester was selected as the H2O2-sensitive masking group in our first-generation prochelator. 11) is another chelator used to selectively bind Cu2+ in cell studies. 3. Zinc Chelators The only relevant oxidation state for zinc is Zn2+. these ligands disfavor octahedral tris-chelate or square-planar bis-chelate coordination modes because of steric interference of the methyl substituents. 3. appropriately designed chelating agents can prevent this reactivity.103 There are several studies that show TPEN affects cellular concentrations of Zn and Cu and that replenishing either metal into TPEN-treated cells will rescue the cells from TPEN-induced apoptosis. but addition of simple iron salts to standard laboratory buffers.86 Bathocuproine (9) is not very water-soluble. but rather reactivity specific. giving it flexibility in geometric arrangements.1. and Fe2+ is its potential variability in chelate binding geometry.80-86 Unlike phenanthroline. BC-bound Cu(II) is a stronger oxidizing agent by 0. all six are used to chelate the ion in an octahedral geometry.5-tetramethyl-[1. so can be minimized as an interfering species based on ligand donor choice. 2009. Cuprizone (oxalic acid bis(cyclohexylidene)hydrazide. Cu2+. isonicotinic acid [2-(4.100 Another study shows that levels of Fe are affected in addition to Cu and Zn when TPEN is added to cell culture. whereas SIH strongly interacts with most divalent and trivalent metals. thereby triggering metal sequestration.5. which is destabilized by octahedral geometry. copper exists as Cu2+.2]dioxaborolan-2-yl)-benzylidene]-hydrazide). so chelate ring size can be a determining factor.83 however. which converts to SIH (4) in the presence of H2O2 and forms the [Fe(SIH)2]+ complex that prevents iron from Fenton reactivity. Cu2+. it likely exists in the reduced Cu+ oxidation state. 9). it is commonly used in cell studies as an extracellular Cu-limiting agent.3. and bathocuproine disulfonate (BCS. these ligands do have significant interaction with Cu2+ and are known to bind to Cu2+.104 Part of the reason that TPEN can have such significant affinity for Zn2+.

developed by both Tsien and Ellis-Davies. Chelators That Are Selective Based on Reactivity or Localization Haas and Franz exposure to BSIH. Similar treatment of cells with chelating agents SIH and DFO caused significant cell death.110 The study used an iron regulatory protein bandshift assay and a calcein fluorescence assay to show that intracellular labile iron concentrations increase after exposure to UVA. it was shown that a broad-spectrum glucosidase from Agrobacterium faecalis indeed converts prochelators like 15 into their efficient metal-binding hyrdoxypyridinone versions. Another prochelator approach involves masking SIH or PIH (pyridoxal isonicotinoyl hydrazone) with a photoactive nitrobenzyl protecting group to create photocaged iron chelators that release the active ligand only after exposure to UVA light. The most well-developed compounds in this category belong to a class of molecules known as caged calcium. and Mg2+. 109. Vol. Once inside the brain.110 The caged chelators represent another promising tactic of getting the protective benefits of chelating agents without the risks of deleterious side effects associated with prolonged exposure of cells to high-affinity metal chelators.112 Other strategies of targeting chelating agents for specific diseases are covered in the review by Scott and Orvig in this issue. respectively. 2009.4928 Chemical Reviews. The chelating moieties are based on tetracarboxylate chelators BAPTA and EGTA that bind to Ca2+ with high affinity and sufficient selectivity over the most likely competing ions Na+.112 A radiolabeled hydroxypyridinone glucoconjugate was also shown to penetrate the blood-brain barrier in a rat brain perfusion experiment. thereby providing a sudden release of bioavailable Ca2+.110 Exposure of 2NPE-PIH (14) or 2NPE-SIH to UV light cleaves the protecting group to release PIH and SIH. The high concentration of glucose transporters in the brain may provide a pathway to concentrate metal ion chelators across the blood-brain barrier. Gppp) also uses the carbohydrate to mask the metal-binding functionality of the pyridinone. Orvig and co-workers have introduced an alternative prochelator strategy that is based on the premise that glucose transporters can be co-opted to import glucose-tagged molecules into cells.32 5. Skin fibroblast cells treated with 14 were significantly protected against UVA-induced necrotic cell death at UVA irradiation doses that mimic natural exposure levels of a sunny day. the carbohydrate moiety of the glucoconjugate 15 (3-(-D- glucopyranosyloxy)-2-methyl-1-phenyl-4(1H)-pyridinone. presumably due to metal depletion. the glucose masking group would be enzymatically cleaved to release the chelator. whereas the labile iron levels are kept in check if the cells are first treated with the caged chelators prior to irradiation.111 In addition to potentially targeting the molecule to the brain.113-115 These reagents operate by using light to trigger a chemical change that reduces a chelator’s affinity for Ca2+. it is also desirable to manipulate cellular metal ions by increasing their local concentration in a controlled way. demonstrating adequate cerebral uptake. As a proof-of-principle. No. 10 Chart 2. Complexes That Release Metals While the previous sections focused on using chelating agents to reduce the concentration of bioavailable metals.107 These initial findings suggest that BSIH discriminates toxic iron from healthy iron. K+. Constructs . thereby preventing premature metal binding.

There are two general series of caged calcium. therefore. including blood pressure regulation. and in many cases their biological targets are metal centers. thereby resulting in a 40-fold decrease in Ca2+ affinity.121 In both cases. although it was suggested that the affinity may need to be improved to avoid competition with endogenous copper-binding proteins.120 The knowledge of the biological roles of CO is about 15 years behind that of NO.119 6. It is possible. with low concentrations being critical for signaling events and high concentrations being toxic. typically heme iron. Their biosynthesis is metal-dependent. NO and CO The biological effects of carbon monoxide (CO) and nitric oxide (NO) can aptly be described by the general observation of Paracelsus in the early 16th century that “the dose makes the poison”. and cell death. but it appears to play a role in some of the same pathways as NO in addition to other important processes. Metal Complexes that Bind/Release Small Molecules 6. our group recently introduced a photocleavable pyridal amide ligand that binds to Cu2+ with an apparent Kd at pH 7. immune response. An in vitro assay showed that copper-catalyzed hydroxyl radical formation increased 160% following light-induced uncaging. No.117. 109.115 In an effort to broaden the applicability of caged metals beyond calcium. NO is now recognized as an important signaling molecule that impacts a wide range of physiological responses.b. as shown in Chart 3a. Photoactive Caged Complexes That Release Metals Chemical Reviews. These reactions inform us that transition metal complexes in biological environments can liberate and sequester NO and CO by breaking or forming metal-nitrosyl (M-NO) and metal-carbonyl (M-CO) bonds.119 Photolysis of caged copper with 360 nm UV light cleaves the tetradentate ligand to give photoproducts that have only bidentate chelating ability and therefore a reduced affinity for copper. In the nitr series (nitr-7.Application of Metal Coordination Chemistry Chart 3. the concentration dictates the biological effect. as well as act as therapeutic agents.116 In the series of compounds including NPEGTA (17). 16). Vol.1. neurotransmission. . each using a different mechanism to reduce Ca2+ affinity.118 There have been hundreds of studies that utilize caged calcium to explore biology. colorless gases that were long known only as toxic poisons until the discovery in the 1980s of NO as the endothelium-derived relaxing factor. photoactivation converts the 2-nitrophenyl substituent to a more electronwithdrawing group that reduces the donor strength of a metalbinding nitrogen. 10 4929 based on EDTA are also available but do not discriminate Ca2+ from Mg2+ as effectively. thereby demonstrating that light can be used to trigger the availability and reactivity of copper. These were recently reviewed by Ellis-Davies and will not be repeated here. depending on the derivative.4 of 16 pM ([Cu(OH2)(cage)]. Both diatomic molecules are odorless. Caged copper has yet to be validated in a cellular system. to develop artificial small molecule coordination complexes that can detect or manipulate NO and CO in order to study their biological roles. a photoactive 2-nitrophenyl group is embedded into the backbone of the chelator so that illumination with UV light results in chelator fragmentation and a 40 000-600 000-fold loss in Ca2+ affinity. 2009. 18). These gas molecules also have in common rich transition metal coordination chemistry that begets their biological impact.

The recent development of a fluorescein-based copper complex CuFL (Chart 4.135 The fluorescence enhancement observed following reaction with NO was determined to be a similar reductive nitrosylation process as described above.0 to give a nonfluorescent complex that reacts rapidly and selectively with NO to give an 11-fold increase in fluorescence intensity.122. indicating that it is the species responsible for the observed fluorescence signal. MNIP-Cu was used to show NO production in lipopolysaccharide-activated macrophages and in a model of acute liver injury in mice.134 CuFL has subsequently been used as a key tool to decipher the role of a bacterial NO synthase in the pathogenicity of Bacillus anthracis.123 These compounds contained cobalt(II) supported by aminotroponiminate ligands appended with dansyl fluorophores that are quenched due to their proximity to the paramagnetic metal center. whereas the later time point is indicative of NO production by the macrophage. HNO. 19) overcomes many of these challenges and has enabled visualization of NO in living cells.5-fold higher quantum yield than FL itself. In terms of biological applications. Metal-Based NO Sensors The first metal coordination compounds to provide turnon fluorescence enhancement in response to NO were reported in 2000.140 The success of SNP despite its obvious drawbacks associated with its ancillary cyanide ligands has inspired the development of improved coordination complexes for NO delivery. The irreversible. and ONOOgive no response.3-d]imidazol-2-yl)phenol (MNIP. 22). NO2-. a number of metal complexes have appeared that give a fluorescence change in response to NO. reductive nitrosylation of a metal center can produce NO+ that reacts with metal-coordinating amines to give noncoordinating N-nitrosamines with enhanced emission. Reaction with NO forms Co(I)-dinitrosyl adducts and induces dissociation of a fluorescentligand. Select Examples of Metal-Based Fluorescent NO Sensors Haas and Franz 6. Given these positive attributes.128 ruthenium(II). FL-NO no longer binds to Cu(II) or Cu(I) and has a 7.138 Fluorescence imaging of macrophages infected with B.138 Another Cu(II)-based NO sensor has also appeared recently along with in vivo imaging data.2.3. NOinduced reduction of paramagnetic Cu(II) to diamagnetic Cu(I) restores fluorescence of a coordinated fluorophore. other drawbacks of some of these examples include cross-reactivity with O2 or other species and fluorophores that are not ideal for cellular imaging studies. No.4930 Chemical Reviews. it does have an associated risk of cyanide toxicity and must be administered cautiously. Na2[Fe(NO)(CN)5]. the causative agent of anthrax.127. CuFL was then shown to be cell permeable and capable of detecting NO produced by both constitutive and inducible NO synthases in living neurons and macrophages.therebyprovidingemissionenhancement.134 The FL ligand coordinates Cu(II) with an apparent Kd of 1. Many of these complexes are based on multidentate chelating ligands that improve the stability of the metal complex and avoid loss of supporting ligands.134 Neither removal of Cu(II) from the CuFL complex nor addition of Cu(I) to FL gives the dramatic enhancement seen for the reaction product of Cu(II)FL and NO.126 these include systems based on iron(II).124 In addition to cobalt(II). 2009. providing further assurance that the response is NO-dependent and not the result of metal release or simple reduction of the probe compound. Vol. Taken with other control experiments. The ability to visualize NO production in a time-dependent manner by this metal-based NO sensor helped establish that bacterial-derived NO is a key defense mechanism of bacteria against the host immune response. The focus in recent years has been to design agents that deliver a payload of NO in a controlled and targeted manner. these data showed that the major source of NO during early stage infection is from the bacterium itself. NO3-. anthracis spores showed a positive fluorescence response to CuFL within 2 h of infection. NOinduced fluorescence increase was shown through detailed spectroscopic analysis to derive from the N-nitrosated product FL-NO (20) that results from a reductive nitrosylation mechanism. a seemingly simple octahedral coordination complex of Fe(II) with one NO ligand and five cyanide ligands.141.139 The first metal-containing NO-releasing drug was sodium nitroprusside (Chart 5.134 The turn-on response was also obtained by reacting CuFL with S-nitrosothiols. SNP.130 and copper(II)131-137 and achieve fluorescence enhancement by a variety of mechanisms in addition to the fluorophore displacement strategy described above.5 µM at pH 7. 109.125.129 rhodium(II). For example.135 6.134 For most of the early attempts at building metal-based NO sensors. Metal Complexes as NO-Releasing Compounds NO-releasing drugs are a broad class of compounds that release NO in vivo or in vitro to effect a pharmacological outcome.4 water/DMSO of 0. the fluorescence responses were restricted to reactions done in organic solvents with high concentrations of NO gas bubbled into a cuvette.123 Over the past decade.131 Alternatively. while spores lacking their NO synthase gene gave no response to the NO probe at 2 h but a significant response after 18 h. The fluorescent compound 4-methoxy-2-(1H-naptho[2. 10 Chart 4.6 µM. where Cu(I) is released from the complex and one of the nitrogen ligands is nitrosylated.139 Although it is still used clinically for the rapid reduction of hypertension in acute cases. while other reactive nitrogen and oxygen species like H2O2. 21) binds Cu(II) with 1:1 stoichiometry to give nonfluorescent Cu-MNIP with an apparent Kd in pH 7.122.142 Such .

have been used as controlled sources of NO to elucidate the biological and notably neurophysiological roles of NO in cells and tissues.145 In addition to release of toxic side products like cyanide.141.3.142. 2009. The known photosensitivity of many metal-nitrosyl compounds makes light an attractive stimulus to release NO from a metal center.148 particularly those that can be activated by two-photon excitation.Application of Metal Coordination Chemistry Chart 5.144.146 Considerable effort has therefore gone into designing alternative multidentate ligands that support photoactive Mn and Ru nitrosyl adducts.1. 109. the NO is lost prior to photolysis either thermally or by conversion to other NOx products in the case of the Fe-NO compounds. or the metal itself reacts with biomolecules as in the case of Ru forming DNA adducts.144. NO-Releasing Metal Complexes Chemical Reviews. Vol.N-bis(2-pyridylmethyl)amine-N-ethyl-2-pyridine2-carboxamide).145 Metalloporphyrins that were studied for their photolytic properties by Ford in the early 1990s have the advantage of being very stable macrocyclic metal complexes with intense long-wavelength absorptions but suffer thermal instability and oxygen sensitivity in the case of the Fe-NO adducts and complicated back and side reactions in the case of Ru-NO porphyrins. Several photosensitive metal compounds including SNP. 10 4931 directed release of NO could have applications as antiinfectious agents143 or in cancer therapy as sensitizers for γ-radiation or as agents for photodynamic therapy. 6.141 These compounds are shown in Chart 5 and described in more detail in the following sections. and simple Ru complexes like [Ru(NO)(Cl)5]2-. Fe-S-NO clusters known as Roussin’s salts. however.145 as well as water-soluble Fe complexes. which supports octahedral M-NO com- .147. NO Donors Sensitive to Visible and Near-IR Light The Mascharak group introduced the pentadentate ligand PaPy3H (N. No. these complexes all suffer from undesirable side reactions.

Fluor-RSE). and fluorescent NO donors that are activated by visible light to release NO. As illustrated in the PaPy3 examples described above. While the chromophores increase light absorption and improve the rate of NO photorelease at longer wavelengths. 26 has been immobilized into a biocompatible.152 Both the Mn and the Ru compounds activate soluble guanylate cyclase activity in vitro in a light. Adjustment of the ligand frame effectively merges the Ru-NO photoband with the intense absorption of the coordinated dye. The iron complex [Fe(PaPy3)(NO)]2+ can be activated by visible light in the 500-600 nm range.158 Cell culture studies in human breast cancer cells demonstrate the ability to track triggerable NO release from these compounds. Both strategies are further explored below. the advantage of these dyes is their moderate two-photon cross sections that enable the dye-modified clusters to release NO upon twophoton excitation at 810 nm femtosecond pulsed light. including AFchromophore(28. By using this open coordination site to attach a lightharvesting dye directly to the metal. 10 Haas and Franz plexes by providing four nitrogen atoms around the equatorial plane of the metal with a carboxamide group trans to NO.3. therefore the sensitivity to light depends on the energy of the M f π*(NO) electronic transition.158 Control experiments indicated that the red fluorescence is due to intact 25. Ford and co-workers have modified Roussin’s red salt esters (RSE. 6. the compounds showed light-dependent vasorelaxant activity in a rat thoracic aortic ring. ultimately inducing photochemical release of NO. The strong σ-donor character of the negatively charged carboxamide being trans to NO has proven to be a key feature in the photolability of these complexes. However.N′-bis(bipyridine-2-carboxamido)1.2-diaminobenzene) nicely demonstrates how clever alterations to the coordination chemistry of the complex can tune its photophysical properties. However.158. the four-coordinate H2bpb framework allows an additional donor ligand to be installed trans to NO. it is very desirable to have NO donors that are photoresponsive in this region if they are to be used in applications like photodynamic therapy. samples that were subjected to visible light showed signs of nuclear degradation consistent with apoptosis within 4-8 h after illumination (see arrows in Figure 1b). NO Donors Sensitive to Two-Photon Excitation Roussin’s red salt.150 Photolysis only occurs with UV light. not free resorufin. which is only sensitive to low-intensity UV light.162fluorescein(29.156 To demonstrate NO transfer to biological targets. These preliminary cellular studies are consistent with cellular uptake of nontoxic.153 The photoactivity of metal nitrosyls requires promotion of an electron from a metal-based molecular orbital to a π*(NO) antibonding orbital. Resorufin (Resf) is a dye with intense absorption in the visible and a phenolate moiety that enables direct attachment to the Ru center to give Ru-NO complexes with strong absorption bands around 500 nm. While there was no sign of apoptosis for cells held in the dark.4932 Chemical Reviews.158 Because light penetration through mammalian tissue is mostly restricted to the 700-1100 nm region. thereby accessing visible light photoactivation. which may limit future in vivo use. thereby increasing the extinction coefficient and sensitizing the compound to visible light photoactivation. Trackable NO Donors In addition to acting as light harvesters to increase the photosensitization of NO donors. because it is soluble and stable in aqueous buffers and is activated by visible light (500-650 nm) to release NO and form the Mn(II) aqua species [Mn(PaPy3)(H2O)]+. Mascharak reported that replacing one of the pyridal arms of HpaPy3 with a more conjugated quinoline arm to give HpaPy2Q provides a framework that supports Mn-NO complex 26 with near-IR sensitization. one example is the dimethyl derivative [Ru(Me2bpb)(NO)(Cl)] shown in 24. Vol.165 About 85% of the fluorescence intensity is quenched in these compounds.161 To increase the sensitivity of this class of NO donors to visible and near-IR light. No.156 6. which presumably instigates the onset of apoptosis. but it does not have appreciable absorption in the longer visible range desirable for in vivo applications. stable.141 . the Mn complex [Mn(PaPy3)(NO)]+ (23) is an example. indicating energy transfer from the antenna to the Fe-NO cluster. as evidenced by colocalization with the blue nuclear stain DAPI. but the complex is a useful tool for studying fast reactions of NO with heme proteins.3. The ligand H2bpb (N.163.2. Na2[Fe2(µ-S)2(NO)4]. one way to do this is to change the metal. In addition to 26. has been shown to generate NO photochemically and sensitize hypoxic cells to γ radiation.153 They also elicit a concentration-dependent increase in cGMP in vascular smooth muscle cells. [Fe2(µ-SR)2(NO)4]) with a variety of light-harvesting chromophores. polyurethane-coated sol-gel matrix that delivers NO to myoglobin under near-IR light at 780 nm. PPIX-RSE). the M-NO photoband needs to be in this longer wavelength region. Two other strategies are to change the field strength of the ligand or to attach light-harvesting chromophores to the complex. but the complex is not stable in aqueous solutions and transfers its NO to thiol-containing compounds even in the absence of light.158.156 Again by clever alterations of the ligand. Furthermore.160 Both 26 and 27 are diamagnetic species that retain appreciable fluorescence intensity of the coordinated dansyl or resorufin dyes. Several variations of this ligand exist. replacing the pyridal arms with quinolines red shifts the Ru-NO photoband from 380 to 455 nm. demonstrating that the complexes release NO intracellularly under the control of light. because [Ru(PaPy3)(NO)]2+ is soluble in water and stable between pH 5 and 9. additional sensitization is achieved. the incorporation of fluorescent dyes into NO-releasing compounds has the advantage of providing a tracking signal to monitor cellular distribution. 2009.3.164 and protoporphyrin IX (30.149 Replacing Fe with Ru improves the solubility and stability of the complex.159 The combination of the quinoline arms on the ligand and the dye attached to the metal to give [Ru(OMe2bQb)(NO)(Resf)] (25) demonstrates that these effects are additive.151 The Mn analog [Mn(PaPy3)(NO)]+ (23) has proven to be the most interesting among this series.AFX-RSE).160 The bright red fluorescence in Figure 1 indicates that 25 is taken up by live cells and localizes in the cytosol and nucleus. 109. with the nature of the metal center dictating the wavelength of light required to achieve photorelease. the dansyl-containing [Ru(Me2bpb)(NO)(Ds-im)]+ (27) also provides a fluorescent handle. the process is not very efficient.154-156 To push the excitation wavelength into the visible and near-IR.and concentration-dependent manner. Photoinduced loss of NO gives paramagnetic Ru(III) species that quench fluorescence of the coordinated dyes to an extent related to the degree of NO released.157 Furthermore.

The first transition metal complexes to be investigated for their ability to release CO under physiological conditions were [Mn2CO10] and [Ru(CO)3Cl2]2. Chart 6). 10 4933 Figure 1. Examples in this category include Ru supported by bipyridine (bpy) and pyridine (py) spectator ligands. CdSe/ZnS core/shell quantum dots have also served as antennas to sensitize photorelease of NO from [Cr(cyclam)(ONO)2]+ cations that are electrostatically embedded in the nanoparticle assembly.179 Incorporation of glycine onto the Ru(CO)3 core renders this compound water-soluble.173. Reprinted with permission from ref 158. which is upregulated in response to both chemical and physical stress. with the . cystic fibrosis.172 Several protective roles for CO have been identified by increasing the activity of HO-1 or by administering low concentrations of CO gas in cell culture or animal models.172 The basal level of CO in healthy humans is approximately 20 µmol/h. protection against organ transplant rejection.172-174 The expanding possibilities for how heme oxygenase regulates physiological response via CO production along with the possibility that CO could be used therapeutically if appropriately administered has led to a search for small moleculesthatcandeliverCOinacontrolledfashion. antiapoptosis. Copyright 2008 American Chemical Society. FluorRSE has modest solubility in water and therefore has potential as a biological NO donor via two-photon excitation. inhibition of platelet aggregation. The arrows point to evidence of nuclear degradation. Poly(ethylene glycol) units have also been incorporated onto a RSE cluster modified with a two-photon chromophore to instill water solubility.173 In addition to a constitutively expressed HO-2 isozyme. for CO-releasing molecule 1 and 2 (32 and 33. there is also inducible HO-1. Metal Complexes as CO-Releasing Compounds CO is produced endogenously in mammalian cells as a byproduct of heme degradation by heme oxygenases (HO).178 The drawback that neither compound is water-soluble inspired a search for molecules with improved biological compatibility. metal nitrito (M-NO2) complexes can also act as precursors for NO release. Human breast cancer cells loaded with NO-releasing compound 25 for 1 h and either (a) kept in the dark or (b) exposed for 1 min to visible light. which were coined CORM-1 and CORM-2.171 6.3.178 These compounds were shown to transfer CO in vitro to myoglobin and promote vasodilation in an isolated aortic ring model. or CORM-3 (34).176 The aqueous solution chemistry of CORM-3 is in fact complex. The number of biological tests for CORM-3 are extensive and were recently reviewed. neurotransmission.169 alternatively. and protection against ischemic tissue injury. and CORM-2 reduced acute hypertension in rats. which release NO upon visible light excitation. anti-inflammatory. 2009. Metal-Based CO-Releasing Compounds 6. pendant chromophores such as anthracene shown in 31 have been attached to the cyclam ring.4. While PPIX-RSE is only soluble in organic solvents. Vol. These roles include vasorelaxtion. within the past decade this diatomic gaseous molecule has emerged as an important signaling molecule that influences numerous physiological processes.167 and Cr(III) supported by the tetraaza macrocycle cyclam to give the water-soluble trans[Cr(cyclam)(ONO)2]+. and diabetes can significantly elevate this capacity. The most promising CORM to date and the one that has received the most intensive biological evaluation is [Ru(CO)3Cl(glycinate)]. which also release biliverdin and iron as coproducts. No. inhibition of smooth muscle cell proliferation. but diseases including asthma.121. Bottom insets show magnified views of a cell undergoing apoptosis. Other NO Donors In addition to metal nitrosyl complexes. Long thought of as a potentially toxic waste product.168 To increase the absorption cross-section. respectively. The complex was shown to have moderate cancer cell killing ability following two-photon excitation.Application of Metal Coordination Chemistry Chemical Reviews. 109.170. cis[Ru(NO2)(py)(bpy)2]+.4.166 Chart 6.175-177 This work has been pioneered by Motterlini.

Because 40 does not penetrate cell membranes. it does not have significant discrimination among polyphosphate anions including other nucleoside triphosphates.178 The water-soluble [CpFe(CO)3]Cl also suffered from precipitation following CO loss. the DPA-bound Zn(II) centers bind and activate a water molecule for nucleophilic attack on the xanthene ring. as well as other anions. The fact that the rate of CO release can be tuned depending on the trans ligand portends that further optimization of this system could result in compounds where exchange of the trans ligand itself could be manipulated under certain conditions to instigate CO loss.7. Metal Complexes That Bind Phospho Anions The charge and Lewis acid character of metal ions make them attractive components of sophisticated anion receptors wherein a metal ion is anchored by a ligand that allows open coordination sites for anion binding. The homoleptic compound [Fe(CO)5] was dismissed early on as a biologically viable candidate because photolysis to release CO caused undesirable precipitation. photoreactive Mn(CO)3 units stabilized by a tris(pyrazolyl)methane ligand to give [Mn(CO)3(tpm)]+ (37) have been introduced. More recently. The ligand scaffold also provides added molecular recognition for anion selectivity or reporter groups for colorimetric or fluorescence sensing. thymidine nucleotide. Hamachi introduced a xanthenebridged bis(Zn-DPA) compound (40) that has a remarkable fluorescence enhancement upon binding to polyphosphate anions and is effective for fluorescence imaging of intracellular ATP stores in living cells. the concept of using metal complexes as host-guest recognition systems for anion detection has moved beyond detection in aqueous solution to the more complicated intracellular applications.4 µM/min in a DMSO/phosphate buffer. competing ligands like cysteine or glutathione could act as strong trans-labilizing ligands that induce CO loss. bind to Ru to form the sensor.186 Many of these dinuclear Zn-DPA complexes incorporate a chromophore either into the backbone of the ligand or exogenously for indicator displacement that enablesdetectionofanionslikephosphate.190. 38). forming the µ-oxygen atom that destroys the xanthene conjugation.181 A series of CORMs containing 2-pyrone have also been prepared in the hope that the pyrone moiety might facilitate cell membrane transport and intracellular distribution. 2009. In addition to cyclen.and diphosphates in aqueous solution. No.183 Examples include CORM-F3 (35) and the Mo complex CORM-F10 (36). It was also shown that the compound was taken up via passive diffusion by human colon cancer cells and induced photoinitiated cytotoxicity.177 In plasma or cellular environments. the compound was not cytotoxic in the dark.193 pyrophosphate. which has been suggested to involve replacement of the labile glycinate and chloride ligands by other components in solution.185-187 As an example.184 This compound was shown to release two equivalents of CO to myoglobin in solution following irradiation at 365 nm. CORM-F10 is one of the fastest CO releasers to date.182.177 The difference in rates of CO liberation is likely a consequence of the mechanism of CORM-3 CO loss. photoactive CO-releasing molecules are attractive for directed and triggerable CO release in high concentration at a localized site.184 Importantly. 10 Haas and Franz major species present at physiological pH 7. they have been applied for measuring phosphate concentrations in biological fluids like serum and saliva by using an indicator-displacement strategy where phosphate displaces fluorescein from the host-guest complex to provide the signal readout.4. In Jurkat cells 40 showed a particulate pattern of fluorescence that matched images obtained with quinacrine. the rate and extent of CO release could be tuned by varying the halogen directly attached to the 2-pyrone ring. The Ru core acts as both a template to arrange the Zn sites optimally for IP3 binding and as a luminescent reporter for IP3 sensing. and other biologically relevant anions in aqueous solution.194-197 and ATP198.199 in aqueous solution.192 Three of the cyclen-appended bipyridal ligands. suggesting that it is indeed the Zn-bound form of the probe that operates as the ATP recognition domain. and inositol triphosphate. Anslyn introduced a tris[(2-pyridyl)methyl]amine Cu(II) complex that positions guanidinium groups in ideal positions to recognize a phosphate anion above the metal center (Chart 7. images of cells exposed only to the xanthene-DPA ligand without Zn showed a fluorescence response that was spread over the entire cytosol.and diphosphates.200 The fluorescence turn-on mechanism of 40 derives from the recovery of conjugation in the xanthene ring upon polyphosphate binding to the diZn site. which result from attack of hydroxide onto a coordinated carbonyl. Fe carbonyl complexes are also being investigated for their CO-releasing properties. but substitution of the cyclopentadienyl ring results in water-soluble compounds of the type [(C5H4CO2Me)Fe(CO)3]+ with promising biological activity. it was modified by acetylation at both hydroxyl groups to generate a cell-permeable version that converts to 40 intracellularly by esterases. Work by Kimura showed that Zn(II) complexes of the macrocyclic tetraamine cyclen (1. Notably.5. shown as 39 in Chart 7.180 The half-life of CO loss from CORM-3 varies dramatically depending on the solution conditions: in distilled water. The supramolecular compound suffers from photodegradation but shows a creative approach for the selective detection of an important second messenger for intracellular signal transduction.10-tetraazacyclododecane) form 1:1 complexes with various anions in aqueous solution at neutral pH.200 Instead of incorporating a fluorophore into the bis(ZnDPA) construct. Vol. whereas it shortens to under 4 min in human plasma. With a CO release rate of 3.or [Ru(CO)2(CO2H)(OH)(glycinate)]-. 109. 6.188 While these compounds have not been used in a cellular setting. While this agent has significant discrimination for polyphosphates over mono.189 Coordination complexes of Zn(II) are more broadly explored than those of Cu(II) and offer the advantages of being non-redox-active and typically nonquenching toward attached fluorescence dyes. Toward this goal. it is 98 h.182 As with the case of photoactive NO-releasing molecules.4934 Chemical Reviews.121. Importantly. dipyridylamine (DPA) Zn(II) complexes are also promising recognition sites for biological anion sensing. Modifications of this motif have led to receptors for phosphate. an existing probe of ATP stores. The probe can detect low micromolar concentrations of ATP.191 A supramolecular sensor that arranges six Zn2+-cyclen units around a Ru(bpy)3 core is capable of discriminating inositol triphosphate (IP3) from mono. receptor 41 works as a probe of flavin adenine dinucleotide (FAD) by taking advantage of the intrinsic fluorescence of the isoalloxazine ring system of the .4 conditions expected to be [Ru(CO)2(CO2)Cl(glycinate)]2. In the absence of polyphosphate and in neutral buffered solutions. pyrophosphate. In these compounds. In addition to Mn and Ru.

10 4935 flavin. which are known to have a high FAD content. The selectivity is again based on the recognition of anionic phosphates by dinuclear Zn-DPA constructs. Hong and co-workers showed that it caused a significant enhancement in the autofluorescence of granules in human white blood cells. although small molecule probes could overcome some limitations of the protein-based assay. Fluorescence imaging of Jurkat cells treated with the apotosis-inducing anticancer drug captothecin and subsequently with 42 showed selective staining of cell surfaces on the apoptotic cells. indicating that 42 does not permeate membranes. Vol.Application of Metal Coordination Chemistry Chart 7. which is normally quenched by intramolecular ring stacking with the adenine. 2009. intracellular fluorescence was not observed. No. whereas normal cells showed no staining. To demonstrate that 41 could be used as a cellular probe. The assay is frequently done with the protein annexin V. A related Zn-DPA construct has also been used to selectively label bacterial cells over mammalian cells. which therefore provides a platform for the selective targeting of FAD over other flavin-containing species. The autofluorescence of riboflavin and flavin mononucleotide (FMN) are not affected by 41 because they do not contain an appropriate binding site for the dinuclear Zn-DPA probe. Examples of Phosphate Anion Binders Chemical Reviews. Whereas mam- .202 Furthermore. The Smith group discovered that Zn-DPA coordination complexes have a selective affinity for mem- branes enriched in anionic phosphatidylserine and that fluorescently modified versions like 42 could be useful for assays of programmed cell death.201 The utility of the dinuclear Zn-DPA systems for binding phosphate anions has further been explored for the selective detection of anionic phospholipids for several biological applications.201 Binding of FAD to 41 disrupts this interaction and increases the quantum yield approximately 7-fold. 109.202 The appearance of phosphatidylserine on the outer surface of the cell membrane is an early sign of apoptosis that provides a means for differentiating healthy vs apoptotic cells.

The receptor is not maximally fluorescent unless both sites are saturated with Zn2+.203 Fluorescence imaging studies of the fluorophore-labeled version 43 show that staining of both S. which suggests that these complexes might be useful for detecting and imaging bacteria. and Ln(III) complexes. Vol.206.204 6. The antibiotic action appears to be caused by depolarization of the bacterial cell membrane. Metal complexes in particular are attractive components of kinase/phosphatase sensors because they can couple a strong metal-phosphate interaction (as demonstrated in the previous section) to a change in fluorescence as a spectroscopic readout of phosphorylation state. tissues.205. No. The dinuclear design of these receptors is critical to their function. 6. An X-ray crystal structure of 45 bound to phenyl phosphate shows the phosphate unit bridging the two Zn(II) centers by providing a separate O ligand to each metal210 (as indicated by the binding mode of p-Tyr to 45 in Chart 8). aureus infection in the left rear thigh muscle 18 h after tail-vein injection of carbocyanine-modified bis(ZnDPA) 44. proving that the phosphate functionality is required for molecular recognition by the probe. Copyright 2006 American Chemical Society. a probe designed to detect anionic phospholipids found in bacterial cell walls. 109. the surfaces of bacterial cells contain a high percentage of anionic phospholipids. Binding of the d10 ion Zn2+ to the receptor increases fluorescence by canceling the photoinduced electron transfer of the benzylic amine that otherwise quenches the appended anthracene fluorescence. A comparison of a series of short peptides with sequences based on various kinase recognition domains revealed that receptor 45 has the highest affinity for peptides with the greatest overall net negative charge. The Smith group therefore explored the differential interactions of a family of complexes based on the 2.6-bis(zinc(II)-dipicolylamine)phenoxide construct. sulfonate. The difference between the negatively charged.1. or tyrosine amino acid side chains by protein kinases and removed by phosphatases.203 An NBD-tagged version is shown in 43.210 The Kapp of 45 for NaH2PO4 and phosphotyrosine is ∼105 M-1.208 The first probes contained two Zn-DPA units spanned by an anthracene bridge. which is proposed to occur as a result of large fluctuations in local charge following the binding of cationic Zn-DPA complexes to anionic phospholipids head groups.4936 Chemical Reviews.203 Indeed. moderately sterically demanding phosphomonoester compared with the neutral hydroxyl group of the unmodified amino acid provides a means of using chemistry to differentiate phosphorylated peptide or protein sequences from their unphos- . Chart 8 shows examples of metal-dependent sensors for protein phosphorylation that are based on Zn(II).210 malian cell surfaces are composed mostly of zwitterionic phospholipids.206 Phosphate groups are installed onto a protein’s serine.204 Nude mice were infected with S. a hydrophilic version that contains just a methyl substituent off the phenoxide is not toxic to mammalian cells but is very effective at killing the pathogenic bacterium Staphylococcus aureus. Phosphoprotein Detection by Zinc Complexes In furthering the anion receptor capability of the Zn-DPA unit. and even whole organisms. and anesthetized animals were irradiated with 720 nm light for imaging. The cooperativity of the dinuclear site is also critical for the fluorescence sensing mechanism of 45. thus providing a structural basis for the cooperativity of the dinuclear site in recognizing phosphomonoester functional groups. The unphosphorylated version of the v-Src peptide does not induce a fluorescence change of 45. whereas its affinity for select phosphopeptides increases 1 or 2 orders of magnitude depending on the peptide sequence. The NIR fluorescent probe 44 was introduced 6 h postinfection into the bloodstream via a tail-vein injection. with the maximum signal contrast reached after 18 h (Figure 2).207 While this ultimate goal has yet to be realized. the consensus sequence of the protein kinase. but other derivatives have been made in which the alkyl substituent trans to the phenoxide is varied. 45 has a Kapp of 9 × 106 M-1 for the phosphotryosine (pTyr)-containing sequence Glu-Glu-Glu-Ile-pTyr-Glu-Glu-Phe-Asp. coli is localized to the cell walls. Optical image of a mouse with a S. acetate. one example is shown in 45. For example. Reprinted with permission from ref 204. Ca(II). incorporating a carbocyanine dye that is sensitive to tissue-penetrating near-IR light onto a Zn-DPA construct enabled in vivo imaging of bacterial infection in a living animal. a situation that is facilitated by the presence of phosphate derivatives. The goal is to create probes that can selectively recognize a phosphoprotein of interest and thereby visualize the activity of specific protein kinases and phosphatases in real time in living cells. it is not surprising that 45 also has a high affinity for ATP and ADP (Kapp > 107 M-1) in addition to phosphate monoesters but weak affinity for other anions including carbonate. since mononuclear versions show very little affinity for phospho species. 2009. Whereas lipophilic versions partition into zwitterionic vesicles and are moderately toxic to mammalian cells. v-Src.209. the probes described below have achieved some success in biological applications.6. aureus and E. Metal Complexes for Phosphoprotein Detection The interest in developing sensors that can selectively detect protein phosphorylation stems from the central role that this post-translational modification plays in signal transduction pathways and a host of other biological processes. and chloride. Given the similarity of 45 to the ATP sensors described above. aureus by injection of bacteria into the thigh muscle. nitrate. This correlation between overall net charge of the phosphopeptide and its affinity for the probe presumably arises from a favorable electrostatic attraction between the tetracationic dinuclear Zn-DPA receptor and negatively charged peptides. threonine. 10 Haas and Franz Figure 2. Hamachi has developed a family of probes that recognize and sense phosphopeptides. Scale represents relative fluorescence in arbitrary units.6. phorylated analogs. The fluorescent probe accumulated at the site of infection. Mg(II). Peptides with fewer negative charges or overall positive charge elicit weaker fluorescent changes by the Zn-DPA probes.

7 Å distance found between the phosphate groups on Ser6 and Ser9 on the C-terminal domain of RNA polymerase II (CTD peptide). decreased over time upon addition of protein tyrosine phosphatase 1B.213 Indeed. Vol. they have been used to monitor phosphatase activity in vitro.213 This work demonstrates that the well-placed metal-ligand interactions between the Zn-DPA coordination complexes and target phosphorylatedregionscanbeutilizedtodisruptprotein-protein interactions and may be effective for disrupting signaling cascades in living cells that rely on such interactions. Metal Complexes for Phosphoprotein Detection Chemical Reviews.211 The method was able to brightly stain phosphoovalbumin and phospho-R-casein without staining four other nonphosphorylated proteins under photoillumination of a polyacrylamide gel that had been soaked with a solution of the Zn-DPA probe. the anthracene-linked Zn-DPA probes have also shown utility as selective phosphoprotein stains for gel electrophoresis analysis. A second strategy for increasing selective detection of specific phosphoproteins is a hybrid approach that uses a combination of an artifical Zn-DPA receptor coupled to the Pin1 WW domain. In addition. a fragment of the epidermal growth factor receptor. 109. as demonstrated in 46.212 They found by X-ray crystallography that the biphenyl unit of 46 disposes the two Zn centers at a distance of 11.214 The doubly phosphorylated CTD peptide binds cooperatively to the hybrid sensor by interacting one of its . No.211 In order to increase the selectivity of the Zn-DPA probes for specific phosphoproteins.Application of Metal Coordination Chemistry Chart 8. 2009. Hamachi optimized the length of the spacer connecting the Zn-DPA units so that each Zn(II) could bind to an adjacent phosphomonoester. which matches fairly well with the 9.8 × 106 M-1. it was found that 46 effectively inhibited the protein-protein interaction between the CTD peptide and the Pin1 WW domain with a Ki of 1.210 The emission intensity of 45 in the presence of a substrate peptide Asp-Ala-Asp-GlupTyr-Leu-Ile-Pro-Asn-Asn-Gly.5 Å. 10 4937 While the anthracene-linked Zn-DPA sensors have yet to be utilized in cell culture. By using a fluorescence polarization assay with a fluorescently labeled CTD peptide substrate. The relatively simple staining method could find general applicability for deciphering the phosphoproteome by using 2D gel electrophoresis. 46 binds the CTD peptide in a 1:1 fashion with a Kapp of 8 × 106 M-1 as determined by isothermal titration calorimetry.

The quantum efficiency of the uncaging photoreaction was determined to be 0. two molecules of γ-aminobutyric acid (GABA) coordinate to a Ru(II) center supported by two bipyridine ligands to form the caged complex [Ru(bpy)2(L)2]2+. thereby releasing one equivalent of the free amine. including serotonin. 109. Zn-DPA complexes have also shown an ability to deliVer phosphopeptides and other phosphate derivatives intracellularly. the photoreaction is clean.220 cases.223 In these . As monitored by NMR. and tyramine. In these peptide examples.2.227 Conventional.215 The intracellular fluorescence of fluorescently tagged phosphopeptides was found to increase 4-6-fold in the presence of 47 compared with the absence of the metalloreceptor. Optimization of the linker connecting the fluorophore and the peptide provided probe 48 with a 23-fold increase in fluorescence after phosphorylation. Incorporation of triphenylphosphine (PPh3) into the coordination sphere to form [Ru(bpy)2(PPh3)(GABA)]2+ with only one amine ligand coordinated increases the quantum yield to 0.218 The original designs required a conformationally constrained peptide β-turn to position Sox appropriately for cooperative binding of Mg2+ with the phosphorylated amino acid. Phosphoprotein Detection by Calcium and Magnesium Complexes In addition to Zn2+. but phosphorylation increases the binding affinity of the receptor and turns on the signal. butylamine. it 6.21. they have a strong preference for negatively charged. Being hard acids.221. Not surprisingly.219 More recently. as in DKNADGWIDRA(pS)LA. In a slightly different application from phosphoprotein detection.6. Phosphoprotein Detection by Lanthanide Complexes Long-lived emission lifetimes and sharp emission bands are some of the photophysical properties that make lanthanide (Ln) ions attractive for incorporation into biological probes.224 The peptide-based probe can detect both kinase (phosphorylation) and phosphatase (dephosphorylation) activity by diluting aliquots of the enzyme assay into buffers containing either Tb3+ or Eu3+. This strategy forces the amino acid ligand to bind through its amine functional group and avoid a bidentate mode that would be recalcitrant to photorelease. To increase the sensitization and the metal affinity. The trivalent Ln3+ is the most common oxidation state across the series.1. An advantage of the Ru-based caging groups is their longer wavelength photoreactivity with visible light. Subsequent rupture of that metal-ligand bond by a specific trigger could in principle release the desired molecule in a controlled way. Photorelease of Bioactive Molecules Coordinating a bioactive molecule to a metal center is an attractive way to mask the activity of a molecule of interest. the peptides are short sequences resembling calciumbinding loops where the phosphorylated amino acid either is embedded within the peptide.226 Excitation of these molecules with 450 nm light into a metal-ligand charge transfer band of the Ru(bpy) core induces heterolytic bond cleavage of one of the monodentate ligands.221.223 where pY and pS are phosphotyrosine and phosphoserine. Etchenique and colleagues have applied this general principle in their development of ruthenium polypyridine complexes as inorganic photolabile caging groups for several amine-containing neurotransmitters. Sames incorporated a carbostyril chromophore and an iminodiacetate chelating motif onto peptides that contain a tyrosine residue within a recognition domain for src and abl protein tyrosine kinases (50). organic photocaging groups require UV excitation that could cause cellular photodamage.4938 Chemical Reviews.222 As a consequence.7. Tb3+ can be an effective reporter of phosphorylation status. Our lab has shown that the phosphorylation state of a peptide can dramatically increase its affinity for metal ions. sensitizing chromophores are required to achieve luminescence.224 6.221 or is the anchoring metal-ligating residue near the end of the peptide. Imperiali and co-workers have developed a family of kinase probes that incorporate a sulfonamido-oxine amino acid (Sox) strategically embedded in kinase recognition domains such that phosphorylation improves the chelation of Mg2+ to enhance Sox fluorescence 4-12-fold. Metal Complexes That Release Bioactive Small Molecules 6. The probe may be useful for in vitro evaluation of kinase and phosphatase inhibitors. as in DPDNEA(pY)EMPSEEG. In addition. where L is GABA or one of several amine compounds tested. they have greatly expanded the scope of Sox-based kinase sensors by developing a Sox derivative called C-Sox that can alkylate a cysteine residue with the chromophore (49 in Chart 8). because they are sensitive under conditions of physiological Mg2+ levels. Since lanthanides are poor absorbers. Vol.6.03. tryptamine.220 This strategy has allowed the probe to be incorporated into a larger number of kinase recognition domains with improved specificity. respectively. No. 10 Haas and Franz pSer residues with the WW domain and the other with the Zn-DPA unit.225 Amino acids like glutamate can also be caged by replacing one of the bpy ligands with a tridentate tris(pyrazolylmethane) ligand to give a Ru complex with only one available open coordination site.3. Lawrence and co-workers replaced one of the iminodiacetate arms with a serine-containing peptide that is a substrate for protein kinase C. The probes are amenable for high-throughput screening for kinase inhibitors and.207 These probes have been validated for the measurement of kinase activities in crude mammalian cell lysates.217 The unphosphorylated probe has low affinity for Ca2+ and is therefore weakly fluorescent. The highly cationic tetranuclear complex 47 with sufficient lipophilic character was found as the optimal carrier vector to shuttle anionic phosphorylated peptides into cells via endocytosis. 2009. providing only the monoaquo complex [Ru(bpy)2(L)(H2O)]2+ (52) as the other photoproduct. may be amenable to future in vivo applications. Ca2+ and Mg2+ are also effective at enhancing the fluorescence signal of designed probes via phosphorylation-dependent chelation.7.216 To convert this idea into a kinase sensor. The design of 48 is based on Ca2+ indicators developed by Tsien that rely on Ca2+ coordination to turn on fluorescence. oxygen-rich coordination environments. In the example 51 shown in Chart 9. the pY itself or a tryptophan (W) residue provides sufficient sensitization to observe luminescence. which is slightly higher than that of the more conventional nitrobenzyl ester photolabile protecting groups that have been used to cage GABA and other other biomolecules. 6. they interact strongly with phosphatecontaining groups.

flow only upon activation by GABA. both free marimisat and the metal-bound prodrug also potentiated tumor metastasis relative to controls. a hydroxamic acid derivative of coumarin-343 mimics the hydroxamic acid motif of marimistat. action potentials of a leech neuron were measured by a micropipet electrode inserted into isolated neurons that were exposed to [Ru(bpy)2(4AP)2]2+. Because the intact metal-fluorophore emmission is weak. be it by reduction or ligand exchange.227 In another example. kinetically inert metal ions like Co3+ could be used as chaperones to deliver bioactive molecules intracellularly by releasing them upon reduction to the more labile Co2+ oxidation state.2.Application of Metal Coordination Chemistry Chemical Reviews. and more recent work suggests that ligand exchange is the more likely mechanism.228 To date the Ru-based cages have been applied in two biological settings. Action potentials increased when the neurons were exposed to both the Ru compound and flashes of visible light. however. the intensity of the images suggests that the fluorophore is released from the metal. Reductive Activation and Ligand Exchange Reductive activation or ligand exchange is an alternative strategy of releasing a bioactive compound that is deactivated by coordination to a metal center. the fluorescence of the metal-bound coumarin is quenched but is restored upon ligand release. complexes with fluorescent ligands such as 54 were developed.233 Unfortunately.234 In the example shown.231. In principle. Given the promise of using cobalt as a drug chaperone and the clear need to understand its mechanisms of cellular distribution and prodrug activation in order to rationally design effective agents. Furthermore. a ligand-gated ion channel that opens for Cl. a compound known to increase neuronal activity by blocking certain K+ channels. while the different staining patterns of the metal complexes vs the free coumarin suggest that metal complexation influences its cellular distribution. Vol. suggesting that the more reducing environment of hypoxic tumors favors reduction of the metal complex and release of the cytotoxic agent. Metal Complexes That Release Bioactive Amine-Containing Small Molecules has been shown that the Ru-based caged compounds can undergo two-photon excitation at 800 nm. In the Co(III) form. These studies importantly demonstrate that metal complexes can effectively chaperone bioactive molecules and traffic them in different ways compared with the uncomplexed agents. Current flow was measured by voltageclamp recordings of frog oocytes expressing the GABAc receptor. Pulses of visible light. consistent with release of GABA. consistent with photorelease of 4AP to stimulate neuronal response.230 More recently.230 This strategy has been explored with a variety of Co(III) coordination complexes with amine-containing mustard compounds that are known alkylating agents.7. No. where 4AP is 4-aminopyridine. which may have important ramifications for altering the pharmacology of small molecules. Details of this mechanism are still not clear.229 6. resulted in measurable increases in current flow. the Ru-caged GABA complex [Ru(bpy)2(PPh3)(GABA)]2+ caused no change in Cl.232 Some of this family of compounds show hypoxia-selective cytotoxicity. Fluorescence microscopy studies showed that the subcellular distribution of the fluorophore was altered by coordination to the metal complex. which is then free to activate the GABAc receptor. 10 4939 Chart 9. When applied in the dark. 109.currents. 2009. these studies provide a strategy for monitoring the cellular trafficking and reactivity . Hambley has shown that the matrix metalloproteinase inhibitor marimastat can be incorporated into a Co(III) coordination complex (53) and used as a prodrug to inhibit tumor growth.

hydrogen bonding. dipyridylamine.239 These examples. While most inhibitors are organic compounds. while less effort has focused on the zincbinding group.1. Additional interactions like hydrogen bonding and hydro- phobic contacts between the inhibitor and the protein can increase specificity of the inhibitor for the protein pocket. The inhibitor-protein interaction can be further strengthened if an amino-acid side chain from the protein binds directly in the inner sphere of the metal complex to anchor the inhibitor in place. 109. Rather than relying on breaking a metal-ligand bond to release the small molecule. a bis-allyloxycarbonyl-protected rhodamine (56) was incubated with cultured HeLa cells. No. thiopyrones.236. The use of metal complexes as enzyme inhibitors was reviewed by Louie and Meade in 1999. and aza-macrocycles. hydrophobic. the following discussion provides select examples to highlight four strategies for using inorganic and organometallic chemistry for targeted enzyme inhibition: (1) metalloprotein inhibitors that use metal binding interactions as a significant component of their molecular recognition. as emphasized in a recent perspective on the topic. non-native one can help in studying an isolated metalloenzyme in vitro. Addition of thiophenol to the cell medium increased the fluorescence 10-fold. substituting a native metal ion with an inactive.240 Inhibitors of these mononuclear Zn(II) enzymes typically contain two components: an organic backbone that forms noncovalent. which is typically a hydroxamic acid moiety. and anthrax lethal factor are some of the examples of zinc(II)-dependent hydrolytic enzymes that are attractive drug targets for a wide range of diseases. If the enzyme is involved in disease. 7.235 To show that such a transformation could be conducted in a mammalian cell.243 . but is a difficult strategy to implement in the complex environment of a cell or whole organism. they are developing organometallic complexes as biocompatible catalysts to carry out intracellular organic transformations. (3) inert metal complexes that provide novel scaffolds for binding protein active sites. hydroxypridinones. but metal complexes can also target nonmetalloproteins. hydroxypyridinethiones. 10 Haas and Franz of cobalt coordination complexes. Indeed. removing or replacing the metal are very effective means of abrogating activity. as shown in 58. strategies for enzyme and protein inhibition. aerobic environment of a cell presents significant challenges for most organometallic complexes. 2009. the sophistication in terms of the design and understanding of metal complexes as enzyme inhibitors has increased substantially for a handful of examples.237 7. Because the metal cofactor of a metalloprotein is critical for function.4940 Chemical Reviews. however.3. and perhaps too often overlooked.238 Since that time.241 In recent years. which comprise one-third of all proteins. as observed by confocal fluorescence microscopy.5-cyclooctadiene) (55) was found to be tolerant of such conditions and able to catalyze the cleavage of allylcarbamates in the presence of excess thiophenol to give amines. Inhibitors with Metal-Binding Headgroups for Targeting Metalloproteins Carbonic anhydrase. compounds that bind directly to an active site metal ion can inhibit enzyme activity by blocking access of the substrate to the catalytic site. are obvious targets for inhibition based on coordination chemistry. organic inhibitors. and a metalbinding headgroup that coordinates the active-site Zn(II). Treatment of the cells with the Ru catalyst caused a 3. 6. are usually nonspecific and provide little advantage for developing inhibitors targeted for a specific enzyme. and electrostatic interactions with the protein surface. The protected rhodamine derivative is not fluorescent. and (4) metal complexes that catalytically inactivate proteins. however. the metal ion may not be part of the administered compound but is instead picked up endogenously from the biological milieu. Finally. inorganic coordination chemistry provides attractive. In contrast. The structures of compounds discussed below are shown in Chart 10. Catalytic Release of Bioactive Molecules Meggers and co-workers have developed a different strategy for releasing amine-containing molecules inside cells.5-fold increase in fluorescence within the cytoplasm. Metal-binding compounds and metal complexes themselves both offer unique properties that contribute to enzyme inhibition and are not found in conventional organic molecules. the reactivity of metal complexes can be harnessed to develop catalytic inhibitors that incorporate aspects of molecular recognition for specificity along with hydrolytic or oxidative reactivity to inactivate proteins through chemical modification. still represent only a small minority of compounds compared with conventional. as shown in Chart 9. Further studies aimed at understanding how to control the localization and release of active agents from these kinds of complexes remain an important goal. Cohen and co-workers have been reversing that trend by taking a bioinorganic approach to identify alternative zinc-binding groups. matrix metalloproteinases (MMP). or the caged rhodamine.235 These encouraging results suggest that there is significant scope for further development of organometallic complexes used in biology. Although the aqueous. including pyrones. A significant amount of work has gone into optimizing the backbone fragments to impart specificity for a particular enzyme isoform. cod ) 1. Likewise. Metal Complexes as Enzyme Inhibitors Small molecules that are capable of selectively inhibiting a particular enzyme are powerful tools for deciphering an enzyme’s biological role. In some cases. the growing field of bioorganometallic chemistry offers exciting opportunities to uncover compounds with unique biological properties. Crystal structures of small chelators coordinated to a Zn(II) model complex that mimics the three histidine ligands in the active site of MMPs were used in conjunction with computational modeling to identify promising zinc-binding groups that could be extended into full-length MMP inhibitors. For proteins that do not contain a metal site. Chelators that strip metals from their active sites. intact metal complexes can take advantage of their shape and organic framework to bind to proteins with high specificity through outer-sphere interactions. [Cp*Ru(cod)Cl] (Cp* ) pentamethylcyclopentadienyl.33. its inhibitors become important drug candidates. Vol. Rather than provide exhaustive coverage of all chelator-inhibited enzymes. (2) compounds that recruit endogenous metal ions to increase their affinity for protein binding and inhibition. Metalloproteins.242 A range of chelating moieties have been investigated.7. thiophenol. but treatment with the Ru catalyst and thiols deprotects it to give highly fluorescent rhodamine 57. Cell viability was not affected by the treatment with the Ru compound. histone deacetylases (HDAC).

2.248 7. One of the drawbacks of conventional.244 Interestingly. having equivalent or higher affinity for iron and copper compared with zinc. 10 4941 An example of a potent. nonhydroxamate inhibitor of MMPs that came out of this process is 1. Examples of Metal Complexes As Enzyme Inhibitors Chemical Reviews.246. when a series of inhibitors containing the same biphenyl backbone but different hydroxypyridinone or hydroxypyrone headgroups was evaluated for selective inhibition against eight different MMP isoforms. Inhibitors That Recruit Endogenous Metals To Increase Potency AMD3100. No.249 It was first identified for its potency as an anti-HIV agent. a general problem with any chelator-based approach. hydroxamate-based inhibitors is their indiscriminant nature. the bicyclam compound 61 containing an aromatic bridge connecting two tetraaza-macrocycles. Vol. The results of this study highlight the importance of understanding both the inorganic and organic aspects of inhibitor design. and the strength and orientation of the zinc-bound chelator. In order to identify potential cross-reactivity of chelator-based inhibitors. It also found that 1. Srivastava and Mallik connected iminodiacetic acid (IDA) units to aryl sulfonamides to create two-prong inhibitors such as 60. where a hydroxypyridinone is the zinc-binding group. a cellbased screening method was developed in a macrophage model that compares the relative activities of five different zinc and iron metalloenzymes in the presence of inhibitors. Crystal structures of 60 bound to human carbonic anhydrases I and II validate this strategy and demonstrate that a metal-binding headgroup can couple with a protein-binding metal complex to enhance the overall effect of the inhibitor. the pKa of the chelating units. while a hydroxypyrone analog did not. 109.245 This protocol identified dipyridylamine and triazacy- clononane as promising zinc-specific binding groups.244 The factors influencing the altered selectivity are likely a combination of altered sterics and hydrophobicity of the substituted pyrone/pyridinone rings.2-HOPO-2 (59) showed some cross-reactivity with heme-dependent enzymes. has a history of over 20 years in drug development.247 Arylsulfonamides have been known since the 1940s to inhibit carbonic anhydrase by binding directly to the Zn(II) and displacing the coordinated hydroxide in the enzyme active site. MMP inhibitors are known to suffer from nonspecific interactions with off-target metalloenzymes. The second prong of 60 was added to bind Cu2+ so that the Cu-IDA unit could bind to surface-exposed histidine residues that are unique to select isozymes.2-HOPO-2 (59). which will influence the positioning of the backbone into the protein site. 61 (now marketed as Mozobil) . Although clinical trials as an AIDS treatment were discontinued due to severe side effects. with a bulky biphenyl backbone.Application of Metal Coordination Chemistry Chart 10. 2009. it was revealed that even subtle differences in the zinc-binding group can influence isoform selectivity. In order to develop isozyme specific inhibitors of carbonic anhydrases.

Further interactions are formed by hydrogen bonding between the cyclam NH and protein carboxylates. The biological effects of several Rubased inhibitors have been tested in mammalian cell culture.or RNAtargeting agents with metal complexes for inducing damage to nucleic acids. were inserted into the bicyclam rings.251 While there are no X-ray structures available for the transmembrane CXCR4 receptor.252 The structures demonstrate that carboxylate side chains can indeed directly bind to open sites on the metal complex. Pak-1. including Pim-1. Xenopus embryos. in particular for hematopoietic stem cell transplantation for treatment of non-Hodgkin’s lymphoma and multiple myeloma.264 While the focus here is on protein inhibitors. a folded cis configuration and a planar trans configuration. and 50-fold when Cu2+. the crystal structure reported by Sadler and co-workers of Cu-cyclam and Cu2bicyclam bound to lysozyme provides some insight into protein-metallocyclam interactions. cocrystal structures of inhibitor-bound enzymes reveal the purely structural role of the metal and the variation in contacts between the substituents and enzyme active sites. Vol. they still work by a conventional mechanism of competitive inhibition at a protein active site. as revealed by mass spectrometry.4. While cyclam has a higher thermodynamic binding affinity for Cu2+ than Zn2+ (log K ) 27. This strategy of catalytic inactivators couples a protein-targeting moiety for molecular recognition with a metal complex capable of either hydrolytic or oxidative reactivity.267 For example.239 Over 500 protein kinases with conserved ATPbinding sites.258. Metal Complexes as Catalytic Protein Inactivators In the previous examples.269 Four Co(cyclen) conjugates decorated with benzothiazine derivatives were found that cleave Aβ monomers and oligomers at submicromolar concentrations. 36-. and P13K. The affinity of 61 for CXCR4 was shown to increase 7-. and zebrafish embryos. It is possible. are encoded in the human genome making selective inhibition very challenging.257. all with low nanomolar binding. The elements of molecular recognition responsible for the tight binding of 61 to CXCR4 were initially thought to derive principally from hydrogen-bonding interactions between the bicyclam N-H groups and specific carboxylic acid side chains on the protein. It was suggested that such a strategy could be used to reduce the amount of toxic protofibrils of Aβ that are implicated in amyloid formation in Alzheimer’s disease. Suh conjugated a Co(III) cyclen complex known to effect amide bond hydrolysis266 to a variety of protein-targeting domains that were uncovered by screening combinatorial libraries. a target for antibacterial drugs.257. Subtle optimization of the ligands oriented around Ru has enabled this family of compounds to complement the ATP binding sites in unique ways to differentiate one active site from another. 10 Haas and Franz continues to show promise for other diseases. Metal-bound cyclam complexes can adopt several configurations that depend on the cis or trans arrangement of coordination sites not used by the ring nitrogens as well as the stereochemistry around each chiral nitrogen donor atom. a transmembrane protein in the G-protein-coupled receptor family that assists entry of HIV into cells and anchors stem cells in the bone marrow. In many cases. Variations of 63 have been shown to target other kinases. and Ni2+.265 In order to direct such reactivity to a specific protein of interest. the cyclam rings are found in two configurations. Zn2+.254 of very potent and selective protein kinase inhibitors from a family of related organometallic Ru core structures.258 The selectivity in this case was achieved as a result of a structure-activity study that identified substituents on the ligands that improved GSK-3 selectivity over a panel of 57 different kinases. No. Inert Metal Complexes as Enzyme Inhibitors Instead of using a coordinate covalent bond between a metal center and a protein side chain to anchor a metal complex into a protein active site.4942 Chemical Reviews.264 The ability of metal ions and complexes. MSK-1.262 7.260 Figure 3 shows the crystal structure of GSK-3 with a derivative of 63 bound. the higher relative concentration of available Zn2+ in blood plasma (reported at 19 µM) coupled with the faster kinetic rate for cyclam-Zn2+ complex formation at physiological pH suggests that AMD3100 likely exists as the Zn-bound form in vivo. configurationally restricted tetraazamacrocycle 62 competes effectively against antiCXCR4 monoclonal antibodies and may be useful in drug screening assays.249 Binding of 61 to CXCR4 therefore blocks viral entry into the host cell and also releases stem cells from the bone marrow into the peripheral blood where they can be harvested for transplantation. While their inorganic nature makes them unique compared with organic inhibitors. Compound 61 has been shown to be a highly specific antagonist for the CXCR4 chemokine receptor. The power of this strategy has been successfully demonstrated by Meggers.269 The same four compounds were subsequently found to be effective cleavage 7. it is also possible to use a substitutionally inert metal center as a structural building block to orient organic ligands in three-dimensional space to create architectures that are difficult to obtain with organiconly scaffolds. 64 was discovered from a library of candidates that was screened for the ability to cleave peptide deformylase.5). Furthermore. especially strongly Lewis acidic species.2 vs 15. to use the reactivity of metal complexes to cause irreversible destruction of a protein target with substoichiometric amounts of the inhibitor.250 suggesting that the metal complexes provide additional binding interactions that improve affinity. as well as hydrophobic contacts to adjacent tryptophan residues.263. which are also inhibitor target sites. it is important to mention that similar strategies have been used to couple DNA.259. Archibald has introduced configurationally fixed macrocycles that in principle present only one configuration to the protein to optimize metal-aspartate coordination that is the key to the increased protein binding interaction of the metal-loaded form of 61.253 They have recently shown that the Cu(II) complex of fluorescently tagged.239.3. All of these interaction types are likely present when 61 binds to its target CXCR4. however. a thorough review on this topic recently appeared.255. respectively. it was shown that a substoichiometric amount of the compound caused cleavage of the target protein.268 While the rate of reactivity was modest. to hydrolyze unactivated amide bonds is well-known.268 A similar combinatorial approach was used to identify Co(cyclen) conjugates to target Aβ peptides and oligomers. 109.256 The example shown in 63 is highly selective for glycogen synthase kinase 3 (GSK-3). 2009. who has assembled a number . structural aspects of metal-ligand bonding provide functionality to these metal-based inhibitors.261.

109. however. The observation that thermolysin. the inhibitory activity of the metallopeptide was enhanced several fold. which can be adjusted to fit into the base stacks and grooves of targeted DNA sequences. can provide an element of enantioselectivity. However. Toward this goal. The enzymeinhibition profile supports a catalytic multiturnover inactivation pathway. for example. if applicable. is not inhibited by [KGHK-Cu]+ but is inhibited by [CGHKCu]+also implies that the amino acid sequence provides target selectivity to the metallopeptides. respectively).272 In this respect. and Rh can be exploited for their interesting photophysical properties as . which uses a carboxylate to coordinate directly to the active-site zinc(II) and a lysine that fits into a protein subpocket. Crystal structure of glycogen synthase kinase 3 with the ruthenium compound (RRu)-NP549 (a derivative of 63) bound in the ATP-binding site: (a) overview of the complete structure.275 Further research along these lines is warranted in order to develop metal-based agents to a point where they could be used in a cellular or animal model system. because DNA itself is chiral. It was therefore hypothesized that the Cu(II)-bound form mimics some of the molecular recognition elements of the popular ACE inhibitor lisinopril. eliminating the possibility of hydrolytic reactivity. Metal Complexes for Probing DNA An ongoing effort toward unlocking biological information contained within DNA is the pursuit of molecules that can specifically interact with.Application of Metal Coordination Chemistry Chemical Reviews. The complexes that have shown the most utility as DNA probes are the ones with interesting chemical and photophysical properties. the authors suggest that a metalbased oxidant such as a Cu2+-associated hydroxyl radical or a Cu3+ oxo may be the agent responsible for catalytic inactivation. as fluxional behavior would negate selectivity based on shape recognition.271.272 Further evidence for protein oxidation as the mechanism of enzyme inactivation was provided in a study of the Cu(II) complex of a Gly-Gly-His peptide tagged with a sulfonamide for targeted inhibition of carbonic anhydrase. it was recently shown that an iron-based catalyst could oxidize amino acids in water. [KGHK-Cu]+ demonstrates competitive inhibition of ACE with a KI of 4 µM. There remains significant scope in further developing such compounds. The stereochemistry of the complex. serving as DNA footprinting agents or as specific luminescent probes for DNA sequence or structure.270 In an alternative strategy.273 Under normal conditions in an in vitro inhibition assay. Reproduced with permission from ref 257. agents against human islet polypeptide. suggesting a change in its mechanism of action. in vivo probes must also be substitutionally inert so that they remain intact within the cell. (b) close up of (RRu)-NP549 in the binding pocket. or cleave oligonucleotide sequences. Vol. Copyright 2008 Wiley-VCH Verlag GmbH & Co. unique chemical properties of metal ions can be exploited to label or cleave selected DNA sites. DNA sequences of interest can be specifically targeted by taking advantage of sequence-dependent changes in overall three-dimensional shape and surface electronic distribution. another Zn(II)-dependent metalloprotease. No. mostly histidine side chains that are 5-20 Å from the active site.274 In this case. another amyloidogenic peptide. whereas Lewis acidic Zn can promote hydrolytic cleavage.276.271. where the inhibitor is presumed to localize.271. 10 4943 Figure 3. both important in cardiovascular and blood pressure regulation. Redox-active Cu and Fe. No protein cleavage was observed. 2009. 8. when the assay is performed with subsaturating concentrations following a preincubation step and addition of ascorbic acid. label. and Ru.272 None of the apo-peptides were effective and neither were metallopeptides lacking the C-terminal lysine. mass spectrometric analysis of the protein following treatment with the inhibitor and ascorbic acid revealed that specific protein residues were oxidized. Os.and endothelin-converting enzymes (ACE and ECE.277 Metal complexes that are useful for structural recognition of DNA must have rigid three-dimensional structure. Although the mechanism is not fully elucidated. The peptide Lys-Gly-His-Lys (KGHK) bound to Cu(II) as shown in 65 was found to inhibit angiotensin. the metallopeptide behaves as a classical inhibitor. can induce oxidative strand scission. KGaA. Metal complexes are especially well suited for DNA interaction because the electropositive metal center is innately attracted to the negatively charged phosphate backbone of DNA. Metal centers also serve as unique threedimensional structural scaffolds.274 This is a promising result that demonstrates the feasibility of metalbased reagents selectively oxidizing and inactivating target proteins. Cowen has shown that Cu(II) complexes of short peptides based on the ATCUN (aminoterminal copper/nickel) motif are effective inhibitors of several zinc metalloenzymes. In addition.

2009. 10 Chart 11.277 Intercalation is a DNA binding mode in which a planar aromatic group inserts and π-stacks between two adjacent bases in the core of a DNA double helix. Sequence-Specific DNA Probes Barton and co-workers have taken advantage of the small variations in shape and functionalities of the DNA major groove in order to design octahedral intercalating complexes that select for specific DNA sequences.276. Coordination complexes that contain at least one large planar aromatic ligand are capable of this noncovalent interaction. 8.4944 Chemical Reviews. Vol. The following section will focus on novel metal complexes (see Chart 11) that have potential for in vivo use to recognize specific DNA sequences or structures. Metal Complexes for Probing DNA Haas and Franz probes. as first demonstrated by Lippard in . 109.1. No.

which shares the same recognition site. however. like [Rh(bpy)2(chrisi)2]3+ (69).R)-Me2trien)(phi)]3+ (67) was designed to recognize 5′-TGCA-3′ via hydrogen bonding between the axial amines and the O6 of guanine. can bind mismatched base pairs at least 1000 times stronger than matched base pairs.281 These contacts are highlighted in the crystal structure shown in Figure 4. Chart 11) readily intercalates B-DNA at the sequence 5′-CTCTAGAG-3′. hydrogen bonding with the endocyclic N of dppz deactivates the excited state and quenches fluorescence. Note the intercalation of the aromatic phi group.277 In addition to their impressive site-selectivity.289 Barton has extended the application of octahedral Ru and Rh coordination complexes into the design of metal coordination compounds that are selective for DNA base-pair mismatches.286 Rh has been exploited for its photoactivated DNA strand cleavage abilities.287 This reaction is useful because it enables direct footprinting of the site of intercalation.Application of Metal Coordination Chemistry Chemical Reviews. the complex shows its characteristic luminescence by intercalation into the base stack. the duplex is thermodynamically destabilized.288. Small ancillary ligands like phenanthroline and bipyridine are nondiscriminatory.2. leading to direct strand scission.3. these complexes employ the useful photophysical properties of Ru and Rh to visualize and cleave DNA. These structures are formed by hydrogen bonding between guanines of the same or different strands to form a planar G-quartet (70). The enantioselectivity of a chiral complex for right-handed B-DNA over left-handed Z-DNA is mostly steric in nature and is achieved through bulky ancillary ligands. 2009. while the Λ-enantiomer is almost completely inactive toward B-DNA. Irradiation with short wavelength light (313-325 nm) in the presence of dioxygen prompts formation of an intercalating ligand-based radical that abstracts a hydrogen atom from an adjacent deoxyribose ring.4′-diphenylbipyridine) are enantioselective. respectively. the hydrogen-bonding interactions between the axial amines and the O6 of guanine. with the ∆-enantiomer binding and photocleaving well. The rational design of a mismatch-selective complex is to use a planar aromatic ligand that is too wide to fit into a normal DNA major groove. The larger ligand cannot intercalate into normal matched DNA. 1976 for a square planar platinum complex.279. A Rh mismatch complex similar to 69 attached to a cellpenetrating polyarginine peptide and fluorophore was tested in cells and shown to localize in the nucleus. The resulting structure can be stabilized by the presence of a monovalent cation bound in the center of the tetrad and can be further stabilized by π stacking with other G-quartets. For example. but in the case of a mismatch. When DNA is added to solution. This destabilization allows the large ligand to eject the mismatched bases and replace them with its own inserting ligand. Octahedral Ru complexes containing a dppz intercalating ligand show A particularly impressive achievement in the area of DNA recognition has been in the selective recognition of mismatched DNA. Copyright 1993 American Chemical Society. While cells have mismatch repair machinery. the Λ-enantiomer will bind to left-handed (and less stereoselective) Z-DNA.282-284 One of the best studied examples of these molecular light switch complexes is [Ru(phen)2(dppz)]3+ (68). Mismatched base pairs are a result of errors in DNA replication.280 Sequence selectivity is also achieved by matching the functional group on the ancillary ligands with those positioned in the intercalated major groove. Metal Complexes That Recognize G-Quadruplex DNA Certain guanine-rich sequences in DNA are known to form higher order structures called G-quadruplexes (G4-DNA).293 .280 On the other hand. even in 1000-fold excess.278 Octahedral metal complexes can be designed to bind specific sequences by using a large planar aromatic intercalating ligand for DNA binding and ancillary ligands for shape and functional group recognition within the major groove. Metal Complexes That Recognize Mismatched DNA Figure 4. as well as by van der Waals contacts between the pendant methyl groups and the methyl groups of thymine. whereas complexes with bulkier ligands like DPB (4. Barton has developed an entire series of site-specific DNA binders. unchecked mismatches lead to permanent genetic mutations. Using both shape and functional group recognition. In aqueous solvent. For example. 10 4945 solvatochromatic luminescence in organic solvents. No. 109. Almost 80% of hereditary nonpolyposis colon cancers and 15-20% of biopsied solid tumors are linked to mutations in the cell’s mismatch repair machinery.279. Metal complexes using the expansive 5. It is noteworthy that 66 will also inhibit the XbaI restriction enzyme. ∆-[Rh (DPB)2(phi)] (66.291 The question of whether the complex is specifically binding to DNA in vivo is yet to be answered. but the Λ-enantiomer is inactive.6chrissene quinone (chrisi) ligand.292. Ru-dppy complexes have good cellular uptake but so far show little selectivity for DNA over other hydrophobic cellular components.290 In addition. enantioselectivity is observed. Crystal structure of the metallo-intercalator 67 bound to target sequence 5′-TGCA-3′. Reprinted with permission from ref 281. shielding its dppz ligand from any interaction with the H-bonding solvent. 8. Vol.285. ∆-R[Rh((R.287 8. and the contacts between the pendant methyl groups of the ligand with the methyl groups of thymine.

4946 Chemical Reviews. which shows nearly 10-fold preference for G4-DNA over duplex DNA and modest telomerase inhibition. Other instances of ss/ds junctions occur during replication. and are known to occur at hairpin loops and Holliday junctions. On the other hand.317.(known commercially as Magnevist) in 1988. and duplex DNA may not be flexible enough to accommodate the required conformation. Metal-Responsive MRI Agents Magnetic resonance imaging (MRI) provides threedimensional images of opaque biological structures with relatively high spatial resolution (∼10 µM) in a noninvasive procedure. Addition of Ca2+ shifts the coordination mode and opens the door for H2O to interact with the Gd3+ centers directly (78) and .310. 8.304 Although these ss/ ds targeting complexes have not yet been fully developed for in vivo application. Cu-ttpy (74) is highly selective for G-quadruplex in comparison to square-planar Pt-ttpy (75).318 enzymatic activity.295 The enrichment of telomeres and promoter regions with quadruplex-forming potential sequences makes them likely candidates in genetic regulation. Notable examples include Mn-TMPyP4 (71). A class of binuclear and trinuclear copper complexes has demonstrated exquisite selectivity in oxidizing DNA at ss/ ds DNA junctions. 9. apical ligands of square-pyramidal complexes prevent intercalation into duplex DNA without interfering with its G-quadruplex association. known to have quadruplex-forming potential. While not used clinically. which can be significantly reduced if the spins are in contact with a local paramagnetic center. motivation to study these structures and their biological implications has been spurred by recent evidence of their in vivo significance.296-298 Development of new methods not only to distinguish G4-DNA from duplex DNA but also to discriminate between the polymorphs of G4-DNA is necessary. Several metal complexes have been reported to interact with G-quadruplex with some selectivity. preferring to oxidize guanine at the n and n + 1 position of the single-stranded DNA directly extended from a duplex region. The myriad of possible conformations of ss DNA may effectively compete against its productive association with the metal complex. but also high-spin Fe3+ and Mn2+ with five d electrons) are therefore excellent candidates as agents that enhance MR images via a T1 mechanism. as well as insert into double-stranded DNA.302 Just as the unique properties of metal complexes have been successfully used to probe duplex DNA. the metal complexes discussed above show promise for probing quadruplex DNA to help elucidate the potential structures that exist and the significance of G-quadruplex structures in live cells. Site specificity derives in part from the coordination of at least one copper to a guanine. extracellular agents. MRI Agents for Calcium The first metal-responsive probe was introduced by Meade in 1999. The time required for the spins to realign with the field is characterized by T1. Single/double strand junctions are yet another DNA structure with implications in disease and represent a potentially rich source of new understanding of in vivo DNA function.293.308 A radio-frequency pulse inverts the magnetization vector of water protons from their preferential alignment with the external magnetic field. Despite their rigorous study in vitro.1. 77) consists of a BAPTA Ca2+ chelating motif onto which Gd(DOTA) macrocycles are fused. Specifically. In the absence of Ca2+. Metal Complexes That Recognize Single-Double Strand Junctions Single strand (ss)/double strand (ds) DNA junctions are another type of structure existing in telomere regions in a wide variety of species. A ss/ds junction may offer unique proximity of the two single strands yet offer flexibility to assemble with the copper complex. No. nor is cleavage observed for GG-GG mismatches flanked on both sides by matched duplex DNA.304 The authors predict that the flexibility of the DNA strand is a factor in the ss/ds junction selectivity.309 Contrast agents that are currently widely used in clinical medicine are nonspecific. efficient cleavage is not observed for ss or ds 9. with 50. Since the approval of [Gd(DTPA)(H2O)]2.319-322 and specific metal ions. thereby brightening the image. Interestingly. For example. 370 000294 such sequences have been recognized in humans and 40% of all known promoter regions have at least one.311 Activateable contrast agents have appeared in the research literature that respond to changes in pH. DNA alone. the simple square pyramidal complex.299 followed by Ni(II)-salphen (72)300 and Mn(II)-porphyrin (73)301 complexes.316 small molecules like glutathione317 and sialic acid. the potential application of these and similar metal complexes that can recognize unique DNA structures is apparent.302 The ttpy ligand in the Pt complex can stack on the outside of G-quad. 2009. research on designing new Gd coordination complexes as improved MR imaging agents has increased dramatically. Metal complexes are especially promising for Gquadruplex targeting. Vol.303-307 Complexes such as 76 are also sequence-selective. Coordination complexes that leave open coordination sites for water molecules to access the inner sphere of paramagnetic metal ions (particularly Gd3+ with seven unpaired f electrons.308 The desire to use MR imaging to understand physiological events has sparked a trend in the design of “smart” contrast agents that change their relaxivity (and therefore their level of contrast enhancement) in response to a biological stimulus or change in environment. however. Some DNA-binding proteins that recognize ss/ds junctions in telomeres are known to have functions in telomere maintenance and capping. the contrast in the image depends on the local concentration of water and on the longitudinal (T1) and transverse (T2) relaxation times of its protons. 109. Square-pyramidal geometry of the metal complex has been shown to be a major factor in the selectivity for quadruplex over duplex DNA. are a major component of RNA secondary structure. Because a MR image derives from the nuclear magnetic resonance of water protons.4.312-315 O2 partial pressure. it is important to note that these complexes are only in the beginning stages of development and are not as evolved as metal complexes that bind duplex DNA. making it an increasingly popular imaging modality.323 The design of DOPTA-Gd (Chart 12. Other sequences with quadruplex-forming potential are overexpressed in promoter regions of diverse organisms.and 1000fold selectivity. respectively. the carboxylate arms of the central BAPTA bind to the Gd3+ centers and occlude H2O from direct coordination (77). these kinds of responsive agents may become increasingly useful for studying molecular biology and intracellular chemistry. 10 Haas and Franz All vertebrate chromosomal DNA ends with telomeres containing repeats of the sequence d(TTAAGGG).

326 In addition to these Gd-based agents. No. Zn-Gd-daa3 (82.O-triacetate) (80).326 The apparent Kd values for Ca2+ in buffered aqueous solution are 400. MRI Agents for Zinc and Copper One strategy for generating Gd-based probes that are sensitive to metal ions like Zn2+ and Cu2+ has been to modify the general template of the Ca-responsive probes by decreasing the number of carboxylic acid arms to lower the affinity for Ca2+ while retaining affinity for these d-block divalent metals. This general concept of flanking a metalrecognition motif with Gd3+ chelates in such a way that binding of the target metal ion induces a shift in coordination.325 Even with this diminished relaxivity. one by Meade and co-workers. either Zn2+ or Cu2+. CG1 (Copper-Gad1.329 and another by Chang. The chelating motifs for the Ca2+ recognition unit for these probes are derived from EDTA (79).323 In order to develop probes that would be responsive to extracellular Ca2+ in the millimolar range. 109.1-10 µM range. that shows a 41% increase in relaxivity in the presence of Cu2+. as shown in 84. but are insensitive to Mg2+ and Ca2+. The impermeability of these reagents to cell membranes is a current limitation for their use. DOPTA-Gd has an apparent Kd of 0. that shows a 115% increase in relaxivity in the presence of Zn2+. the Ca2+ response of the EGTA-based probe (81) in a complex cell culture medium designed to mimic the brain extracellular medium gave a ∼10% change in relaxivity over the 0. a different approach that uses superparamagnetic iron oxide nanoparticles for Ca2+ imaging has been presented by Jasanoff.N.333 The disadvantage with this strategy is that Zn-binding . 9. 83). Chart 13).8-1.96 µM and was shown to be sensitive to in vitro Ca2+ concentrations in the 0.325 Competition from other metal ions was not reported. The aggregation of the nanoparticles in the presence of Ca2+ results in a change in T2 signal.331 An alternative approach for a Zn-dependent MR sensor was introduced by Nagano wherein the Gd-chelate forms the central core of the molecule. which increases water access to Gd. and 200 µM. The DOTA-based probes all suffer from a loss of relaxivity in biological media. 11. with flanking Zn2+-binding units composed of either dipicolylamine or a combination of pyridal and carboxylate ligating arms.3+ has provided the template for a host of other metal-responsive contrast agents.328.Application of Metal Coordination Chemistry Chart 12.325 and EGTA (81). although 82 was shown to be nontoxic to NIH/3T3 cells and to accumulate intracellularly to a concentration of ∼10-14 mol Gd/cell.2.327 The particles are linked to the Ca-binding protein calmodulin and calmodulinbinding peptides. Two very similar designs have appeared. Calcium-Responsive MRI Probes Chemical Reviews.328 Changing the metal receptor from a carboxylate-based ligand as in CG1 to a softer thioether-based ligand set results in later-generation CG sensors with high selectivity for Cu+ over competing ions and turn-on responses up to 360%. All three probes are reported to be selective for Ca2+ over Mg2+. a response that is likely due to anions in the buffers displacing H2O from Gd3+ in the Ca2+ loaded form. 10 4947 increase relaxivity. Intracellular imaging has not yet been reported for these compounds. 2009. Vol.332.330 As anticipated from the similarity in the structures of these two probes. both in vitro. Logothetis and Toth introduced three families of probes with diminished affinity for Ca2+ so that the probes would not be saturated under the target conditions.2 mM Ca2+ concentration range that might be expected in the brain. while compound 81 is also shown to be sensitive to Ca2+ in the presence of Zn2+. however.324 APTRA (o-aminophenol-N. respectively. both compounds suffer from interference of the nontarget metal ion.

high-resolution technique for biological imaging.336 Fluorescent proteins337-339 and heterocyclic aromatic dyes like fluoroscein. MRI Sensors Sensitive to Zn2+ and Cu2+ Haas and Franz displaces H2O from the Gd3+ center. resulting in a decrease in relaxivity that provides a less appealing bright-to-dark signal.334 Yet another approach of placing Zn binding sites in proximity to a paramagnetic center was recently introduced by Zhang et al.334 Unlike the Gd3+-based agents that rely on changes in the water relaxation rates to achieve contrast. It seems likely that 86 could be interacting with other biomolecules inside cells in a Zndependent fashion that results in the increased relaxivity not seen in aqueous solution. No. Luminescent Metal Complexes for Cellular Imaging Fluorescence microscopy is a powerful. In aqueous solution. wherein it localizes predominantly in the nuclear fractions rather than the cytosol.4948 Chemical Reviews. 10 Chart 13. The compound was shown to have little cytotoxicity to HEK-293 cells. addition of Zn2+ results in a darker MR image. Vol. 109. These somewhat conflicting results indicate that the mechanism of the Zn-induced relaxivity changes is not at present well understood. 10. if the probe has a long excited-state lifetime on the order of hundreds of nanoseconds to milliseconds.e.341. This problem can be avoided if the molecular probe absorbs and emits light at distinctly different wavelengths (i. rhodamine.. this is the first reported example of a cell-permeable MRI sensor for intracellular Zn2+. and luminescent molecular probes make it particularly useful for probing cellular biology. A different take on designing a Zn binding site above the plane of a lanthanide chelate was introduced by Sherry as a PARACEST contrast agent.340 However. PARACEST reagents (paramagnetic chemical exchange saturation transfer) respond to changes in water exchange rates at paramagnetic centers. whereas Mn3+ insertion into the porphyrin converts the molecule to a MRI sensor with relaxivity changes sensitive to Zn2+ (86). time-resolved luminescence microscopy (TRLM) can be used to eliminate autofluorescence. dipicolylamine units are situated above and below a water-soluble porphyrin ring. and others have been indispensable tools for observing the cellular localization of biological and other molecules of interest. Alternatively. 2009. Semiconductor quantum dots have been used in tracking cell mobility and . The metal-free form operates as a fluorescence sensor for Zn2+. The increase in the water exchange rate is attributed to a Zn-bound water or hydroxide positioned above the Eu3+ that catalyzes proton exchange between the Eu3+-bound water molecule and bulk solvent. Importantly. A significant advantage of this sensor is its water solubility and cell-permeability. large Stokes shift) such that autofluorescence can be easily differentiated from signal luminescence. a continuing quandary is in distinguishing between autofluorescence of the analyzed sample and that of a luminescent dye meant to image a particular cellular component.335 In this case. in cells the T1-weighted images increase brightness. however. Incorporating two dipicolylamine units across a Eu3+-chelated macrocycle to give 85 provides a compound that shows a significant change in the CEST spectrum upon addition of Zn2+.342 Quantum dots and several luminescent metal complexes have these characteristics.

Application of Metal Coordination Chemistry Chart 14. and they have characteristic properties of a large Stokes shift (187 nm). Metal complexes. Studies of 87 in yeast cells and human MCF-7 cells showed that this membranepermeable dye localizes to the mitochondria. Luminescent Transition Metal Complexes Some examples of membrane-permeable luminescent Ru complexes that target DNA by noncovalent interaction were .343-348 discussed previously in section 8. and can be designed for low toxicity.351 The application of a 3 MLCT luminescent agent for specific targeting of a cellular component has been recently demonstrated with the tricarbonyl rhenium(I) cation 87 (Chart 14). good cellular uptake.1. however their large size limits their application in probing small cellular organelles and structures. can emit over a broad range of the visible spectrum. A 10.350 Their luminescence stems from a metal-to-ligand charge transfer triplet excited state (3MLCT). Rhenium tricarbonyl complexes have luminescence properties that have long been postulated but only recently demonstrated as useful in vivo probes. Several examples of luminescent metal complexes targeted to cellular components or biological receptor sites by covalent linkage to biological recognition moieties have been reported and are promising for biological imaging and biochemical assays. are usually small in size. In addition. Over the past 15 years. 2009. and are relatively resistant to photobleaching. No.349. can have large Stokes shifts and long excited-state lifetimes. 109. however they have demonstrated nonspecific luminescence of various hydrophobic cellular components and could potentially serve as more general in vivo luminescent stains. Vol. there has been great progress in the design of luminescent metal complexes for biological imaging. metal complexes have easily recognizable line spectra. and targeted intracellular localization. These complexes are specifically designed to target DNA. on the other hand. 10 4949 have the advantage of tunable luminescence based on particle size. and good quantum yield.350 The selective localization is likely due to reaction of the chloromethyl group with thiols that are concentrated in mitochonria.345. Luminescent Metal Complexes for Cellular Imaging Chemical Reviews. long fluorescence lifetime (131 ns).

In one example (91).8 GM. Two-photon excitation is advantageous because low-energy light is less damaging to biomolecules.6′-[methylenebis(1-methyl-1H-benzimidazole-5. variations in the R group allow for tuning of emission over a wide range of the visible spectrum from blue-green to orange. their exclusion from the nucleus and specific staining of the cytoplasm was observed. with an estimated error of 0.358.c) substituents resulted in fast uptake and showed no cytotoxicity.360 The two-photon cross section for 92 was measured to be 20. Luminescent Lanthanide Complexes In their mission to develop optical probes that report on conditions of the intracellular environment and that can be targeted to specific cellular locations. because biological samples have increased optical transparency357 at near-infrared (NIR) wavelengths. and have low cytotoxicity up to 100 µM over 24 h. 109. Helicates formed with Eu(III) display bright luminescence with millisecond excited-state lifetimes. the in vivo behavior of cyclen lanthanide probes is defined by the nature of the chromophore.365 At higher pH. Parker and colleagues have developed luminescent lanthanide complexes capable of signaling changes in pH. The emission spectrum varies as a function of analyte-induced changes in the Ln coordination environment. whereas the protonated species emits more strongly at 587 nm.363-366 carbonate. with localization in cytosolic macropinosomes and perinuclear endosomes/lysosomes. In addition.2. and human nasopharygeal cell lines showed strong green luminescence after incubation with the complex (5 µg/ mL) for 5 min.365 Each probe is based on a lanthanide coordination complex of a cyclen macrocycle equipped with a Lnsensitizing antenna chromophore and an analyte-sensing moiety held in close proximity or directly coordinated to the Ln metal center.1 GM).g) caused membrane destabilization resulting in high toxicity. Very clearly. The luminescent properties of these complexes are attributed to an excited state of predominantly triplet intraligand π-π* character.2 pH units. An incubation time of only 5 min was necessary to achieve maximum intracellular emission. In contrast. allowing for ratiometric analysis that is independent of probe concentration. A luminescent probe sensitive to changes in pH between 6 and 8 in biological media is shown as an example (94). the luminescent cyclen complexes can show relatively slow (>30 min) or fast (<15 min) uptake and efflux and localize in various cellular compartments. Both luminescent platinum complexes 91 and 92 represent a new way to visualize cells using two-photon excitation. and excellent spatial resolution can be attained. efflux. cervical carcinoma.366 The probe shows good cellular uptake and low toxicity.361 and also greater than that reported for 91 (4 GM). and localization. have low susceptibility to photobleaching. Although this sensitivity should be improved before it is applicable to measuring intracellular pH for analytical purposes. The Ln ions are bound in an ideal nine-coordinate. Another cyclometalated Pt(II) complex 93 has shown application in staining proteins in 1D and 2D SDS-PAGE analysis as well as live cell imaging.4950 Chemical Reviews.359 Application for live-cell imaging of 91 was tested in three cell lines and showed no cytotoxicity at 10 µM over 24 h. when the substituent was a negatively charged carboxylate (95d) or methyl amide (95e).369 Figure 5 shows two examples of different cellular distribution of these related complexes. 90. which is higher than the value suggested for biological samples (0. In addition. Vol.386.356 The Pt complex 92 also shows two-photon luminescent properties with a relatively long excited-state lifetime on the order of 1 µs in organic solvent.356 The R-group also allows a site for potential conjugation to biomolecules. Both complexes have excited-state lifetimes on the order of 1 µs.371 and urate372 concentrations in live cells.385 Bunzli and co-workers have developed a class of ¨ highly luminescent bimetallic helicate complexes suitable for in vivo fluorescent imaging. deeper penetration.355 Several nontoxic. contains weakly coordinated solvent molecules that readily exchange with histidine ligands and thereby allow for protein staining of histidine-rich proteins in SDS-PAGE gels.378-380 or another group380-384 or varying the linker also altered the cellular uptake.353 The first demonstration of phosphorescent iridium(III) complexes used as cell-permeable dyes for imaging live cells was also reported recently.365 The chromophore in these Ln-cyclen complexes not only influences the luminescent properties but also functions as a recognition element for cellular uptake. with low cytotoxicity observed over 6 h. the complex can be excited by absorbing two low-energy photons in the 780 nm range. respectively.384 Toward the development of bifunctional lanthanide probes. Also.or 4-positions of the pyridines or on the .352. slow cellular uptake was observed.362 10. and cytotoxicity. A systematic study compared the in vivo profiles of a number of complexes of core structure 95 with variations in substitutions on the azaxanthone sensitizer moiety. The complex is photostable and has two-photon excitation luminescence properties. the deprotonated species has a higher ratio of europium luminescence intensity at 680 nm over 587 nm. No. chemically stable luminescent Pt complexes have recently been reported as suitable for live-cell imaging with two-photon excitation and TRLM. The complexes readily diffused across membranes and localized preferentially in the nucleus.374 By design.354 Another cyclometalated Ir(III) complex.370. localization. respectively.354 The two Ir(III) complexes 88 and 89 emit bright green and bright red light. Hydrophobic substituents (95f. are membrane-permeable. changing the azaxanthone moiety to azathiaxanthone (94)369.2-diyl)]bis(4-[2-[2(2-methoxyethoxy)ethoxy]ethoxy]pyridine-2-carboxylic acid) (96) self-assemble with Ln3+ ions to form complexes having the form [Ln2(L)3]. and the authors hypothesize that protein binding to the sensitizing moiety regulates these processes.375-377 Depending on the chromophore identity.376 Carboxymethyl (95a) and cationic peptide (95b.391 The complexes can easily be substituted with polyethylene groups in the 2.376.375. but with sufficient contribution from the metal to achieve long luminescent lifetimes greater than 100 ns. Instead of absorbing one high-energy photon in the near-UV. these complexes report on the target analyte via changes in at least two emission bands. 2009. the complexes are promising for use in two-photon microscopy. and both complexes have lifetimes sufficient for timeresolved techniques. tricapped trigonal prismatic environment and are well-shielded from quenching effects of solvent or other high vibration oscillators.356 Experiments using human lung carcinoma. this probe is a promising step toward an in vivo pH meter. They are thermodynamically stable with negligible dissociation in biological media. 10 Haas and Franz similar mechanism is suggested for the commercial mitochondrial stain MitoTracker.386-390 Derivatives of the homoditopic ligand 6. and have large Stokes shifts (>170 nm) with emission in the visible region following excitation at 360 nm.367-369 citrate.373 In addition.

Using Coordination Chemistry To Label Proteins In addition to in vivo probes targeted to specific cellular compartments.394.Application of Metal Coordination Chemistry Chemical Reviews. However. FlAsH also displays some undesired background fluorescence despite use of excess EDT and washing steps.3. Alternatively.394 Biarsenical dyes such as 4′. Three human cell lines were incubated with the complex for up to 24 h.392 The probe is noncytotoxic at 20 µg/L.390 The low cytotoxicity of the helicates is demonstrated by HeLa cells being unaffected when treated with up to 500 µM for 24 h.374 A new luminescent stain for the cytoplasm of living cells has been generated by using three-photon excitation of a terbium (Tb) complex. metal-ligand bonding joins together the peptide-tagged protein and the fluorescent label. 98) bind specifically with high affinity to the short peptide sequence (CCPCC).2-dithioarsolan-2-yl)fluorescein (FlAsH. λem ) 528) upon peptide binding. Interestingly. In this way. benzimidazole N’s to achieve water solubility or for potential covalent coupling to biological molecules. FlAsH displays fluorescence (λex ) 508. however their large size can interfere with . Copyright 2007 and 2008. there are four types of metal affinity tags that have been developed to label proteins with luminescent reporters.392 protein folding. unlike the cyclen complexes discussed above. and must absorb at least three photons at 800 nm to demonstrate multiphoton-induced emission. the cells demonstrated green fluorescence in the cytoplasm in as little as 1-2 min of exposure to high intensity light. and function. substitution in the 4-pyridyl position results in higher overall quantum yields than complexes substituted at the benzimidazole position.395 The spacing of the two arsenic (As) ions matches the spacing on the two bicysteine (CC) groups in an R-helical structure so that the thiols of CC may replace the thiols of ethanedithiol (EDT) that mask the fluoresceinbound As. It is important to note that this approach is not limited to the use of a fluorescent marker. variation among these substituents has little effect on cellular localization or uptake profiles. The metal coordination properties of specific amino acid sequences have been exploited to create much smaller fluorescent labels via affinity tags.389 One drawback of these helicates is their short excitation wavelength (330-365 nm). and examples are shown in Chart 15. 11. No.1. Localization in vesicles indicates uptake via a lysosomally directed or recycling endosomal pathway. as in the case of lanthanide-binding tags (LBT). although they are photoluminescent with two-photon excitation. In a metal-chelation approach to fluorescent labeling. peptides with high affinity for metal ions are paired with fluorescent labels containing a chelated metal ion with open coordination sites available for simultaneous binding to the peptide tag. Due to nonspecific interaction with membranes and hydrophobic pockets or thiols. trafficking. The ligand N-[2[(bis(2-[(3-methoxybenzoyl)amino]-ethyl)amino)ethyl]-3-methoxybenzamid (97) forms [Tb(L)(NO3)3] polymeric complexes. Each peptide tag consists of a dense region of metal-binding amino acids.389 Bright Eu(III) emission can be seen in cells after incubation with concentrations above 10 µM in as little as 15 min. bearing a carboxymethyl-substituted azaxanthone moiety show localization of the probe to cytosolic macropinosomes. 10 4951 Figure 5. Microscopy images of NIH-3T3 cells loaded with two Ln-cyclen complexes bearing different sensitizing moieties and demonstrating different cellular distribution: (a) cells incubated with the azathiaxanthone complex 94 show typical localization to nucleosomes and ribosomes. Polycysteine Tags Tsien developed the first metal-chelating affinity tag system in 1998 by pairing a cysteine-rich peptide tag with a biarsenical fluorescein derivative. because any group can be attached to the metal-binding synthetic probe. Reprinted with permission from refs 366 and 376. For this reason. each will be discussed further below. particularly cysteine.5′-bis(1. histidine. After incubation. Royal Society of Chemistry.396 11. resistant to photodegradation. Fluorescent proteins of many colors have been extremely popular and useful for tracking expression and localization of fusion proteins. Small peptide affinity tags can be easily fused to a protein of interest without interfering significantly with the protein character. site-specific labeling of proteins is another powerful technique for visualization of cellular processes. luminescent metal ions themselves are the reporting functionality.393 In general. although up to 60 min of exposure was needed for some cells to be visualized. Vol. the 4-pyridyl-substituted helicates are more promising for in vivo application. or aspartic acid or combinations (LBT). (b) cells incubated with Tb-95a. 2009. respectively. 109.

a Cy3-based biarsenical dye (AsCy3.399. 2009.398 FRETpartners.4952 Chemical Reviews.398 improved photochemical properties. showing superior photostability and minimal environmental sensitivity and providing a FRET partner to FlAsH.395. structural requirements of both the fluorophore and As ions . 99) was developed with a larger interatomic distance (∼14. No.401 The development of various colors of biarsenical dyes is expected to promote widespread use of this labeling technique. To address this problem. 109.398 andimprovedaffinitytagsequences.5 Å) between As ions capable of binding a peptide sequence with larger spacing between the target CC groups.401 AsCy3 enables high-affinity binding to the sequence CCKAEAACC. Although many biarsenical dyes have been reported. 10 Chart 15 Haas and Franz Since the development of FlAsH.397. there have been several variations of the polycysteine tag/biarsenical pair with various colors of emission. Vol.400 The conserved interatomic distance of the As ions (∼6 Å) in FlAsH derivatives limits the complementary use of two dyes to uniquely label two separate protein sites.

The 108 monomer shows emission at 379 and 407 nm. Labeling in live cells was not as efficient as labeling in vitro. Zn2+. the use of paramagnetic Ni2+ results in partial quenching and a decrease in fluorescence. Polyhistidine Tags Peptide tags with six or more sequential histidine residues (His-tag) are known to interact with transition metal complexes and are commonly used for purification of expressed protein by Ni2+-NTA affinity chromatography. where NTA is nitrilotriacetic acid.410 Notable here is the similarity in design of these constructs with Zn-DPA constructs mentioned in sections 6. and upon binding to a D4 tag. After the probe binds to its His-tag target via Ni coordination. the probe is fluorescent whether or not it is bound to the peptide tag. The binding of Ca2+ to CaGF results in a 10-fold increase in fluorescence. 107) takes advantage of the local acidic pH of polyaspartate tags to create an affinity probe that changes emission wavelength upon binding. Compared with the binuclear Zn(II) complex. which combines a FlAsH moiety with a Ca2+ chelator. In the first example. Goldsmith et al.413 The in vivo utility of this system was demonstrated in Escherichia coli cells with CAAAAAADDDD-tagged maltose binding protein paired with 109. A recent advance in biarsenical dyes has come in the form of the Ca2+ sensor Calcium Green FlAsH (CaGF.7′-bis(pyridyl-2-sulfonamido)4′. 2009. a binuclear Zn-DpaTyr construct 109 bearing a fluorophore and a thiol-reactive chloroacetyl group both targets and reacts with a D4 tag containing a cysteine thiol. HisZiFiT has been used successfully in live cells to label surface-exposed His-tagged proteins without compromising the integrity of protein structure.413 Hintersteiner et al.411. With the Ni-NTA complex present in solution as opposed to being in large excess on a solid support as is the case for protein purification. which have affinity for poly(aspartic acid) sequences. 10 4953 has made the scope of compatible dyes narrow and difficult to predict. UV-light is used to photoactivate the arylazide and form a covalent bond to the protein of interest. Vol. where covalently bound probe 11. Covalent Protein Labeling Facilitated by Metal Chelation A drawback to imaging proteins tagged with metal affinity peptides is the high background generated by unbound fluorophores and the reversibility of the metal-tag interaction. In this way.403 CaGF-labeled calcium channels have demonstrated that CaGF can be used for precise and fast detection of intracellular Ca2+. Increasing the number of NiNTA appendages on the fluorophore increases affinity by 4 orders of magnitude in going from a mono-Ni-NTA probe to one with four Ni-NTA groups. 101). a fluorescent moiety is separated from the biarsenial targeting moiety in order to remove any restrictions on its structure. In bulk solution.5 and 6..407 In most cases. however. No. Hamachi and co-workers developed multinuclear Zn(II) luminescent complexes con- . in this case by using a His-tag as the affinity target. but when two probe complexes bind to the D4 tag simultaneously. covalent labeling strategies have been developed that take advantage of the peptide tag to direct metal complexes containing a reactive functional group to a protein of interest. To further improve the applicability of the polycysteine/biarsenical system. a Ni-NTA complex. Tsien developed a novel chelating molecule 2′.412 Ratiometric analysis is highly desirable because it helps reduce background signal due to the unbound fluorophore thereby facilitating precise detection and imaging of the protein of interest. as shown in Chart 16.408 A strategy for avoiding paramagnetic quenching is to replace Ni2+ with diamagnetic Zn2+ as the metal.402 Based on a modular approach. The affinity of His-tags for metal complexes can also be applied to fluorescently label proteins or other molecules in vitro404. and a photoactive arylazide crosslinker.410 The utility of the D4/Zn-complex system was expanded by creating probes capable of ratiometric analysis.Application of Metal Coordination Chemistry Chemical Reviews. The fluoroscein-based biarsenical targeting moiety. The reactive group is used to covalently attach the probe thereby achieving site-specific protein labeling without the requirement of an enzyme.4. Miller developed a strategy for recruiting any fluorophore to a polycysteine tag.6. resulting in a covalently labeled protein. the relatively low affinity between a Histagged protein and Ni-NTA (Kd ≈ 1-20 µM) becomes a limitation to this approach. which holds two Zn2+ ions in a coordinately unsaturated environment and has affinity for His6-tags comparable to bis-NTA-Ni2+ ligands (Kd ≈ 40 nM). but improvements in the affinity between the tag and probe are expected to improve labeling efficiency.3. The affinity of the binuclear probes for aspartic acid stretches increases by an order of magnitude with each additional aspartic acid residue up to four (D4 tag).412 11. but it has the advantage that any fluorophore can be used. excimer formation results in emission at 472 nm. 103). This system was applied to label His-tagged proteins on the surface of living cells.409 sisting of a fluorescein or Cy5 fluorescent moiety attached to one (104) or two (105.414 Probe 110 consists of a rhodamine reporter group.408 This tag does not enter cells either by itself or as its Ni(II) complex. 106) pendant binuclear Zn-DpaTyr groups.411 In a second example. can be conveniently attached to a variety of fluorophores to create different colored emission. called SplAsH (100). 109.1 that target phosphate-containing molecules.405 and in vivo406-408 by introducing a Ni-NTA complex where NTA is derivatized with a fluorophore.2. therefore it is useful for imaging extracellular proteins that are expressed with His-tags. protonation of the phenol shifts emission to 628 nm. the tetranuclear Zn(II) complexes 105 and 106 have affinity 3 orders of magnitude higher when paired with a longer tag (D4-G-D4) capable of interacting with both Zn-DpaTyr groups. will also help avoid heavy metal toxicity that can be caused by Ni2+.5′-dimethylfluorescein (histidine-zinc fluorescent in vivo tag. the phenolate form of SNARF emits at 586 nm. In this method. To overcome this problem. which is ubiquitous in biological systems. also developed a nonenzymatic covalent labeling technique. a binuclear ZnDpaTyr attached to a pH-sensitive fluorophore (seminaphthorhodafluor (SNARF).403 11. have reported that the fluorescence of NTADCF (102) is not significantly influenced by the presence of Ni2+. a peptide containing two D4 tags attracts two separate binuclear Zn(II) complexes containing pyrene moieties (108) into close proximity.404 Increasing the length of the His-tag from His6 to His10 also improves affinity. Polyaspartate Tags Runs of negatively charged amino acids provide another target as metal affinity tags. called HisZiFiT.

By incorporating nonnatural amino acids directly into prokaryotic and eukaryotic organisms. and millisecond luminescent lifetimes. Schultz demonstrated a new way to introduce metal sites into proteins without the use of oligopeptide tags. including metal-binding centers. These new metal sites are usually limited to the Cor N-terminus of a protein of interest so as not to disrupt the protein’s native structure and function. 10 Chart 16.4 of 19 nM and achieves bright luminescence by occluding inner-sphere water molecules that quench emission. where the coordinated Ln itself is the luminescent reporter. Imperiali and coworkers developed lanthanide binding tags (LBTs) as genetically encodable peptide sequences optimized for binding Ln ions. the properties of protein-bound Ln ions enabled by LBTs also facilitate macromolecular structure determination by both NMR419-421 and X-ray crystallography. time-resolved luminescencemeasurementswereusedtointerrogatetheprotein-protein interactions between Src homology 2 (SH2) domains expressed with an LBT and phosphopeptides labeled with chromophores suitable for luminescence energy transfer with the LBT-bound Tb(III). chemically and photochemically active groups. site-specific labeling of proteins. 2009. including sharp emission spectra. Lanthanide Binding Tags Rather than using a synthetic chelator attached to a fluorophore to accomplish protein labeling. No. 109. large Stokes shifts.417 The utility of LBT-tagged proteins has so far been demonstrated in vitro.416 The best sequence to date is FIDTNNDGWIEGDELLLEEG. Tb(III) is particularly important because its emission can be sensitized by native tryptophan or tyrosine chromophores. For luminescent LBTs. Peptides with high affinity and bright emission for Tb(III) were discovered from combinatorial screens of sequences based on known calcium-binding motifs under 20 amino acids in length. 111) was incorporated into defined sites in a DNA binding protein to directly map its binding site on duplex DNA.415 In the proper coordination environment. Vol. Ln ions have very attractive luminescent properties for biological applications.426 Importantly. coli catabolite activator protein (CAP).4954 Chemical Reviews. can be introduced site specifically into proteins.6. Constructs Used for Nonenzymatic Covalent Protein Labeling Haas and Franz remained on the cell surface while reversibly bound probe was easily washed away. Unnatural Metal-Binding Amino Acids Labeling systems that employ metal coordination chemistry to unite a synthetic probe with a genetically encoded peptide tag are applicable to a wide variety of proteins and provide general tools for in vivo. the ability to genetically encode a protein with a nonintrusive and multifunctional tag is attractive for future cellular applications.418 11. which binds Tb with an apparent Kd at pH 7.418 Beyond luminescence applications.423-425 The metal-binding nonnatural amino acid bipyridylalanine (BpyAla. For example.415.414 11.422 Although the challenge of incorporating Ln ions into tagged proteins in a living cell remains.5. In the case of E. the replacement of one lysine in the DNA recognition site did not significantly perturb its interaction with its DNA binding . introduction of the metal binding site involved replacement of only one amino acid in the entire protein sequence.

O-triacetate amino-terminal copper/nickel adenosine triphosphate (1. Abbreviations 4AP ACE ADP APTRA ATCUN ATP BAPTA BC BCS BPS bpy CAP CDK9 CEST cod CORM Cp* CTD Cy3 cyclam DFO DMSO DOTA DPA DTPA ECE EDT EDTA EGTA FAD FMN FRET G4-DNA GABA GSK-3 HDAC HSAB IP3 IDA LBT LFSE MLCT MMP MNIP MR MRI NIR NMR NTA PARACEST PET PIH PPh3 pS pSer py pY Resf RNA ROS RSE SH2 SIH SNARF SNP 4-aminopyridine angiotensis-converting enzyme adenosine diphosphate aminophenol-N. Conclusions As discussed in the previous sections.7. we hope to inspire further research in the area. . As these kinds of reagents become more frequently used in biological studies. which is used as a chelator to alter cellular metal content. and organometallic complexes in cell biology offers diverse opportunities for manipulating biological processes.N′-tetraacetic acid) bathocuproine bathocuproine disulfonate bathophenanthroline disulfonic acid bipyridine catabolite activator protein cyclin-dependent protein kinase 9 chemical exchange saturation transfer 1. like an enzyme inhibitor or catalytic protein inactivator. as well as endogeneous chelating agents that can compete for the metal ion or form unanticipated ternary complexes that might alter their reactivity.8.10-tetraacetic acid dipyridylamine diethylenetriamine pentaacetic acid endothelin-converting enzyme ethanedithiol ethylene diamine tetraacetic acid ethylene glycol tetraacetic acid flavin adenine dinucleotide flavin mononucleotide fluorescent resonance energy transfer guanidine quadruplex DNA γ-amino butyric acid glycogen synthase kinase 3 histone deacetylases hard and soft acid and base inositol triphosphate iminodiacetic acid lanthanide binding tag ligand field stabilization energy metal-to-ligand charge transfer metalloproteinase 4-methoxy-2-(1H-naptho[2. In particular. at what point does the cellular machinery differentiate the cargo from the carrier. functionality. and reactivity of inorganic complexes in this context. In many cases. In describing the principles and trends involved in the design. Small molecule chemical tools that manipulate cellular metal content and distribution are beneficial for understanding metal trafficking pathways. as we better understand these pathways we can develop better strategies for intercepting them. Likewise. recognize phosphorylated proteins and other phosphate-containing biomolecules.2-bis(o-aminophenoxy)ethane-N. There is an obvious synergy between inorganic chemical biology and the biology of metal trafficking.N.4. reducing a metal overload. The breadth of these different biological applications for inorganic complexes is exciting.5-cyclooctadiene carbon monoxide releasing molecule pentamethylcyclopentadienyl C-terminal domain cyanine dye 1.N′. 10 4955 sequence. redox-active metals could be placed almost anywhere in a protein to map protein-RNA or protein-protein interfaces. questions of cross reactivity and specificity are likely to increase.10-tetraazacyclododecane-1. 13.7. 109. this area will benefit greatly from advances in understanding cellular metal trafficking and regulation.Application of Metal Coordination Chemistry Chemical Reviews. a creative approach can extend a seemingly simple motif across several applications. in addition to factors of stability and metabolism.4. The examples that have been highlighted here are only representative of the significant opportunity that metal complexes offer for exploring the chemical biology of living cells. The cellular reactivity of any of these complexes will depend on their distribution and relative concentration with respect to competing metal ions that may induce transmetalation. CAP-K26Bpy-Ala showed site-specific oxidative cleavage at two different sites on the DNA sequence.11-tetraazacyclotetradecane desferrioxamine dimethylsulfoxide 1. but also presents ongoing challenges.N. or bind to specific DNA sequences. or hijacking a native metal into an artificial complex with non-native function. No.4.426 Because introduction of non-natural amino acids requires only simple mutagenesis and because there are numerous potential metal binding non-natural amino acids. coordination compounds. the application of metal chelating agents. At the same time. This basic motif appears in TPEN. and in constructs that use their coordinated metal ion to tag proteins. how do cells handle unnatural metals and metal complexes? For intact metal complexes that are applied to cells or organisms. The dipicolylamine chelating unit is one example. whether it be for overcoming a metal deficiency. All of these complex equilibria. as well as fluorescence and MRI-based Zn(II) sensors. will affect the specificity of the reagent for the intended biological interaction. and how does this influence where metal complexes are distributed in a cell? Finding answers to these questions will be an exciting journey into the frontier of inorganic chemistry and cell biology. 2009. Future development will therefore need to focus on understanding these complex processes. Vol.3-d]imidazole-2-yl)phenol magnetic resonance magnetic resonance imaging near-infrared nuclear magnetic resonance nitrilotriacetic acid paramagnetic chemical exchange saturation transfer positron emission tomography pyridoxal isonicotinoyl hydrazone triphenylphosphine phosphoserine phosphoserine pyridine phosphotyrosine resorufin ribonucleic acid reactive oxygen species Roussin’s red salt esters Src homology 2 salicylaldehyde isonicotinoyl hydrazone seminaphthorhodafluor sodium nitroprusside 12.

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