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Chuma.cas.usf.edu

Structure and Function Department of Chemistry and Institute for Biomolecular Science, University of South Florida, Tampa, Florida 33620-5250 DOI 10.1002/med.10052 Abstract: Although most antibiotics do not need metal ions for their biological activities, there are anumber of antibiotics that require metal ions to function properly, such as bleomycin (BLM),streptonigrin (SN), and bacitracin. The coordinated metal ions in these antibiotics play an importantrole in maintaining proper structure and/or function of these antibiotics. Removal of the metal ionsfrom these antibiotics can cause changes in structure and/or function of these antibiotics. Similar tothe case of ‘‘metalloproteins,'' these antibiotics are dubbed ‘‘metalloantibiotics'' which are the titlesubjects of this review. Metalloantibiotics can interact with several different kinds of biomolecules,including DNA, RNA, proteins, receptors, and lipids, rendering their unique and specificbioactivities. In addition to the microbial-originated metalloantibiotics, many metalloantibioticderivatives and metal complexes of synthetic ligands also show antibacterial, antiviral, and anti-neoplastic activities which are also briefly discussed to provide a broad sense of the term‘‘metalloantibiotics.'' ß 2003 Wiley Periodicals, Inc. Med Res Rev, 23, No. 6, 697–762, 2003 Key words: albomycin; aminoglycosides; anthacycline; antibiotics; aureolic acid; bacitracin;bleomycin; cisplatin; function; gramicidin; ionophore; metalloantibiotics; quinolones; side-rophore; streptonigrin; structure; tetracycline Antibiotics can interact with a variety of biomolecules, which may result in inhibition of thebiochemical or biophysical processes associated with the biomolecules. This can be illustrated in theinteraction of the peptide antibiotic polymyxin with glycolipids which affects membrane function,1 inthe intercalation of the anthracyclines (ACs) into DNA base pairs which stops gene replication,2 in the Contract grant sponsor: American Cancer Society—Florida Division (Edward L. Cole Research grant); ContractGrant number: F94US F-3. Contract grant sponsor: University of South Florida, Research and Creative ScholarshipGrants and PYF Award.
Correspondence to: Li-June Ming, Department of Chemistry and Institute for Biomolecular Science University of South Florida,Tampa, Florida 33620-5250. E-mail: ming@chuma.cas.usf.edu Medicinal Research Reviews, Vol. 23, No. 6, 697 762, 2003ß 2003 Wiley Periodicals, Inc.
imbedding of the lipophilic antibiotic gramicidin3 and the insertion of the amphiphilic antibioticprotein colicin A into cell membrane4 which disturb normal ion transport and trans-membranepotential of cells, in the inhibition of transpeptidase by penicillin which affects cell wall synthesis,5and the inhibition of aminopeptidase by bestatin, amastatin, and puromycin which impairs manysignificant biochemical processes.6 While most antibiotics do not need metal ions for their biologicalactivities, there are several families of antibiotics that require metal ions to function properly. Insome cases, metal ions are bound tightly and are integral parts of the structure and function of theantibiotics. Removal of the metal ions thus results in deactivation and/or change in structure of theseantibiotics, such as bacitracin, bleomycin (BLM), streptonigrin (SN), and albomycin. In other cases,the binding of metal ions to the antibiotic molecules may engender profound chemical andbiochemical consequence, which may not significantly affect the structure of the drugs, such astetracyclines (TCs), ACs, aureolic acids, and quinolones. Similar to the case of ‘‘metalloproteins,''these families of antibiotics are thus dubbed ‘‘metalloantibiotics'' in our studies and are the titlesubjects of this review.
The term ‘‘antibiotic'' was originally coined by Selman A. Waksman and was used in the title of a book of his, Microbial Antagonisms and Antibiotic Substances published in 1945, and was defined as‘‘ . . produced by microorganisms and which possess the property of inhibiting the growth and evenof destroying other microorganisms.''7 However, many clinically useful ‘‘antibiotic drugs'' nowadaysare either synthetic or semi-synthetic, including many b-lactams, (fluoro)quinolones, and amino-glycosides. These (semi-)synthetic drugs and many synthetic metal complexes and organometalliccompounds that exhibit ‘‘antibiotic activities'' can be considered ‘‘synthetic antibiotics'' as thecounterparts of the originally defined ‘‘microbial-originated antibiotics'' from a broad sense of theterm. In this review, we focus on those nature-occurring metalloantibiotics and also briefly discuss afew synthetic metalloantibiotics to provide a broader view of the term ‘‘metalloantibiotics.'' Thestructures and anti-microbial, anti-viral, and/or anti-cancer activities of these natural and syntheticmetalloantibiotics will be discussed to provide further insight into their structure–functionrelationship.
Metal ions play a key role in the actions of synthetic and natural metalloantibiotics, and are involved in specific interactions of these antibiotics with proteins, membranes, nucleic acids, andother biomolecules. For example, the binding of Fe/Co –BLM, Fe/Cu–SN, Mg–quinolone, Mg–quinobenzoxazine, Mg–aureolic acid, and cisplatin with DNA impairs DNA function or results inDNA cleavage (Section 2); the involvement of Mg/Fe in the binding of TCs to the regulatory TetRprotein turns on the mechanism for bacterial resistance to TCs (Section 3); the binding ofmetallobacitracin to undecaisoprenyl pyrophosphate prohibits the recycling of the pyrophosphate tophosphate which in turn inhibits cell wall synthesis (Section 4); and the binding of metal ions toionophores or siderophores allows their transport through cell membrane which can cause disruptionof the potential across the membrane, enables microorganisms to acquire essential iron from theenvironment, or delivers antibiotics to foreign microorganisms (Section 5). The structural andfunctional roles of metal ions in metalloantibiotics have been further advanced in recent years fromextensive biological, biochemical, and physical studies,8 which are discussed herein to provide anoverview of this important and unique group of antibiotics.
DNA can bind many different biomolecules and synthetic compounds, including proteins, antibiotics,polyamines, and synthetic metal complexes and organometallic compounds.9 In the case of the veryspecific protein–DNA interaction, transcription is regulated to turn on or off a specific biologicalprocess. DNA is also a target for therapeutic treatment of disorders and diseases, such as cancers, viadirect ligand binding to it or binding to DNA-regulating biomolecules which in turn imparts DNA function indirectly. Several clinically used anti-cancer antibiotics, such as BLM and the ACs, areDNA-binding (and cleaving) agents. A better understanding of the structure of these antibiotics andtheir DNA complexes, and a better understanding of the relationship of structure, function, andtoxicity of these drugs can provide information for the design of more effective but less toxic drugs fortherapeutic treatments. The investigation of the interaction between DNA and synthetic compoundsor metal complexes can also further our understanding of DNA–ligand binding specificity whichwould provide clues for rational design of DNA-specific drug in the future.10 The structure andfunction of a few natural and synthetic DNA-targeting metalloantibiotics are discussed in this section.
Bleomycin (BLM, also known as Blenoxane) was first isolated as a Cu2þ-containing glyco-oligopeptide antibiotic from the culture medium of Streptomyces verticullus,11 and was later found tobe also an antiviral agent.12 It was soon found to be an anticancer agent and has ever since become oneof the most widely used anticancer drugs,13 most commonly used in treatment of testis cancer,lymphomas, and head and neck cancer, as well as the AIDS-related Kaposi's sarcoma in combinationwith cisplatin and adriamycin. However, it can cause life-threatening side effect, including lungfibrosis. BLM contains a few uncommon amino acids, such as b-aminoalanine, b-hydroxyhistidine,and methylvalerate, two sugars (gulose and mannose), a few potential metal-binding function-alities such as imidazole, pyrimidine, amido, and amino groups, and a peptidyl bithiazole chainconsidered to be the DNA recognition site (Fig. 1). Similar to many other nature products, BLM isproduced as a mixture of several analogues with BLM A2 and B2 being the most abundant.11 BLM isthe most extensively studied metalloantibiotic from several different view points, such as its metalbinding property, structural studies with a variety of spectroscopic methods, mechanistic study of itsoxidative DNA cleavage, investigation of its structure–function relationship, and its use as a non-heme model for investigation of dioxygen activation and DNA recognition/cleavage.14 There are afew BLM-like antibiotics which exhibit similar physical, structural, and biochemical characteristicsas BLM, which have been previously reviewed.15 1. DNA/RNA Binding and Cleavage The antibiotic mechanism of BLM has been proposed on the basis of the results from the better studiedFe and Co derivatives (however, it is the Fe form that is considered the active form in vivo because ofits higher abundance in the biological systems).14 In the presence of reducing agents, the metal ion inFe2þ- or Co2þ-BLM binds dioxygen and converts into an ‘‘activated form'' HOO-MIII-BLM probably Figure 1. Schematic structure of bleomycin (BLM) A2 and B2.The proposed metal-binding ligands based on spectroscopic studiesare in bold-phase.
via an superoxide-MIII-BLM intermediate. DNA cleavage by Fe–BLM is proposed to be carried outby the active O = FeV–BLM or O = FeIV–BLM species generated by O–O bond cleavage in theactivated form14,16 via oxidation at C4 0 and C2 0 –H proton abstraction from the deoxyribose of DNAimmediately following 5 0GC and 5 0GT sequences.14f,17 The damaged deoxyribose then breaks down,and cleavage of DNA strand occurs. The mechanism for DNA cleavage by Co–BLM has beensuggested to follow a similar mechanism via photo-activation.18 Recent studies indicated that thesequence GTAC is a hot spot for double-stranded DNA cleavage by Fe–BLM at site T.19 The sugarmoiety of BLM is important in determining the specificity of the cleavage since Fe–BLM anddeglycosylated Fe–BLM were reported to cleave d(CGCTAGCG)2 at different sites.20 In the presenceof H2O2, Fe3þ–BLM generates hydroxyl  OH free radical in the vicinity of DNAwhich is expected tocause DNA cleavage in vivo.21 More detailed discussion on the mechanism of DNA cleavage14 andassociated cytotoxicity22 can be found in the cited review articles.
Several studies indicate that Fe2þ–BLM can also bind and cleave RNA molecules,23 including tRNA and its precursors and rRNA.24 The cleavage occurs mainly at the junctions between double-stranded and single-stranded regions in RNA molecules,25 such as at C26 and A32 in E. colitRNA His .25d However, not all RNA molecules can be cleaved by Fe2þ–BLM, which include E. coli tRNATyr and tRNACys.25 These studies reveal that RNA cleavage by Fe2þ–BLM shows much higherselectivity as opposed to DNA cleavage that occurs at all 5 0GC and 5 0GT sites. Another significantdifference is that the rate for RNA cleavage is significantly slowed in the presence of Mg2þ andabolished at 0.5 mM (but not in DNA cleavage at even 50 mM25a), which has been attributed tostabilization of RNA structure by this metal ion.25d It is interesting to note that a DNA moleculeanalogous to the T-stem loop of yeast tRNAPhe is cleaved by Fe2þ–BLM at 5 0GT site as in the case ofnormal DNA cleavage, whereas the yeast tRNAPhe loop is cleaved at G53 after the corresponding GUsequence with a rate 16-times slower.25a Fe2þ–BLM can cleave DNA–RNA hybrids as well.23b,26However, the cleavage sites on the RNA strand are different from that of the RNA alone, and isinhibited at slightly higher Mg2þ concentrations equal or greater than 1 mM. In the meantime, theDNA strand in the hybrids is cleaved at all 5 0GT sequences and 5 0GC sequences to a less extent,similar to a regular double-stranded DNA.26b 2. Metal Binding and Coordination Chemistry BLM was originally isolated as a Cu2þ complex which has since been extensively studied.11 It hasalso been known to be an excellent ligand for binding with several different metal ions,27 includingMn2þ,28 Fe2þ /3þ,29 Co2þ /3þ,30 Ni2þ /3þ,31,32 Cu þ /2þ,33,34 Zn2þ,33 Cd2þ,35 Ga3þ,36 and Ru2þ 37 ionsas well as the radioactive 105Rh for use in radiotherapy.38 The d–d transitions of the Cu2þ complexesof BLM and analogues are detected at 600 nm with a molar absorptivity 110 M1 cm1. Theenergy of the d–d absorption is higher than those of many ‘‘type 2'' Cu2þ centers in the range of 650–750 nm, suggesting the presence of a strong ligand-field in a distorted 5- or 6-coordination sphere.39The metal coordination became clear after the structure of a Cu2þ complex of a biosyntheticintermediate of BLM was determined with crystallography.40 This intermediate contains all themetal-binding moieties, but lacks the sugars and the peptidyl bithiazole moiety. In this complex,the Cu2þ is bound to the ligand via imidazole, pyrimidine, the amines of b-aminoalamine, and theamide nitrogen of b-hydroxyhistidine.
The identification of nitrogen-containing ligands in Cu2þ , Co2þ , and Fe3þ–BLM complexes has also been achieved by means of electron spin-echo envelope spectroscopy through the detection of 14Nhyperfine coupling.41 This metal-binding mode has been considered to be conserved in Fe2þ–BLM. Aprevious observation of a perturbation on the ligand field and ligand-to-metal charge transfertransition bands in a few BLM congeners suggested the presence of an axial ligand which might beexchangeable.42 A later nuclear magnetic resonance (NMR) study of the diamagnetic CO adduct ofFe2þ–BLM suggested a similar metal binding site as previously determined, except that the amide group of a-D-mannose was considered to be involved in metal binding.43 Despite the disagreement, astructure of the metal center with five coordinated ligands in a distorted octahedral geometry hasemerged which leaves an open coordination site or an exchangeable site for oxygen binding.
3. Zn2þ and Co2þ /3þ Complexes and Their DNA Binding The diamagnetic Zn2þ–BLM complex of BLM has been utilized as a structural model for theparamagnetic Fe2þ–BLM complex owing to the difficulty in high-resolution NMR studies of theparamagnetic species. Previous 2D-NMR studies of Zn–BLM strongly suggested that the metal isbound to BLM through the secondary amine of b-aminoalanine, the amido-N and imidazole of b-hydroxy histidine, pyrimidine, and the carbamoyl group of mannose.44 This coordination chemistryof Zn–BLM has been suggested to be similar to that of the diamagnetic CO complex of Fe2þ–BLMbased on NMR studies.43 However, this metal coordination has recently been challenged by an NMRstudy of an analogous complex Zn–tallysomycin,45 in which five N-containing donors are suggested,including the primary amines of b-amino-Ala, pyrimidine, and the peptidyl amide and imidazole of b-(OH)His with the pyrimidine at the apex and an SS chirality. This study also excludes the binding ofthe carbamoyl group. Instead, the disaccharide covers the sixth binding site. This disagreement inaxial binding has also been raised in the study of HOO–Co3þ complexes of BLM and analoguesdiscussed below.
BLM forms a complex with Co2þ under anaerobic conditions at pH 6.8, which exhibits a nearly axial electron paramagnetic resonance (EPR) spectrum with g? ¼ 2.272 and g// ¼ 2.025 and shows alarge hyperfine coupling of A// ¼ 92.5 G attributed to the 59Co nucleus of I ¼ 7/2 and threesuperhyperfine-coupled lines of 13 G because of coupling with one 14N (I ¼ 1).46 The sharp EPRfeatures of this complex at 77 K with g 2 and g? > g// reflect the presence of a low-spin Co2þ centerof S ¼ 1/2 with the unpaired electron in the dz2 orbital overlapping with one N-containing ligand,since a high-spin Co2þ center of S ¼ 3/2 can only be observed at liquid He temperatures (andshowing g 2 and 4 features) because of its fast electron relaxation rates.47 The observation of alow-spin Co2þ center also concludes the presence of a strong ligand field as suggested based on theelectronic spectrum of Cu2þ–BLM above. Upon oxygen binding at 77 K, the EPR spectrum isdramatically changed to give g// ¼ 2.098 and g? ¼ 2.007 and a very small hyperfine coupling with 59Co of A// ¼ 20.2 G. The similar g values of 2 and the small coupling with the 59Co center stronglysuggest that the unpaired electron density is not located at the Co2þ center, but very possibly on thebound oxygen, i.e., a ligand-centered EPR spectrum.46a The study of Co2þ binding of the less activedeamido-BLM by means of EPR revealed that the fifth ligand is the amino group of b-amino-Ala.
Upon the introduction of DNA, the spectrum of Co2þ–BLM is not changed whereas the spectrum ofoxy-Co2þ–BLM is noticeably changed to give g// ¼ 2.106, g? ¼ 2.004, and A// ¼ 18.9 G.46 Thisspectral change indicates that the binding of BLM to DNA via the bithiazole rings should affect theorientation of the bound O2 molecule where the unpaired electron resides.
The Co2þ in Co2þ–BLM can form an activated ‘‘green species'' HOO–Co3þ–BLM and an inactive ‘‘brown species'' H2O–Co3þ–BLM upon treatment with peroxide.48 These low-spindiamagnetic Co3þ complexes of BLM and analogues have been extensively studied by means of 2D-NMR spectroscopy.49,50 The coordination chemistry of BLM has further been established from thesestudies (Fig. 2A). The overall structure is similar to that revealed in the crystallographic study of theCu2þ complex of the BLM bio-intermediate,40 despite the lack of consensus regarding the axialligands49,50 (i.e., alanyl-NH2 vs. mannose-CO–NH2 binding).
These Co3þ complexes can bind double-stranded DNA to form DNA2–Co3þ–BLM and DNA2–(HOO)Co3þ–BLM ternary complexes. Several ternary complexes have been investigated bymeans of 2D-NMR techniques, and their structures determined.51,52 A representative structure ofthe ternary complex (CGTACG)2–Co3þ –(OOH)–deglycopepleomycin is shown in Figure 2. Thecoordination chemistry of the Co3þ–deglycopepleomycin complex in the ternary complex is very


Figure 2. Top: The superimposed structures of the activated ‘green species' HOO-Co3þ-deglycopepleomycin (green ball-and-stick structure; Protein Data Bank ID 1A02) and the complex upon binding with d(CGTACG)2 (red stick structure without showingthe oligonucleotide) and (bottom) deglycopepleomycin-Co3þ (OOH)-(CGTACG)2 derived from NMR studies and moleculardynamic calculations (Protein Data Bank ID1A01.pdf).The Co3þ deglycopepleomycin complex is shown in red color, DNA in gray,Co3þ in pink, and the metal-bound peroxide in blue.
similar to that of the DNA-free complex; however, the peptidyl bithiazole tail is pointed away from themetal center (Fig. 2A). The metal coordinated moiety is sitting in the minor groove of DNA duplex,and the bithiazole rings are found to interact with DNA double helix via intercalation which exposesthe bound peroxide to the DNA. This intercalation binding mode was also observed in the binding ofmetal-free BLM to calf thymus DNA by means of low-frequency Raman spectroscopy.53 The bindingof these Co3þ complexes to DNA has brought the terminal oxygen of the Co3þ-bound peroxideclose to the 4 0-H of the scissile ribose (<3 A ˚ ), and has also resulted in several specific perturbations on the DNA structure. For example, in the case of d(CCAGTACTGG)2–(HOO)Co3þ–BLM,51a thebithiazole rings intercalate into the base pairs between T * 5 A and A6 T and the configuration of the T5–A6 riboses and the region C2 through C4 are found to deviate from B-form configuration.
The bithiazole was observed to span in the minor groove in the case of Zn2þ–BLM–DNA,54 different from the intercalation binding mode in the Co3þ–pepleomycin–DNA complex.52 Thisgroove-binding mode was suggested to be probably the initial binding of the metal –BLM complexwith DNA, prior to the more specific binding at 5 0-GC or 5 0-GT sequences (and intercalating into thebase pairs next to the sequences).52 However, intercalation has been suggested not necessarily arequired interaction for DNA cleavage in a study wherein the bithiazole terminus of Fe–BLM istethered to a porous glass bead, which shows similar efficacy in DNA cleavage as free Fe–BLMin solution.55 The importance of the lesion of one strand and the role of the bithiazole in the cleavage mechanism of double-stranded DNA have recently been very elegantly investigated. The binding ofHOO-Co3þ–BLM to a double-stranded DNA with a lesion site was studied and structure determinedwith 2D-NMR.56 This DNA has the sequence d(5 0-CCAAAG6 _ A8CTGGG)*d(5 0-CCCAG-T19ACTTTGG), in which the underlined site has a 30-phosphoglycolate lesion next to 5 0-phosphatomoiety and this ‘‘cleaved strand'' is connected to the other strand of a complementary sequence (with the corresponding lesion site occupied by an A) via two 5 0-3 0 hexaethylene glycol linkers. The metal-binding domain of Co3þ–BLM is located in the minor groove in close proximity of T19 and thebithiazole is most likely to partially intercalate between T19 and A20 according to nuclear Overhausereffect interactions (NOE, which is a function of the molecular rotational correlation time and inter-nuclear distance57). The structural model derived from this study suggests that the metal centerinteracts with G on the second strand at the 5 0 end of the cleaved site on the first strand as well as areorientation of the bithiazole rings upon cleavage of the first strand.
4. Paramagnetic Fe2þ/3þ Complexes The binding of Fe3þ to BLM at slightly alkaline conditions forms a low-spin complex of S ¼ 1/2(g ¼ 2.41, 2.18, and 1.89),58 in which the sixth position is occupied by a hydroxide based onresonance Raman spectroscopy.59 An oxy-form of Fe–BLM is formed by introducing dioxygen toFe2þ–BLM in the absence of reducing agent and DNA. This oxy form has been determined to be asuperoxide O  –Fe3þ–BLM complex based on its 57Fe Mo¨ssbauer spectrum.60 In the presence of a reducing agent, the activated hydroperoxide HOO–Fe–BLM complex is formed which is the activespecies for DNA cleavage.58b A recent theoretical study suggested that both heterolytic and hemolyticcleavage of the O–O bond in the active peroxo complex are not favorable based on energetics andreaction specificity, which concludes a direct attack on DNA by the hydroperoxide of the activecomplex.61 The Fe2þ–BLM complex is paramagnetic (S ¼ 2), which has been studied by means of NMR techniques.62,63 In paramagnetic metal complexes, the NMR signals of nuclei near the metal centercan be hyperfine-shifted outside the ‘‘regular'' spectral range (i.e., 13 ppm for 1H-NMR and200 ppm for 13C-NMR) by the unpaired electron(s) to afford a large spectral window that may reachmore than 100 ppm for 1H-NMR. In the meantime, the nuclear relaxation times are dramaticallyshortened which are proportional to the sixth power of metal-nucleus distances.64 This paramagneticcomplex exhibits many hyperfine-shifted 1H-NMR signals in a spectral window of 230 ppm (Fig. 3),which have been assigned to the protons of the coordinated ligands or the protons near the metal by theuse of 1D- and 2D-NMR techniques.63 A structural model of this metal complex has been built by theuse of the distance-dependent nuclear relaxation times as constraints. This structural model turns outto be similar to the structural models built on the basis of the Co3þ–BLM complexes and their ternarycomplex with the oligonucleotide duplex discussed above. The Fe2þ complex of a BLM congenerpeplomycin and its derivatives have been studied with X-ray absorption spectroscopy which revealsthat an axial ligand may affect the Fe(II) dp ! pyrimidine back-bonding as previously observed42which may stabilize the superoxide intermediate, consistent with the auto-oxidation rate of the metalcenter in the complexes.65 A recent 1H-NMR study of the paramagnetic Co2þ–BLM complex at pH 6.566 corroborates the metal coordination chemistry obtained in the Fe2þ–BLM study.63 A possible involvement ofmannose-amido group as the sixth ligand was proposed. Co2þ–BLM at slightly higher pH of 6.8 waspreviously determined by means of EPR to have a low-spin Co2þ center,46 which would exhibit onlybroad hyperfine-shifted 1H-NMR features.64 The observation of sharp 1NMR features in Co2þ–BLM Figure 3. Hyperfine-shifted1H-NMR spectrum of high-spin Fe2 þ BLMin D2Oat pH meter readingof 6.5.The shifted signals outsidethe regular 0 10 ppm window are because of protons in close proximity of the paramagnetic Fe2þ ion.
reflects the presence of a fast-relaxing high-spin Co2þ center. The NMR and EPR studies suggest apossible presence of a high-spin to low-spin transition that is controlled by pH. However, thishypothesis cannot be verified because of the lack of cross investigations of the complex at lower pHwith EPR at liquid He temperatures and the complex at higher pH with NMR in these studies.
5. Synthetic Analogues and Biosynthesis A number of BLM-analogous compounds have been synthesized that contain the metal bindingmoieties of BLM.67–69 These synthetic analogues form complexes with several metal ions, includingCu2þ and Fe2þ/3þ , and are able to cleave DNA molecules similar to the cleavage pattern by BLMcomplexes. A recent revisit of the Fe–BLM mimicking complex Fe–PMAH67c (PMAH ¼ 2-[N-(aminoethyl)amino)methyl]-4-[N-[2-(4-imidazolyl)ethyl]carbamoyl]-5-bromopyrimidine) showedthat HOO–Fe3þ–PMA (with low-spin features of g ¼ 2.22, 2.17, and 1.94 and a noticeable high-spin feature at g ¼ 4.3) can be formed by reacting Fe3þ–PMA with H2O2, but not with iodosyl-benzene and a base that was previously reported.67c This result is consistent with the observation in anearly study of Fe3þ–BLM.70 Several lipophilic ligands analogous to the metal-binding moiety of BLM have been synthesized that comprise a 4-alkoxypyridine with methylhistamine or methylethylenediamine moiety and a longhydrocarbon chain in the alkoxy moiety for the lipophilicity.71 These ligands bind Cu2þ and formmicelles with critical micelle concentration in the range of 0.9–1.4  104 M. The catalyticproperties of these complexes were not tested in this study. Some pyridine-containing BLM analogueswere synthesized, and investigated with spectroscopic and crystallographic techniques.72 The lmax of650 nm of Cu2þ complexes is significantly longer than that of Cu2þ–BLM11 which indicates aweaker ligand field in the complexes of these analogues, whereas the g values of 2.21–2.22 and 2.04–2.05 of these complexes are close to those of Cu2þ–BLM and its analogues (2.21–2.25 and 2.06)which suggests the presence of a similar axially symmetric magnetic environment of the Cu2þ centerin these complexes.27 BLM is a natural peptide–ketide hybrid (Fig. 1), like the cyanobacterial hepatotoxins such as cylindrospermopsin. The biosyntheses of many peptides and polyketides and their hybrid conjugates(including a number of antibiotics such as BLM, ACs, bacitracin, and some ionophore antibiotics)follow a nonribosomal pathway catalyzed by large clusters of peptide and ketide synthases/synthetases and peptide/ketide ‘‘hybrid'' synthetases, respectively.73,74 The genes of the synthases/synthetases of peptides and polyketides from microorganisms have recently been analyzed andcloned and the enzymes further studied,73–75 including those of BLM and bacitracin (Section 4).
BLM has been verified to be produced by synthetase clusters comprised of polyketide synthase andpeptide synthetase modules.76 These peptide and polyketide synthetases are comprised of a multi-domain modular structure for the catalysis of the initiation of the synthesis via ATP-activatingformation of thioester linkage to the enzyme, elongation mediated by condensation of the thioester-linked amino acid and/or peptide on the peptide carrier domain following a mechanism not yet fullyunderstood, and termination of the peptide or polyketide chain by a thioesterase domain via transfer ofthe final product to a serine in the thioesterase followed by hydrolysis.77 The reactant amino acids orcarboxylates are specifically recognized and covalently linked to the different domains beforetransferred to an intermediate peptide or polyketide chain. Changing of the stereochemistry is carriedout by epimerization domains in the enzyme complex. The studies of several peptide and polyketidesynthetases and their hybrids, including crystallographic studies of the adenylation domain and anNMR study of a peptidyl carrier domain,78 have greatly enhanced our understanding of the structureand mechanism of this superfamily of ‘‘mega enzymes.'' Since these synthetase complexes possessenzymatic activities toward the syntheses of secondary metabolites,75 thus are potential targets fordrug discovery in the production of potential bio-active peptides and polyketide as well as theirhybrids.79 B. Aureolic Acids The glyco-antibiotic aureolic acid family produced by Streptomyces species is comprised of severalmembers with similar structures, including chromomycin A3 (ChrA3), mithramycin (Mit, producedby S. plicatus and also known as plicamycin), olivomycin, and variamycin, which exhibit activitiestoward Gram-positive bacteria, DNA viruses, and tumors.80 However, high toxicity has limited theiruse as clinical antibiotics and anti-tumor agents. Mithramycin has been tested against severalmalignant diseases since its discovery,81 and has a limited use for the treatment of the Paget'sdisease82 and for the treatment of hypercalcemia83 (however, controversies have also raised84). BothChrA3 and Mit have recently been found to be potent inhibitors of neuronal apoptosis induced byoxidative stress and DNA damage in cortical neurons.85 Thus, these antibiotics may be effectiveagents for the treatment of apoptosis-associated neurological diseases, which suggests that sequence-selective DNA-binding drugs may serve as potential neurological therapeutics.
1. Structure of Aureolic Acids These antibiotics contain a metal-binding b-ketophenol chromophore, a highly functionalizedaliphatic side chain, and a disaccharide and a trisaccharide chains important for DNA binding andinhibition of DNA transcription (Fig. 4). The identity of the sugar chains and the sequence of the sugarlinkage of this drug family were first established by partial hydrolysis of olivomycin A86 and Mit.87The structure of ChrA3 has later been further studied by means of 1H- and 13C-NMR spectroscopy.88The structure of Mit has also been studied by the use of synthetic and NMR techniques,89 and hasrecently been fully determined by means of 2D-1H and 13C homonuclear and heteronuclear methodswhich is shown in Figure 4.90 The structures of the DNA complexes of the drugs have also beeninvestigated by the use of 2D-NMR techniques in recent years, which are discussed in a later section.
2. Role of Metal Ions in the Action of Aureolic Acids A divalent metal ion, such as Mg2þ, Co2þ, Zn2þ, or Mn2þ, is required for aureolic acid to bind to adouble helical DNA to form a drug2–metal–(DNA)2 ternary complex.91–93 The metal–drug2complex in the ternary complex is bound to DNA in the minor groove with a high preference to GCsites and a length of approximately six base pairs based on 1H-NMR,94 DNA footprinting,95 andbiochemical96 studies. Consequently, these antibiotics can inhibit transcription of the genes that haveG-C-rich promoter sequences,96 such as the c-myc proto-oncogene97 (this binding might be non-specific98) that regulate cell proliferation and also controls the expression of b-galactosidase.97,99These studies led to further investigations of the interaction between this antibiotic family and doublehelical DNA of different sizes and sequences.100 In addition to the above metal ions, Mit has been Figure 4. A schematic structure of mithramycin. The metal binding b-ketophenol moiety is shown with thick lines. All drugs in theaureolic acid family have the similar metal binding moiety, but vary in the sugar chains which have been suggested to cause differentinteractions with DNA.
determined to bind several other metal ions, including Ca2þ, Cd2þ, Tb3þ, Gd3þ, and alkali metals.101Metal:drug4 complexes are suggested to form for Ca2þ, Tb3þ, and Gd3þ; however, which does notassist the binding of the drug molecules to DNA.
Some early studies of Mg2þ binding of Mit showed that two different complexes can be formed which exhibit different absorption and CD spectra, in which the better known 1:2 Mg2þ–Mit2complex formed at low Mg2þ concentrations and a 1:1 Mg2þ–Mit complex formed at higher Mg2þconcentrations.93 Interactions of these two complexes with bulk DNA, polynucleotide, and anoctanucleotide are observed to be different. For example, while the 1:1 complex interact with theB-DNA-representing poly(GC)  poly(CG) and the A-DNA-representing poly-G  poly-C in a similarfashion, the 1:2 complex shows distinct interaction patterns with the two DNA forms. Since cellularMg2þ concentration varies significantly in neoplastic tissues,102 these two complexes can be expectedto form to certain extents and are considered important for in vivo action of the drug.93e 3. Role of Sugars The importance of the sugar moiety in antibiotic activity of this family has been established in earlystudy of the different congeners and derivatives of this antibiotic family.103 Removal of the sugarmoiety E (Fig. 4) from olivomycin A (affording olivomycin D) and ChrA3 (affording ChrA4) results insignificant loss of antibiotic activity. Moreover, the derivatives with only one sugar and the aglycones(without any sugar) are inactive. The involvement of the sugar chains in stabilization of the 1:2complexes M2þ–(ChrA3)2 (M ¼ Mg and Ni) in methanol solution has been suggested.104 On thecontrary, the deglycosylated chromomycinone forms 1:1 complexes with these two metal ions. Inaddition, Ca2þ was determined to form only a 1:1 complex with ChrA3 in methanol as opposed to aprevious observation in aqueous solution.101 The metal complexes of ChrA3 and Mit are found to interact differently with A- and B- representing DNA sequences. Since these two congeners differ in the sugar moieties (the sugars ofChrA3 are acetylated), this observation indicates the significance of the sugar chains in the interactionof these antibiotics with DNA.93 The difference between ChrA3 and Mit has also been shown in theirbinding with the oligonucleotide d(ACCGGGT)2, wherein ChrA3 forms a drug2–Mg2þ–(DNA)2ternary complex whereas Mit has been proposed to afford a (drug2–Mg2þ )2–(DNA)2 ternarycomplex based on their NMR spectra.105 This difference has also been attributed to the differencein the sugar chains in these two congeners. The role of the sugar chains has further been investigat-ed with a simple synthetic analogue, in which a simple triethylene glycol chain is attached to ab-ketophenolate aromatic ring structure as the aglycone of ChrA3.106 This simple model forms M2þ–ligand2 complexes (M ¼ Co and Mg) similar to ChrA3. Preliminary study by these authors shows thatthis complex can bind DNA. The above studies further corroborate the significance of the sugar chainsin metal binding and in the binding of this drug family to double-stranded DNA.
Total synthesis of aureolic acid has been attempted,107 wherein stereoselective syntheses of aryl 2-deoxy-b-glycosides and the A-B disaccharide of olivomycin have been achieved. Since manyantibiotics are found to be glycosylated, such as BLM, aureolic acid, aminoglycoside, and ACfamilies discussed in this review, further exploration of glycosylated metal complexes and their DNAbinding properties should be encouraged.
4. (Aureolic Acid)2–Mg2þ–(DNA)2 Ternary Complexes The requirement of divalent metal ions for the binding of aureolic acid to DNA has been fullyestablished by means of 1H- and 31P-NMR techniques, in which the spectral features of DNA arechanged upon the binding of the drug in the presence of Mg2þ.94 Several palindromicoligonucleotides have been used for NMR studies, wherein the addition of 1:2 Mg2þ–(Mit)2 toDNA afford ternary complexes whose 1H- and 31P-NMR spectra are completely different from thoseof the parent DNA molecules. The interactions between the drug and DNA and between the two bound


Figure 5. Stereo view of the structure of the ternary complex Mit2 Mg2þ (TCGCGA)2 obtained with 2D-NMR techniques (ProteinData Bank ID 146D.pdb). The complex has a 2-fold symmetry with the two drug molecules residing in the minor groove of the DNAduplex.
drug molecules in the drug2–Mg2þ–(DNA)2 ternary complexes have been revealed with 2D-NMRtechniques, from which the structures have been built as illustrated in Figure 5.
When a DNA sequence contains two GC sites separated by a few base pairs, such as the decanucleotide (TAGCTAGCTA)2, binding of two equivalents of (drug)2–Mg2þ complex to theDNA becomes possible.108 The introduction of two Mit drug molecules and one Mg2þ to thisdecanucleotide forms a complex with half of the DNA molecule bound with the drug complex, i.e., the(TAGCTA . . )2 moiety on one end of the helix is bound with the drug complex whereas the samemoiety on the other end is not. This binding mode breaks the symmetry of the palindromic DNAsequence, which results in doubling the number of NMR signals. Upon the addition of anotherequivalent of Mit2–Mg2þ recovers the palindromic symmetry of the complex. The structure of thisunique ternary complex (Mit2–Mg2þ)2–(TAGCTAGCTA)2 has been determined by the use of 2D-NMR techniques and molecular dynamic calculations, which can be retrieved from the Protein DataBank (PDB ID 207D.pdb). The above studies laid a good foundation for future studies of aureolic acidbinding to DNA of different lengths and sequences.
5. Paramagnetic (Aureolic Acid)2–Co2þ–(DNA)2 Terminal Complexes The diamagnetic Mg2þ can be replaced with a paramagnetic Co2þ for the binding of aureolic acid toDNA duplex to afford a ternary drug2–Co2þ–(DNA)2 complex.91a Because of the paramagnetism ofCo2þ, protons near the metal center are hyperfine-shifted64 to afford a 1H-NMR spectrum with a widespectral window of 100 ppm as represented in Figure 6 for the complex Mit2–Co2þ–(ATGCAT)2.91a There are more than 50 signals well resolved in the large spectral window, Figure 6. Hyperfine-shifted 1H-NMR spectrum (360 MHz) of the ternary complex Mit2 Co2þ (ATGCAT)2 at pD 8.0 obtained at40C.
representing a rare ‘‘high resolution'' 1H-NMR spectrum of a paramagnetic species. The good signalresolution allows further extensive study of this complex. The ternary complexes (ChrA3)2–Co2þ–(TTGGCCAA)2 and other complexes of longer oligonucleotides109 allow nuclear Overhauser effect(NOE)57 to be clearly detected for better signal assignment. However, information about through-bond nuclear interaction cannot be obtained because of the large signal widths of the hyperfine-shiftedsignals attributable to large molecular size of the ternary complex. Nevertheless, combining thedistance constrains derived from nuclear relaxation times and the geometry-related dipolar shift,64 astructure of the ternary complex has been constructed (Protein Data Bank ID 1EKH.pdb and1EKI.pdb)109 which is similar to the structures derived from the previous 2D-NMR studies of thediamagnetic Mg2þ complexes of this antibiotic family discussed above.
Streptonigrin (SN, also known as rufochromomycin and bruneomycin) is a metal-binding quinone-containing antibiotic produced by Streptomyces flocculus110 (Fig. 7). This antibiotic has been shownto inhibit several tumors and cancers (e.g., lymphoma, melanoma, and breast and cervix cancers) aswell as viruses in some early in vitro and clinical observations.111,112 While SN is active towardmammalian cells at the chromosome level, it is found to be much less effective against insect celllines.113 A recent study shows that SN also exhibits ionizing radiation-like damage toward Ataxistelangiectasia heterozygote cells.114 Despite the potency of SN, high toxicity and serious side effectsof this antibiotic have reduced its clinical value, and limit its use only as an experimental anti-tumoragent.111,112 Nevertheless, because of its anti-tumor potency and unique structure, SN has served as alead drug molecule for chemical modification and synthesis of new compounds to correlate thestructure features with the biological activity and toxicity of this potent antibiotic.115 1. Action of Metallo-SN SN is known to bind different transition metal ions to function properly.116,117 The interaction ofmetal –SN complexes with DNA has been proposed on the basis of some optical studies.118 A redoxactive metal ion such as Fe and Cu is required for this antibiotic to exhibit full antibiotic and anti-tumor activities.119,120 The redox-active Fe and Cu complexes have been shown to accelerate SN-mediated DNA scission in the presence of NADH, thus enhance the anti-tumor activity of thisantibiotic.121–123 These results indicate that metal ions are possibly directly involved in the action ofSN. However, the metal binding mode and structure of these metal complexes could not be definitelydetermined in these studies. Particularly, two different configurations of the drugs are possible formetal binding (Fig. 7) with the metal bound through either the quinolinequinone-aminefunctionalities based on the crystal structure124 or the quinolinequinone-picolinate functionalitiesthat requires a significant twist of the crystal structure.
Figure 7. Schematic structures of streptonigrin (SN). The structure A is metal-free drug determined by means of crystallography,whereas the structure B represents the configuration upon metal binding as determined by means of NMR relaxation.The formationof structure B requires a dramatic twist of the C2 C2 0 bond in structure A.
Since SN contains a quinone moiety, it may share some common mechanistic characteristics with other quinone-containing antibiotics125 such as the ACs (discussed in Section 2.D ‘‘Anthracyclines'')in terms of in vitro and in vivo DNA and RNA cleavage and inhibition of cancer growth via inter-ference with cell respiration and disruption of cell replication and transcriptional control.116,119,126The metal –SN complexes can be reduced to their semiquinone forms by NADH, which then caninduce cleavage of DNA. This process is inhibited by superoxide dismutase and catalase, indicatingthe involvement of superoxide and peroxide.119,121 Reduction of this antibiotic in the presence of abound metal ion is also confirmed by the detection of EPR signals attributable to the reducedsemiquinone form.127 Metal chelators and an antioxidant are found to prevent SN-induced DNAdamage and cytotoxicity,128 which supports the involvement of metal ions in the action of SN.
2. Metal Complexes of SN Zn2þ binds SN to afford a few different complexes with different metal binding modes at varioustemperatures, in which a 1:1 metal –drug complex is the predominant complex.129 A recent study ofthe crystal structure of a Zn2þ complex that mimics the metal-binding moiety of SN showed thebinding of the metal to the quinolinequinone-picolinate functionalities,130 corroborating the struc-tures of several paramagnetic metal complexes of the drug determined by means of NMR techniquesdiscussed below. The interaction of Zn2þ–SN with DNA and oligonucleotides has been investigatedwith 1H- and 31P-NMR spectroscopy. This study concluded the requirement of metal ion for SNbinding to DNA131 and revealed sequence preference in DNA binding of this antibiotic, in which thebinding of Zn2þ–SN to d(GCATGC)2 shows noticeable spectral changes whereas the complex doesnot affect the spectra of d(ATGCAT)2.
SN can bind several different paramagnetic metal ions, including Co2þ, Fe2þ, and Yb3þ ions, with large formation constants to form 1:1 metal –SN complexes.132 The paramagnetic Fe2þ, Co2þ,and Yb3þ complexes of SN have been studied with 1H-NMR spectroscopy and relaxation, and theirstructures have been determined.132 The study of Fe2þ–SN complex is particularly important since itis considered an active form of this drug that exhibits enhanced activity toward DNA destruction bothin vitro and in vivo.122 The hyperfine-shifted 1H-NMR signals of these paramagnetic complexes havebeen fully assigned. The proton-metal distances derived from the relaxation times of the hyperfine-shifted signals in these complexes match those of the complex with the metal located at thequinolinequinone-picolinate site (structure B, Fig. 7), but not the quinolinequinone-amine site basedon the crystal structure (structure A). This configuration requires a significant twist of the C2–C2 0bond by 180 in the crystal structure124 of the drug.
The introduction of poly[dA-dT] to reduced Cuþ–SN complex causes some small changes in chemical shift of the 1H-NMR signals of the complex (0.22–0.31 ppm), which was suggested to beattributed to the binding of this complex to the DNA duplex.123 The hyperfine-shifted 1H-NMR signalsof Co2þ–SN complex are found to be significantly changed upon addition of calf thymus DNA orpoly[dA-dT] (the chemical shifts of two hyperfine-shifted signals are shifted by 20–40 ppm),132which are also indicative of direct binding of the complex with DNA. Along with the DNA bindingstudy of Zn2þ–SN complex, these studies indicate the significance of metal ions in the action of thisantibiotic.
D. Anthracyclines Anthracycline (AC) antibiotics133 are produced by Streptomyces species. Soon after their discovery,they were found to exhibit a wide spectrum of antineoplastic activity toward both solid andhematologic tumors and cancers.134 In addition, an AC antibiotic has recently be found to exhibitantifungal activity.135 Despite their severe cardiotoxicity136 (e.g., cardiomyopathy) and otherside effects,137 these antibiotics have been widely used as dose-limited chemotherapeutic agents forthe treatment of human cancers such as acute leukemia. The side effects have been attributed to the toxicity of these drugs toward mitochondria,138 leading to disturbance of bioenergetics, inhibitions ofenzymes, oxidation of lipids, disorders of membrane, and oxidative stress. The less toxic adriamycin(doxorubicin) has currently been widely prescribed as a chemotherapeutic agent in association withother antineoplastic agents, such as BLM and cisplatin. In addition, new AC antibiotics and theirchemical derivatives are still found or synthesized,139,140 which may provide potential clinical use inthe future.
The antineoplastic activity of AC antibiotics has been mainly attributed to their strong interactions with DNA in the target cells. The AC family members possess a quinone-containingchromophore and an aminoglycoside side chain.133 The structures of the representing members of thisfamily daunomycin (daunorubicin) and adriamycin are shown in Figure 8.141 There are a fewmembers of the AC family that contain more extensive sugar chains, such as b-rhodomycin contains amonosaccharide and a trisaccharide, cinerubins, marcellomycin, and rhodirubins have a trisac-charide, and musettamycin has a disaccharide chain.133 The redox activity of the AC ring plays a keyrole in the action of these drugs. In addition, the metal ion bound to the 11,12-b-ketophenolate site isalso thought to be involved in some actions of these antibiotics.
1. Action of AC and Metal –AC Complexes The action of this drug family has been considered to be attributable to their redox activity and DNA-binding capability.133,142,143 Two pathways have been proposed for these drugs to deform DNAstructure and terminate biological function of DNA:2 (a) intercalation of the drugs into the base pairswith the sugar chain sitting in the DNA minor grooves which involves hydrogen bonding,electrostatic, van der Waals, and hydrophobic interactions (a representing AC–DNA structure isshown in Fig. 9); and (b) a free radical damage of the ribose. The intercalation of AC drugs to DNAdramatically distort the DNA structure which thus prohibits transcription. A number of crystal144 andNMR145 structures of different AC–DNA complexes have been resolved. A representing AC–DNAstructure nogalamycin2-d(TGTACA)2 is shown in Figure 9.144h These structural studies allowdetailed comparison of sequence specificity of the drug binding and the different modes for thebinding of different drugs with DNA.
These antibiotics can be reduced to their semiquinone forms by biological reducing agents, such as NADH and NADPH. Superoxide anion radical (O) and H 2O2 can be produced from dioxygen upon receiving electrons from the semiquinone. Then, hydroxyl radicals can be generated, which canattack cell components, such as membrane and DNA, and impair cell functioning. In the presence ofascorbic acid and H2O2, hydroxyl radicals can also be generated by Cu2þ and Fe3þ–adriamycin.146The radicals generated during the redox cycle of ACs and their Fe complexes have been considered thecause of the cardiotoxicity.147 However, a recent study showed that the capability of producing freeradicals of ACs is not directly related to their cardiotoxicity. For example, although the 13-hydroxyderivatives of ACs are more cardiotoxic, they are less effective producers of oxygen-based freeradicals.148 The 13-hydroxy metabolites of ACs have been found to impair intracellular ironhomeostasis, which provides new perspectives on the role of iron in cardiotoxicity of ACs.149 Figure 8. Schematic structures of daunomycin (R ¼ H) and adriamycin (R ¼ OH).


Figure 9. Crystal structure of nogalamycin2 d(TGTACA)2 complex in which the anthracycline (AC) rings are intercalated into DNAbase pairs.The complex is packed in the crystal lattice as a dimer with the monomers nearly perpendicular to each other.The DNAduplex is shown in ribbon structure in one of the subunits.
ACs are known to bind various metal ions, including transition metal, main group, lanthanides, and uranyl ions.150,151 A number of articles reported that some metal ions, e.g., Fe2þ /3þ, Cu þ /2þ,Pd2þ, Pt2þ, and Tb3þ, play an important role in altering the biochemical properties of ACs.152–155 Thesestudies point a new direction in the pursuit of chemotherapeutic efficacy and lowering toxicity of theseantibiotics. The binding of metal ions may cause a significant influence on the redox property of thesedrugs as shown in their Yb3þ complexes,156 thus affecting their activities. The interactions of DNAand other cell components with metal –AC complexes, and their subsequent damage by the ACcomplexes of redox-active metal ions, including iron and copper,157,158 have been previously studiedby the use of various physical and biochemical methods. Adriamycin has been suggested not toundergo flavo-associated reduction upon intercalation.159 However, a site-specific modification ofDNA bases suggests a possible binding through intercalation,157b although specific electrostaticinteractions cannot be completely ruled out. Pulse radiolysis studies indicate that adriamycinsemiquinone can mediate a long-range electron transfer to as far as 100 base pairs in DNA,160 whichmay also serve as a mechanism toward DNA base modification.
2. Fe–AC Complexes It has been shown that several different metal ions, including alkaline earth metals,161 first-row161 andheavy154 transition metals, and rare earth metals,155,161,162 can bind AC antibiotics very tightly inaqueous and methanol solutions, with the metal bound to one or both of the two b-ketophenolatemoieties depending on the solution conditions. Iron is involved in the actions of several antibiotics,such as BLM discussed in Section 2.A ‘‘Bleomycin'', SN in Section 2.C ‘‘Streptonigrin'', andpossibly ACs,153 which serves as a redox center and can generate free radicals in the presence ofdioxygen under reduction conditions which can damage cell components. The binding of Fe3þ withdaunomycin has been studied by the use of 57Fe Mo¨ssbauer, EPR, and X-ray absorptionspectroscopies, in which several different complexes are seen at mM drug concentrations.153a,163Despite the similar structures of daunomycin and adriamycin, the Mo¨ssbauer spectra of their Fe3þcomplexes are noticeably different which has been attributed to their slight difference in structure andreactivity.164 The binding of Fe3þ with several other ACs has recently been revisited.165 The results suggest that Fe3þ binds these drugs to form 1:1 Fe–drug complexes with the metal bound at 11,12-b-ketophenolate site, and 2:1 Fe2–drug complexes with the metal bound at both b-ketophenolate sites.
The formation of mononuclear, dinuclear, and polynuclear metal –AC complexes are also suggested.
The Fe3þ complexes of these drugs are very complicated systems since their spectra are dependentupon the preparation procedure, equilibrium time, metal-to-drug ratio, and drug concentration.163–165 Different complexes are also formed for lanthanide(III) binding with AC antibiotics observed in anearly study, which is discussed in the next section.
A 1:2 Fe3þ–adriamycin complex was proposed to form a stable complex with calf-thymus DNA in solution. This drug –Fe–DNA tertiary complex is distinct from both the Fe3þ–adriamycin complexand the DNA-intercalated Fe3þ-free adriamycin on the basis of optical and chromatographicstudies.157b In another study, Fe3þ–ACs have been suggested not to intercalate into DNA basepairs until the Fe3þ ion is released, despite the strong binding of Fe3þ with the drugs.157c A recentmutagenesis study indicated that Fe3þ is directly involved in the mutagenicity caused by doxorubicinthrough oxidative DNA damage, which further strengthens the role of Fe in AC action.166 Formationof intracellular Fe–AC complexes have also been confirmed with different methods.167 Furtherstudies are still needed to clarify the mechanistic and structural roles of Fe in the action of this familyof antibiotics.
3. Lanthanide–AC Complexes Lanthanide(III) ions (Ln3þ) have been very widely utilized as substitutes and spectroscopic probes168for biological Ca2þ owing to their very similar ionic radii, binding properties, and coordinationchemistry, yet with much higher affinity constants because of the higher charges of Ln3þ ions (thus isable to probe weak Ca2þ interactions).169 Indeed, both Ln3þ and Ca2þ ions have been reported to bindACs, in which Ln3þ ions show > 3 orders higher in affinity constants.156 Early NMR studies of theparamagnetic Yb3þ–daunomycin complex did not yield useful information for the description of thecoordination chemistry of the complex because of the formation of a mixture and the lack of fullassignment of the paramagnetically shifted 1H-NMR features.170 The binding of several Ln3þ ions,including Pr3þ, Eu3þ, Dy3þ, and Yb3þ, with ACs in both aqueous and methanol solutions underdifferent conditions has recently been revisited by means of electronic spectroscopy, cyclicvoltammetry, and NMR techniques.156,171 Like in the case of Fe3þ-bidning to AC drugs, different complexes are also formed for lanthanide binding with ACs. A 1:1 Yb3þ–daunomycin complex has been successfully prepared in solution,and its hyperfine-shifted 1H-NMR spectrum fully assigned by means of 2D-NMR techniques(Fig. 10).156,171 On the basis of the conclusive signal assignment, the configuration of the complex insolution has been determined to be similar to that of the metal-free drug in solution172 and in thecrystal structure,173 and the metal binding site determined to be the 10,11-b-ketophenolate moiety.
The AC drugs can bind Ln3þ to form complexes in solution with metal-to-drug ratios of 1:1, 1:2, 1:3,and 2:1 depending upon proton activity in the solution.156 All of the complexes have beencharacterized by means of 2D-NMR techniques.156 The complication in the earlier NMR studies hasbeen attributed to the formation of the different complexes at different proton activities, which islikely to be the case for other metal complexes of the ACs.
4. Interactions of ACs and Their Metal Complexes With Other Biomolecules In addition to their DNA intercalation and redox activity, AC antibiotics have been observed tointeract with other biomolecules that may also influence cell functioning and may be the cause of theside effects of these drugs. For example, (a) adriamycin and its Fe3þ, Cu2þ, and Co2þ complexescan cause influence on effector cells of humoral and cell immune response.174 (b) Fe–adriamycincomplex was found to damage erythrocyte ghost membranes, which is attributable to the productionof superoxide and hydrogen peroxide by the complex.157a (c) The Fe2þ, Cu2þ, and Co2þ complexes ofadriamycin are potent inhibitors of propanolol-induced Ca2þ-dependent Kþ efflux, but not Pb2þ-dependent Kþ efflux, whereas Fe3þ–adriamycin can activate Kþ permeability of erythrocytes.
However, the AC rings of adriamycin alone enhances Ca2þ-dependent Kþ efflux from erythrocytes.
These influences are attributed to the influence on cellular Ca2þ transport rather than direct action onKþ channels.175 (d) AC drugs can cause poor wound healing176 as a result of impaired biosynthesis of Figure 10. The 1H-EXSY-NMR spectrum of 1:1 Yb3þ daunomycin complex in methanol. The signals due to the metal complex(top trace) can correlate with those of the free drug (trace onthe left) inthis spectrum, shown as cross peaks inthe‘ 2D map' (labeledwith numbers corresponding to the structure in Figure 8).The spectrum obtained in aqueous solution exhibits similar features as inmethanol.
collagen.177 The inhibition of AC drugs against the Mn2þ-containing prolidase has been observed tobe parallel to the impairment of collagen synthesis.178 The binding of the AC drugs to the Mn2þ in theactive site of prolidase has been suggested to be the cause of the inhibition. Moreover, the higherMn2þ-binding affinity of daunomycin than that of adriamycin has been considered to contribute toits greater potency in inhibition of collagen biosynthesis. (e) The Fe3þ–adriamycin complex isdetermined to be a potent inhibitor of protein kinase C,179 and the Cu2þ–AC complexes are con-sidered to serve as a vehicle to carry Cu2þ to protein kinase C which results in inhibition of theenzyme.180 Direct binding of the complexes with the enzyme has been ruled out. These studiessuggest that the interactions of AC drugs with different bio-targets must be taken into considerationfor further drug design and future studies of the bioactivity and toxicity of these drug family.
E. Aminoglycosides Aminoglycosides form a unique and structurally diverse family of antibiotics (Fig. 11), which includethe famous Waksman's streptomycin and the widely used neomycin (an ingredient in ‘‘tripleantibiotic'' ointment along with bacitracin and polymyxin B). Despite their nephrotoxicity andototoxicity, these antibiotics have remained their clinical values and also serve as lead drugs forrational design of next-generation antibiotics.181 1. RNA-Binding and Aminoglycoside Action Aminoglycoside antibiotics are known to bind RNA which is considered the key mechanism in theirantibiotic activities.181–183 This binding decreases translational accuracy and interferes withtranslocation of the ribosome.184 For example, neomycin-like aminoglycosides bind rRNA near theaminoacyl site, preventing chemical modification on the nucleotides in the aminoacyl site.185Neomycin B has been determined to bind to the transactivation-responsive element of HIV-1 RNA.186Neomycin has also been determined to inhibit the self-cleavage of the ribozyme from human hepatitisd virus by direct replacement of the active divalent metal ions.187 Moreover, aminoglycosides areknown to bind and cleave hairpin ribozyme in the absence of Mg2þ, however, with much smaller rate Figure 11. Schematic structures of aminoglycosides (A) neomycin B (R ¼ NH2) and paromomycin (R ¼ OH), (B) gentamicin C1(R1 ¼ R2 ¼ CH3), gentamicin C2 (R1 ¼ CH3; R2 ¼ H), and gentamicin C1A (R1 ¼ R2 ¼ H), and (C) kanamycin A (R1 ¼ OH;R2 ¼ OH), kanamycin B (R1 ¼ OH; R2 ¼ NH2), and tobramycin (R1 ¼ H; R2 ¼ OH).
constants kcat in most cases except neomycin and apramycin of 18 and 13 times smaller, respectively,than that of Mg2þ-catalyzed cleavage.188 The interaction of aminoglycosides with RNA has been investigated by the use of small RNA nucleotides that contain the drug recognition site.189 The solution structure of a 27-mer RNAmolecule, and the structures of this RNA nucleotide bound with paromomycin and gentamicin havealso been determined with NMR spectroscopy.190 The structures of an E. coli decoding region A-siteoligonucleotide with and without a bound paromomycin have also been resolved by means ofhomonuclear and heteronuclear NMR techniques, wherein the two structures are found similar exceptat the antibiotic binding region.190,191 Crystal structures of ribosomal 30S RNA subunit192 and itscomplexes with paromomycin, streptomycin, and spectinomycin have been resolved (Fig. 12, Top).193These structures have provided structural details about the conserved A1492 and A1493 region aswell as detailed interactions of aminoglycoside antibiotics with RNA (Fig. 12, Bottom) which affordstructural basis for the understanding of the action of aminoglycosides. Another crystal structure ofRNA –aminoglycoside complex has also been recently determined, wherein two ribosomal decodingA-sites are bound with two paromomycin molecules.194 In both solution and crystal structures, therings A and B of the antibiotics (cf. Fig. 11) are found to be involved in specific interactions with RNAvia H-bonding with G and A nucleotides, whereas rings C and D in paromomycin and neomycincontribute to the drug binding affinity to RNA. Consequently, methylation of G or A nucleotide canlead to different bacterial resistances to this family of drugs.195 Nevertheless, different drugs are foundto bind at different locations in 30S RNA, which could be metal-dependent (cf. Section 3 for TCbinding to RNA).
2. Metal Binding and Bioactivities Metal ions have been determined to be involved in some unique activities of aminoglycosides. Thebinding of iron to gentamicin (Fig. 11) has been postulated to induce free radical formation which


Figure 12. Crystal structure of antibiotic-bound 30S rRNA complex (top structure, Protein Data Bank ID 1FJG.pdb) and the detailsabout the paromomycin-binding environment (bottom).The RNA molecule is shown in green, proteins in gray, and the aminoglyco-side antibiotics paromomycin, streptomycin, and spectinomycin are shown in purple, blue, and red colors, respectively in the topstructure.There are 96 Mg2þ ions and 2 Zn2þ ions (bound to the peptide chain) found in this structure (not shown in the figure); how-ever, the metal ions are not involved in the binding of the antibiotics.
causes peroxidation of lipids.196 The Fe2þ/3þ complexes of gentamicin have recently investigatedwith NMR, in which a low-spin 2:1 drug-to-Fe2þ complex as well as a 1:1 and a 2:1 drug-to-Fe3þcomplexes have been proposed to form.197 These redox-active iron complexes were implied foraminoglycoside toxicity.
The macrolide antibiotic erythromycin has a structure different from the streptomycin-like antibiotics, yet it contains two sugar moieties (one being a t-aminosugar), carbonyl, and hydroxylgroups which potentially can serve as metal binding ligands. An erythromycin–iron complex wasobserved to exhibit superoxide scavenging activity that was not seen for the antibiotic without themetal.198 However, the physical and structural properties of the metal binding site and the structure ofthe complex were not determined in the study.
Several other aminoglycoside antibiotics have been determined to bind Cu2þ, including lincomycin,199 kasugamycin,200 kanamycin B,201 tobramycin,202 genticin,203 and the semi-syntheticamikacin204 (Fig. 11). In addition, a few simple amino sugars have also been reported to bind Cu2þ,which serve as simple model systems for metal-binding of aminoglycoside antibiotics.205 In all thecases, the binding of Cu2þ to the aminoglycosides are highly pH-dependent, and afford multi-speciesaround neutral pH based on the results from potentiometric and EPR studies. The Cu2þ–amino-glycoside complexes are observed to exhibit oxidative activity, which can catalyze oxidationof nucleotides in the presence of H2O2.199–201,204 Hydrolytic cleavage of DNA206 and RNAmolecules207 and the RNA of the HIV-1 viral Rev response element207 under physiological conditionsby Cu2þ–aminoglycoside complexes was also observed. The metal ion in these complexes has been proposed to bind to the drugs through a chelating vicinal aminohydroxyl binding moiety of the drugs.
The binding site of Cu2þ in kanamycin A has been determined to be the 3 0 –NH2 and 40–OH groups ofring C (Fig. 11C) by means of 13C-NMR relaxation and potentiometric measurements.201,206 Quinolones are comprised of a large family of antibacterial agents such as nalidixic acid, pefloxacin,norfloxacin, ofloxacin, and ciprofloxacin (Fig. 13).208,209 The first-generation nalidixic acid is activeonly against Gram-negative bacteria, whereas the later generations, such as the fluoroquinolones witha fluorine atom on the number 6 carbon (Fig. 13B), have been modified to become effective anti-bacterial agents which exhibit a broad spectrum of activity highly against Gram-negative bacteria andless active against Gram-positive bacteria and also show significant activity against anaerobicbacteria. Fluoroquinolones have been further modified to produce quinobenzoxazines (Fig. 13C),which are found to show anti-tumor activities (whereas the parent quinolones lack such activities)believed to be attributable to their interaction with topoisomerase II.210 Ciprofloxacin (Cipro1 ofBristol-Myers) is a prototypical fluoroquinolone which has been brought on the stage in recent anti-bioterrorism reactions. It has become ‘‘the antibiotic of choice'' for fighting against anthrax caused byBacillus anthracis prior to the release of significant amount of toxin by the bacterium, despite the factthat several other antibiotics are also effect against this bacterium.211 Since this family of drugs havebecome widely used, one should also bear in mind the risk of serious side effects such as tendinopathyas a consequence of quinolone treatment.212 1. Metal Complexes of Quinolones Quinolones can bind several divalent metal ions, including Mg2þ, Ca2þ, Mn2þ, Fe2þ /3þ, Co2þ, Ni2þ,Cu2þ, Zn2þ, Cd2þ, and Al3þ,213,214 and may result in change in their activity. Mg2þ and Al3þ werefound to decrease the activity of the drugs,215 whereas Fe3þ and Zn2þ complexes were found toexhibit higher activities.216 The crystal structures of the Ni2þ and Cu2þ complexes of cinoxacin andciprofloxacin have been solved, in which the metals are found to bind to the a-carboxylketo moiety toform 1:2 metal-to-drug complexes.214 The complexes have a pseudo-axial symmetry with the twodrug ligands bound symmetrically at the equatorial positions. The axial symmetry is also seen in theEPR spectra of Cu2þ–(drug)2 complexes.213e,214c The drug was also determined by means ofcrystallography to form a 1:3 Co2þ :drug3 complex.217 A few metal complexes (Fe3þ, Cu2þ, and Bi3þ)of quinolones were prepared in acidic solutions, from which crystals were obtained and structuressolved.218 However, the metal ions in these crystals do not bind directly to the drugs owing toprotonation of the carboxylate group, which may not be relevant to the drug action under Figure 13. The structures of (A) nalidixic acid; (B) the prototypical fluoroquinolones (F substitution at position 6) ciprofloxacin (Cipro ); R1 ¼ H; R2 ¼ cyclopropyl, norflozacin; R1 ¼ H; R2 ¼ ethyl, and pefloxacin; R1 ¼ CH3; R2 ¼ ethyl; and (C) a prototypicalquinobenzoxazine, A-62176.
physiological conditions. The formation of M2þ (quinolone)(2,2 0-dipyridine) ternary complexes(M ¼ Co, Ni, and Cu) was observed by means of electrospray ionization and laser desorption massspectroscopy.219 A recent theoretical study suggested that metal binding to these drugs is associatedwith the action of these drugs, and fluorescence quenching measurements indicate the presence of ap–p stacking which has been suggested to be associated with the DNA intercalation capacities of thedrugs and their Cu2þ complexes.220 2. Mechanism of Quinolone Action The binding of quinolones to DNA-gyrase or topoisomerase IV has been considered the key step in theaction of these drugs, which prohibits DNA religation activity and distorts DNA in the complex.221Recent studies on the mapping of the functional interaction domain of topoisomerase II revealed thatthe quinolone-action site on the enzyme overlaps with those sites for the DNA cleavage-enhancingdrugs, including etoposide, amsacrine, and genistein.222 DNA has been considered the target forquinolone drugs, and a cooperative quinolone–DNA binding model of DNA gyrase in the presence ofATP is proposed.223 Norfloxacin exhibits a Mg2þ-dependent binding to plasmid DNA in the absenceof the enzymes,224 wherein metal –drug, metal –DNA, and drug –metal –DNA complexes aredetected. The drug does not bind to DNA in the absence or in the presence of an access amount ofMg2þ. Intercalation of norfloxacin into DNA is proposed in the study, and Mg2þ is proposed to serveas a ‘‘bridge'' for the carboxylate of the drug to interact with DNA. However, DNA unwindingefficiency of 10 by this drug is only marginal for a weak intercalation.224,225 Fluorine-19 NMRstudy of the binding of pefloxacin with double stranded DNA also revealed the participation of Mg2þin the binding.226 Moreover, the Mg2þ-dependent single-stranded DNA binding affinities of several 6-substituted quinolones are found to correlate with the gyrase poisoning activity of these drugs,227confirming the involvement of Mg2þ in such interaction and the significance of the substitution atposition-6 and supporting the mechanism derived from quinolone–DNA interaction.
Quinobenzoxazines have been proposed to bind DNA duplex in the presence of Mg2þ to form a ternary complex in the form of drug2–Mg2þ –DNA, in which one drug molecule is proposed to intercalate into the DNA base pairs while the other is ‘‘externally bound.''228 The two Mg2þ ions serveas salt bridges which interact with both molecules of the drug and the phosphoester backbone of DNA.
These drugs have been determined to form a 1:1 or 2:2 complex with Mn2þ in methanol by means ofJob plot229 (in which absorption is measured against different metal-to-ligand ratios), which alsoimplies a possible formation of ternary complexes between 2:2 metal –quinolone complexes andDNA. The metal –quinobenzoxazine complex interacts with DNA in a cooperative manner, i.e., a 4:4metal –drug complex is proposed to interact with DNA as a unit, in which two drug moleculesintercalate into DNA base pairs while the two ‘‘external'' drugs have p–p interaction and are expectedto interact with the enzyme topoisomerase II or gyrase. The 2:2 metal –drug complex is also suggestedto be assembled in the presence of topoisomerase II based on the results from photocleavage assay, theuse of mismatch sequences, and competition experiments.230 The formation of the 2:2 metal –drugcomplexes suggests that different quinobenzoxazine or quinolone drug molecules should be utilizedto form ‘‘hybrids'' for the pursuit of optimal structure–activity relationship.
The antibiotic activities of the platinum complexes cis-diamminedichloroplatinum (also commonlyknown as cisplatin, cis-[PtII(NH3)2Cl2]; R1 ¼ NH3 and R2 ¼ Cl in Fig. 14) and cis-PtIVCl6(NH4)2were found serendipitously by Barnett Rosenberg to cause dramatic elongation of E. coli during astudy of the influence of electric fields on the growth of the bacterium by the use of a platinum electrodein a buffer solution containing NH4Cl.231 The abnormal growth of this bacterium was later identifiedto be caused by the oxidation of the Pt electrode to form the Pt(IV) salt, which was confirmed viachemical synthesis of the compound. In the meantime, the Pt(II) compound cis-[Pt(NH3)2Cl2] was Figure 14. Schematic structures of (A) cisplatin (R1 ¼ NH3 and R2 ¼ Cl) and ‘ transplatin' (R1 ¼ Cl and R2 ¼ NH3) and (B) theless toxic analogue carboplatin.
also identified to be a potent antibiotic agent, causing the same effect as the Pt(IV) salt. Thus, thesesynthetic metal complexes can be considered metalloantibiotics from a broad sense of the term as theycan inhibit the growth of microorganisms. After its discovery, cisplatin was soon found to be a potentanti-cancer agent and is nowadays one of the most prescribed anti-cancer drugs which has been usedfor the treatment of several different cancers and tumors, including head and neck tumor andtesticular, lung, breast, and ovarian cancers.232 DNA is considered the main biological target ofcisplatin. The coordination chemistry and reactivity of cisplatin and the interaction of cisplatin withDNA have been extensively studied by means of 1H-, 31P-, and 195Pt-NMR spectroscopy and X-raycrystallography, and has previously been reviewed in a number of publications.233 1. Cisplatin–DNA Complexes The chemistry of cis-[Pt(NH3)2Cl2] has been thoroughly investigated in the late 1890s by AlfredWerner.234 The two bound chloride ions in cisplatin are relatively labile, which can undergo exchangewith nucleophiles such as amine bases. Upon introduction of DNA, cisplatin binds to the N7 nitrogenof two adjacent guanidine bases or guanidine–adenine bases in the major groove, or two proximalguanidine bases on different strands in the minor groove which distorts the DNA structures by bendingthe helix by 40–60 and a helical twist of 25–32. This binding pattern and structural perturbation onDNA have recently been revealed by means of crystallography235 and NMR spectroscopy236 (Fig. 15).
The DNA binding mode of cisplatin cannot be achieved by its stereoisomer ‘‘transplatin'' (R1 ¼ Cl; R2 ¼ NH3 in Fig. 14),237 in which the two trans chloride cannot bind to adjacent guanidinebases as in the case of cisplatin. The lability of the Pt–Cl bond still allows nucleophilic substitution tooccur in transplatin which can result in DNA binding. However, the DNA binding of transplatin issignificantly different from that of cisplatin, wherein cross-strand linkage becomes predomi-nant.233,238 The observation of significant cytotoxicity of the trans analogues with pyridine in place ofthe ammonia239 and high anti-tumor activity of trans-imino analogues240 suggest that ‘‘transplatin''analogues are worth further exploration for design of new platinum antineoplastic agents.237 2. Cisplatin Conjugates Cisplatin has been linked to bioactive molecules to form conjugates which exhibit unique proper-ties in terms of DNA binding and anti-tumor activity. For example, adriamycin (Section 2.D‘‘Anthracyclines'') can form complexes with PtCl2 to afford cisplatin-like complexes,154a such as cis-dichloro-t-butylamine-adriamycino-platinum which has been determined to be active againstmurine carcinoma and leukemia.154b This complex has been suggested to interact with DNA byintercalation of the AC rings rather than covalent binding to the Pt center. A hormone-anchoredcisplatin complex has been prepared in which testosterone is bound to cisplatin in place of thediammine groups via a thiosemicarbazone linkage.241 This conjugate exhibits a higher activity thancisplatin against human breast cancer cell line MCF-7. The binding of cisplatin with proteins,including serum albumin242 and transferrin,243 has also been reported which is considered to playimportant role in the metabolism and bioactivity of this drug. The interaction of proteins with cisplatinmay possibly mediate cell response to the drug, which has been recently reviewed.244


Figure 15. Stereo view of the structures of d(CCTGGTCC)*d(GGACCAGG) obtained with NMR spectroscopy (top structure; ProteinData Bank ID1AU5.pdb), in which cisplatin is bound at the center GG nucleotides of one strand, and d(CCTCGCTCTC)*d(GGAGC-GAGAG) obtained with X-ray crystallography (bottom structure; Protein Data Bank ID1A2E.pdb), in which cisplastin is bound to thecenter G from each strand. The binding of cisplatin to DNA significantly distorts DNA structure, particularly in the case of the cross-strand binding.
3. Cisplatin Analogues A large number of cisplatin-like compounds have been synthesized, their molecular propertiesthoroughly characterized, and their anti-tumor activities evaluated.233,245 Of these new analogues, thecompound carboplatin (cis-diammine-cyclobutane-1,1-dicarboxylatoplatinum, Fig. 14B) exhibitslower toxicity than cisplatin and has currently been used clinically for cancer treatment. The othercompounds such as nedaplatin (cis-diammine-1-hydroxoacetatoplatinum) and oxaliplatin (1,2-diaminocyclohexane-oxalatoplatinum) also exhibit potential antineoplastic activities, which havegained approval for clinical use in some countries and are under extensive evaluation.233,245,246 Several cisplatin analogues with two Pt centers have recently been prepared, possessing a general formula of [(trans-PtCl(NH3)2)2-m-L]2þ in which L is a diamine linker.247 Because of the presence oftwo DNA-binding motifs in each molecule, binding of these dinuclear platinum complexes to DNAduplex affords intrastrand and/or interstrand cross-link, wherein the bending of DNA at the bindingsite is much less than that caused by cisplatin.247,248 These dinuclear platinum compounds exhibitanti-tumor activities differently from cisplatin and may also be different from each other, and are potential new anti-tumor agents. Analogous compounds with multi-platinum centers have also beenprepared and show significant anti-tumor activities and a cellular response different from cisplatin,and have been under clinical trial.249 The DNA-binding pattern of these new compounds has also beeninvestigated which shows a similar bifunctional manner as dinuclear platinum compounds.250 Platinum(IV) complexes have been known to exhibit anticancer activities.231 Several Pt(IV) complexes have entered clinical trials;251 however, they have not been widely used because of loweractivities than cisplatin or high toxicity and viability of drug uptake, including cis,trans,cis-[PtCl2(OH)2(isopropylamine)2] (iproplatin, CHIP, or JM9),252 [PtCl4(D,L-cyclohexane-1,2-diamine)](tetraplatin or ormaplatin),253 and cis,trans-[PtCl2(OAc)2(NH3)(NH2C6H5)] (JM216 or satrapla-tin254). The bioinorganic chemistry of Pt(IV) complexes has recently been extensively reviewed.255 H. Organometallics Organometallic compounds are a large family of unique synthetic metal-containing organiccompounds, which are characterized by the presence of direct metal –carbon bond(s). Severalorganometallic compounds have been found to exhibit antineoplastic activities.256 Of these, the‘‘metallocene'' compounds M(IV)Cp2Cl2 (Cp ¼ cyclopentadienyl; M ¼ Ti, V, Nb, and Mo) showsignificant activities toward several experimental animal tumors and human tumors on nude mice,whereas the Zr and Hf analogues do not show anti-tumor activity.257 The Ti compound has enteredclinical trials.258 In addition to the metallocenes, there are a number of non-platinum metal complexeswhich have been extensively studied and tested for their anti-tumor activities and are covered in recentreviews.233e,256 Although metallocenes were originally considered to bind DNA similar to cisplatin, recent studies indicated that they do not bind tightly to DNA at neutral pH.259,260 Nevertheless, DNA is still abinding target of these compounds under certain conditions,256 as suggested by NMR studies.261TiCp2Cl2 has been suggested to exhibit an anti-tumor mechanism different from cisplatin,262 showinginhibitory activity toward protein kinase C and DNA topoisomerase II.259 The hydrolysis of thesecompounds into M(IV)Cp has been proposed to render their anti-tumor activities.259 The a values of the bound water molecules in MCp2(H2O)2 result in different charges on the compounds, which relates to their capability of entering cells. The high acidity of TiCp with pKa values of 3.51 and 4.35, which afford a neutral species at pH 7.0, and its reasonable stabilitywith t1/2 ¼ 57 hr for Cp dissociation263 may account for its high anti-tumor activity.
MCp2Cl2 can form conjugates with adriamycin (Section 2.D ‘‘Anthracyclines'') to give 1:2 metal-to-drug complexes (M ¼ V and Zr) and 1:1 and 1:2 complexes (M ¼ Ti).264 While the Zrconjugate does not show activity toward P-388 leukemia, the Ti complexes exhibit activitycomparable to the free drugs. The structures of these metal conjugates were not determined in theprevious study. The metal ions are suggested to assist the binding of the drug to DNA and red bloodcell membrane. However, these metal complexes do not catalyze electron transfer from NADH todioxygen as does adriamycin (Section 2.D ‘‘Anthracyclines''), which possibly may decrease thecardiotoxicity of adriamycin. Thus, these conjugates seem to serve as bifunctional anti-tumorcompounds, i.e., to release adriamycin and the M–Cp2 complex.
The tetracyclines (TCs) have once been widely used as both external and internal medicines for anextended period of time because of their broad-spectrum activity toward both Gram-positive and-negative bacteria, and also their activity toward rickettsiae, chlamydiae, and protozoans, such as theprototypical TC aureomycin (Fig. 16) produced by Streptomyces aureofaciens.265 The antibioticactivity of TCs is attributed to their binding to the ribosome which inhibits protein synthesis.266 Theirusage has been limited in recent years because of side effects, including staining of teeth and increase Figure 16. Schematic structure of aureomycin (7-chlortetracycline). Substitute OH for 5-H and H for 7-Cl afford terramycin (5-oxytetracycline).
in bacterial resistance. However, recent studies of the mechanism for bacterial resistance of this drughas afforded new insight into rational design of analogues and searching for new analogues of thisbroad-spectrum antibiotic family, such as the novel 9-glycylamido derivatives the ‘‘glycylcyclines,''for defending bacterial infections.267,268 One of the glycylcyclines 9-t-butylglycylamido-minocy-cline (GAR-936, tigilcycline) is currently under phase II clinical trials.269 The metal-binding capability of TCs has been well documented,150 including the binding withalkaline earth and transition metal ions (VO2þ, Cr3þ, Mn2þ, Fe2þ/3þ, Co2þ, Ni2þ, Cu2þ, and Zn2þ) andAl3þ .270–274 TCs have been determined to be present mainly as Ca2þ -bound form (and Mg2þ -boundform to a lesser extent) in the plasma when they are not bound to proteins such as serum albumin.
Thus, the bio-availability of TCs should be dependent upon the physical and biochemical properties oftheir metal complexes instead of their metal-free forms. Metal binding to different TCs are found to beslightly different which has been suggested to be correlated to their pharmacodynamic effect.271b The acidic oxy-groups at positions 1, 3, 10, 11, and 12 of TC are the potential metal binding/ chelating sites (Fig. 16). The acidity of these groups has been determined to follow the order of 3–OH > 12–OH > 4-ammonium > 10–OH.150 The 11,12-b-ketoenol moiety has been considered tobe the primary metal binding site,275 which has also been determined to be the Mg2þ binding site inthe repressor TetR –Mg2þ–TC ternary complex (see later). A recent study indicated that TC forms 2:1TC:metal complexes with 3d transition metal ions in non-aqueous solutions, in which the metal isbound at the 2-amido 3-enol chelating site.270 Moreover, the formation of metal –TC complexes withdifferent stoichiometries, including 2:1, 1:1, and 1:2 metal:TC ratios, has also been suggested in theprevious studies.
The antibiotic activity of TCs is attributed to their binding to the ribosome.266,276 TCs have beenreported to bind different forms of RNA, including the ribosome, bulk RNA, rRNA, and ribozymes.
The studies of TC binding and interaction with RNAs have recently been reviewed.277 The binding ofTCs to bulk RNA is not specific, and may not be significant for their antibiotic activity.278 On the otherhand, this family of antibiotics bind to the ribosome at the 30S subunit with Kd of 1–20 mM(in addition to many other low affinity sites). This binding induces a conformational change thatprevents tRNA from binding to the ribosome and results in interference of protein synthesis.279 Theinteraction of TCs with 16S rRNA has recently been extensively studied with photo-modification,activity assay, mutation, and other methods, from which the TC-binding sites have beenidentified.277,280 The crystal structures of TC-bound small ribosomal subunit have recently been resolved,281 further confirming the significance of such binding in the action of this antibiotic family. Two TCmolecules are found to bind to the RNA (Fig. 17).281a One of the TC molecules may involve a Mg2þion (at the 11,12-b-ketophenolate site that is found in metal binding studies of TC discussed above) in



Figure 17. Top: The crystal structure of 30S rRNAwith two tetracycline (TC) molecules bound (red).There are 96 Mg2þ ions found inthe structure. The one located near 11,12-b-ketoenol moiety may be involved in the binding of the drug to RNA (bottom enlargedbinding site of theTC on the right), as in the case of the binding of the drug toTetR receptor discussed below.
Figure 18. Top: Stereo view of one subunit of the ternary complex formed between class DTetR repressor and Mg2þ aureomycin(Protein Data Band ID 2TCT.pdb). The Mg2þ aureomycin complex is shown in red. The DNA-binding domain is located at the N-terminus on the left.The 2-fold symmetry of theTC TetR dimeric complex allows the binding of the complex to the15-base pair tet-operator. Bottom: Structure of theTC biding site in theTetR Mg2þ TC ternary complex. The drug complex is bound to the proteinthrough His100 via Mg2þ (green), and is also H-bonded with the protein through three amino acid side chains.
binding to RNA through the phosphates of C1054, G1197, and G1198 (Fig. 17). The TC moleculesoccupy the sites that are distinct from those for the binding of aminoglycosides discussed in Section2.E ‘‘Aminoglycosides'' (cf. Fig. 12).
Inhibition of ribozymes by TCs has been studied, including groups I and II introns, hammerhead ribozyme, a ribozyme from hepatitis delta virus, and Neurospora crassa Varkud satellite RNA.187,282The concentration for 50% inhibition (IC50%) of ribozymes has been determined to be 10–500 mMfor several different TCs, with hydrophobic TCs showing higher inhibitions. The large IC50% valuesindicate that these drugs are weak inhibitors for ribozymes, or may even serve as non-specificinhibitors.277 The binding sites, the binding nature, the pattern for the inhibition, and the role of metalions (particularly the RNA-significant Mg2þ ) in the binding with ribozymes were not revealed in theprevious studies.
C. Metal-Dependent Bacterial Resistance Despite the high potency as broad-spectrum antibiotics, TCs are of little use nowadays because oftheir bacterial resistance.265b,283 The predominant TC-resistance mechanism in Gram-negativebacteria is active efflux of the drugs mediated by the antiporter membrane protein TetA which pumpsout TC as a Mg2þ complex coupled with proton uptake.284 The expression of TetA is controlled by therepressor protein TetR, whose binding to operator prevents transcription of both tetR and tetA genes.
A conformational change of the TetR repressor is supposed to occur upon binding of TC in thepresence of divalent metal ions.285 The conformational change results in the release of the repressorfrom the operator and initiates the expression of TetA for active TC efflux. The crystal structures of therepressor TetR and the ternary complex TetR–Mg2þ–TC have been resolved which confirm theinduction of the conformational change of the repressor upon the binding of the Mg2þ–TCcomplex.286,287 A structure of Mg2þ–TC-bound TetR is shown in Figure 18(Top). TetR is a dimeric protein with 10 a-helical structures, of which the first three helical bundles from the N-terminus of each subunitserve as the DNA binding site. The tet-operator is composed of 15 base pairs shown below, which has a2-fold symmetry (boxed sequences) with respect to the center T-A base pair.
Upon Mg2þ -TC binding, significant conformational changes of TetR are observed, including changes in the drug binding site and the DNA binding site.286 Significant changes are also observedat helix-9, suggesting that the opening at the C-terminus of helix-9 serves as the entrance for thedrug as this opening is significantly narrowed after TC binding.286,287 Mg2þ in the ternary TetR–Mg2þ–TC complex is found to bind to the drug at the 11,12-b-ketoenol moiety (as suggested in earlymetal-binding studies, see above) and to TetR via His100, in addition to three water molecules(Fig. 18).
Fe2þ can form a ternary complex with TC and TetR in place of Mg2þ .285,288 An in vitro induction assay shows that Fe2þ–TC is a stronger inducer of Tet repressor than Mg2þ–TC by more than 1,000times, suggesting that Fe2þ may play a role in TC resistance in vivo.288b Specific sites of cleavage ofTetR by the bound Fe2þ is achieved in these studies via Fenton chemistry, and have been identified bymeans of electrospray ionization mass spectroscopy. The cleavages are found to occur at residues104 and 105, 56 and 136, and 144 and 147 in order of preference. This cleavage pattern is consistentwith the geometric locations of the respective residues to the metal center found in the crystalstructures. The determination of the roles of metal ion in the binding of TC to TetR and in the structureof the TC–M2þ–TetR complex is expected to lead to rational design of TC analogues that exhibit broad-spectrum antibiotic activities yet devoid of bacterial resistance, such as the glycylcyclinefamily.267–269 Bacitracin is a metal-dependent dodecapeptide antibiotic excreted by Bacillus species, including B.
subtilis and B. licheniformis. It is a narrow spectrum antibiotic directed primarily against Gram-positive bacteria, such as Staphylococcus and Streptococcus, via inhibition of cell wall synthesis.289Currently, this antibiotic is commercially produced in large quantities as an animal feed additive forlivestock290 and in human medicinal ‘‘triple antibiotics'' ointments (along with polymyxin andneomycin).291 The historical perspectives, the structure of metallobacitracin, and the structure–function relationship of this antibiotic have recently been reviewed.292 A. Congeners and Biosynthesis This antibiotic is produced as a mixture of many closely related peptides, in which bacitracins A1 andB1 are the major components with the most potent activity (Fig. 19).293 Several congeners of thisantibiotic have previously been isolated and characterized by means of amino acid sequence andmass spectroscopy.293 Bacitracin contains four D-amino acids, including D-Glu4, D-Orn7, D-Phe9,and D-Asp11, and a thiazoline ring formed by condensation of the carboxylate of Ile1 with the –NH2and the –SH groups of Cys2 (Fig. 19). A cyclic heptapeptide structure is formed via an amide bondlinkage between the side-chain e-NH2 of Lys6 and the C-terminus of Asn12. These unusual structuralfeatures may protect this peptide from degradation by proteases.
Like those structurally diverse peptides and polyketides and their hybrids such as BLM (Section 2.A ‘‘Bleomycin''), bacitracin congeners are also nonribosomal products of a large peptide synthetasecomplex.294 The structure and mechanism of bacitracin synthetase resemble those of other peptideand polyketide synthetases, which are comprised of a multi-domain modular structure for thecatalysis of the initiation of the thioester linkage to the enzyme, elongation of the thioester-linkedamino acid, and termination of the peptide or polyketide chain by a thioesterase domain.77 Bacitracin synthetase has been known since its early studies to comprise of a complex modular structure as in the case of other peptide/polyketide synthetases. This enzyme catalyzes an ATP-dependent synthesis of bacitracin, starting from the N-terminus based on the observation of a few N-terminal peptidyl intermediates such as Ile-Cys, Ile-Cys-Leu, Ile-Cys-Leu-Glu, and several otherN-Ile-containing peptides.295 The role of ATP has been suggested to be involved in the formation ofthe labile aminoacyl adenosine intermediates. As in the case of other nonribosomal peptide/polyketide biosyntheses, the synthesis of bacitracin has been suggested to involve thioester-linkages Figure 19. Amino acid sequences and N-terminal structures ofa few congeners of bacitracin. Bacitracin A1contains Ile5,Ile8, and R1;A2, Ile5, Ile8, and R2; B1, Ile5, Ile8, and R3; B2,Val5, Ile8, and R1; B3, Ile5,Val8, and R1; F, Ile5, Ile8, and R4; E,Val5,Val8, and R3; H1, Ile5, Ile8, andR5; H2,Val5, Ile8, and R4; H3, Ile5,Val8, and R4; I1,Val5, Ile8, and R5; I2, Ile5,Val8, R5; I3,Val5,Val8, and R4.
based on the observation of covalent peptide –enzyme complexes.77 The thiazoline ring in bacitracinhas been suggested to be synthesized at the stage of Ile-Cys formation on the basis of the detection ofthe oxidized thiazole product of the Ile-Cys intermediate.296 The thiazoline ring and analogousthiazole ring are found in a number of peptide antibiotics and siderophores that are synthesized with asimilar mechanism.76,297 An early study revealed that the activity of bacitracin synthetase is affectedby Mg2þ , Mn2þ , Fe2þ , and Co2þ (Zn2þ was not checked) as well as bacitracin,298 suggesting afeedback control of the synthetase by bacitracin and metal ions.
The bacitracin synthetase operon contains the gene bacA, bacB, and bacC which have been recently cloned and determined to encode three products BA1 of 598 kDa, BA2 of 297 kDa, and BA3of 723 kDa.294 BA1 contains five modules to incorporate the first five amino acids, an epimerizationdomain attached to the forth module for the inclusion of D-Asp4, and a cyclization domain for theformation of the thiazoline ring between Ile1 and Cys2; BA2 is comprised of two modules and anepimerization domain for D-Orn6 incorporation; and BA2 contains five modules for the addition ofIle8-Asn12 with two epimerization domains and the thioesterase domain, consistent with previousstudies.294 A disruption of the bacB gene results in a bacitracin-deficient mutant, confirming theinvolvement of this gene in bacitracin synthesis. Moreover, the expression of a foreign bacitracinsynthetase in a host B. subtilis results in the production of bacitracin at high level, confirming thefunctional role of bacitracin synthetase and its association with bacitracin self-resistance genes.299The available genes of bacitracin synthetase and other peptide/polyketide synthases/synthetasesafford us the tools for possible preparation of different congeners of peptide antibiotics with higheractivities for combating bacterial infections.79 B. Metal Complexes and Antibiotic Mechanism Bacitracin requires a divalent metal ion such as Zn2þ for its antibiotic activity,300 and can form a 1:1complex with several divalent transition metal ions, including Co2þ , Ni2þ , Cu2þ , and Zn2þ .301 TheCo2þ–bacitracin complex binds tightly to C55-isoprenyl (undecaisoprenyl or bactoprenyl) pyro-phosphate with a formation constant of 1.05  106 M1.302 This binding capability of metall-obacitracin presumably prevents the long-chain pyrophosphate from dephosphorylation by amembrane-bound pyrophosphatase, which subsequently inhibits cell wall synthesis because thehydrolytic product undecaisoprenyl phosphate is required to covalently bind UDP-sugars fortransport of the sugars during cell wall synthesis.303 Thus, the binding of metal –bacitracin complexesto undecaisoprenyl pyrophosphate is the key step in the inhibition of cell wall synthesis by thisantibiotic since the sugars become unavailable as building blocks during cell wall synthesis. Althoughthe formation of the Co2þ–bacitracin–undecaisoprenyl pyrophosphate ternary complex wassuggested in previous studies,302 the structure of different metal –bacitracin complexes and thestructure –activity relationship of this antibiotic were not conclusively defined.
C. Coordination Chemistry of Metal Complexes An early NMR study of Zn2þ–bacitracin suggested that His-10 and the thiazoline ring sulfur atomrather than the nitrogen atom were coordinated to the metal,301 but did not implicate other moieties asmetal binding ligands. A later EPR study on Cu2þ–bacitracin revealed a slightly rhombic EPRspectrum with gx ¼ 2.058, gy ¼ 2.047, and gz ¼ 2.261 and a large copper hyperfine couplingconstants of Az ¼ 534 MHz, typical of a tetragonally distorted Cu2þ center.301b The detection of clearsuperhyperfine coupling is indicative of the presence of N ligands. The coordination environment ofthis complex was suggested to be comprised of ligands from thiazoline ring nitrogen, the His10imidazole, the D-Glu4 carboxylate, and the Asp11 carboxylate. The results from a recent X-rayabsorption spectroscopic study via the examination of the extended X-ray absorption fine structure(EXAFS) of Zn2þ–bacitracin in solid form indicated the involvement of three nitrogens and oneoxygen in the first coordination sphere with a tetrahedral-like geometry.304 The ligands are suggested to be thiazoline nitrogen, His10 imidazole, D-Glu4, and possibly the N-terminal amino group. Thebinding of metal through thiazoline sulfur is excluded in this study. This study has provided furtherinsight into the metal binding environment of Zn2þ–bacitracin and corroborates some previousobservations.
The structure of the metal coordination has emerged from the spectroscopic studies discussed above and a recent NMR study of the paramagnetic Co2þ complex.305 The hyperfine-shifted 1H-NMR signals of Co2þ–bacitracin complex have been successfully assigned by the use of both 1D- and2D-NMR techniques as shown in Figure 20. The metal-binding ligands have been conclusivelyidentified in this NMR study, which are assigned to be the Ne of His10, the carboxylate of D-Glu4, andthe thiazoline nitrogen. The N-terminal amino group is not bound to the metal. The identification ofseveral signals attributed to protons near the metal allows a model to be built using relaxation times asdistance constrains (Fig. 21). It is interesting to note in the model that a hydrophobic pocket is formedby the side chains of Ile5 and D-Phe9, which presumably can serve as the binding site for thehydrophobic hydrocarbon chain of the sugar-carrying undecaisoprenyl pyrophosphate.
D. Structure–Function Relationship Further investigation of the Co2þ complexes of several other congeners, including the activebacitracins B1 and B2 and the inactive stereoisomer A2 and the oxidized form F (which have beencharacterized with mass spectrometry and 1D- and 2D-NMR techniques306), revealed that a propermetal binding is essential for bacitracin to exhibit antibiotic activity. That is, all the active congenershave similar metal binding properties and coordination chemistry as bacitracin A1, whereas the metalbinding patterns of the inactive bacitracins A2 and F are different from that of the active forms, inwhich Glu10 in bacitracin A2 and both Glu10 and the oxidized thiazole ring in F are not involved inmetal binding.305 This study thus reveals a relationship between the structure, metal binding, andantibiotic activity of this antibiotic.
The structure of metal-free bacitracin has been determined by means of 2D-NMR spectroscopy, which revealed that the side chains of D-Phe9 and Ile8 are in close proximity of Leu3.307 However, theresult from the study of the Co2þ complex indicates that D-Phe9 and Ile5 are close to each other.305This difference is possibly induced by the metal binding. Bacitracin is known to bind to serineproteases, and the crystal structures of bacitracin–protease complexes have recently beendetermined.308 The protease-bound bacitracin has an extended structure, which prevents metal frombinding to the antibiotic. This structure of bacitracin is different from both the metal-free and metal-bound forms in solution determined by means of NMR. Bacitracin has also been known to inhibitmetalloproteases, presumably because of its metal-binding capability.309 In addition to proteaseinhibition, bacitracin can also inhibit a membrane-bound protein disulfide isomerase,310 and mayserve as a selective inhibitor of b1 and b7 integrin following a not yet known mechanism.311 Thus, the Figure 20. Proton NMR spectrum of the Co2þ complex of bacitracin in H2O at pH 5.5.The signals have been assigned as indicatedby the use of1D- and 2D-NMR techniques.
Figure 21. Stereo view of Co2 þ bacitracin A1produced by means of molecular modeling using nuclear magnetic relaxation ratesas distance constraints.The metal ion is coordinated to the drug through the nitrogen of thiazoline ring, the carboxylate of Glu4, andthe ring Ne of His10.The N-terminal amine is not bound to the metal, but may be hydrogen-bonded to Asn12.The side chains of Phe9and Ile5 are in close proximity and may serve as a flexible hydrophobic binding site for lipid pyrophosphates.This structure is expectedto be similar to the structures of the Co2 þ complexes of bacitracins B1and B2 with high antibiotic activities.
inhibitory property toward proteins may serve as a unique ‘‘alternative activity'' of this antibiotic,in addition to its better documented inhibition activity toward bacterial cell wall synthesis.
Ionophores3,312–316 and siderophores317 are relatively small molecules excreted by microorganismswhich can selectively bind and transport alkali or alkaline earth metal ions and Fe3þ , respectively,across cell membranes and artificial lipid bilayers. These molecules can serve as antibiotics by (a)disturbing the ionic balance across membrane via ion transport (particularly, the transport of alkaliand alkaline earth metal ions), such as nactins, lasalocid, and valinomycin, (b) creating pores onmembranes which results in leaking of cations through the pores, such as gramicidins, and (c)competing for essential iron in the environment, such as ferrichromes. The antibiotic activity ofionophores have also entitled them to be practically used as growth promoters and for increasingagricultural products.318 A. Structure, Cation Binding, and Transport of Ionophores Cation transport across the membrane by ionophores requires the participation of specific membraneproteins and is strictly regulated. The transport of cations results in disturbance of the ionic balanceacross the membrane upon release of the bound metal ions. This disturbance may slow down normalcell growth or even cause cell death. This family of cation-binding microbial products can thus beconsidered antibiotics. As opposed to the metalloantibiotics discussed in previous sections in whichthe metal ions serve as an integral part of the molecules to exhibit antibiotic activities, the metalions themselves in metalloionophores serve as the ‘‘magic bullets.'' The release of metal ions fromthe metalloionophores in the cells can cause imbalance of potential across cell membranewhich engenders antibiotic activities of ionophores. In the case of ion-channeling antibiotics, the‘‘magic bullets'' are transported directly into the cells to result in antibiotic effect. The mechanism ofthis type of antibiotic activity has been adopted in a recent design of channel-forming antibacterialagents.319 1. Structure and Metal Binding Metal binding to ionophores and siderophores is the key step that allows specific receptors on the cellsurface to recognize the metalloforms of the molecules, which results in transport of metal ions acrossthe membrane into the cell. Upon binding of metal ions, conformational changes of ionophores andsiderophores may occur.312 The structure of the metalloforms may vary dramatically, depending onthe bound metal ions. In the case of enniatin (which has a cyclic[L-N-methyl-valine-D-hydroxy-isovalerate]3 structure), the parent ionophore has a structure very similar to its K þ complex, whereasthe Rb2–enniatin complex has a structure quite distinct from that of the metal-free form.320 Thisdifference is attributed to the different ionic radii and binding affinities of alkali metal ions withenneatin. In many other cases, the binding of metal ions results in significant changes of the struc-tures of ionophores from more extended conformations to more folded forms on the basis of thecrystal structures of nactins (e.g., nonactin, tetranactin, and dinactin) and valinomycin and theirmetalloforms.321,322 A common structural feature of this family of antibiotics is the presence of an O-rich metal binding environment, including ether and ketide linkages, the carbonyl group of esters and amides,and carboxylates. Schematic structures of a few ionophores are shown in Figure 22. Such polyketidestructure is synthesized by polyketide synthases via C–C bond (type I and II polyketide synthases) andC–O bond (type III polyketide synthase) formation, analogous to the peptide synthetase for bacitracinsynthesis (Section 4) and peptide/ketide synthetase for BLM synthesis (Section 2.A ‘‘Bleomycin'').
Type III polyketide synthase has been demonstrated to be involved in the synthesis of the C–Oformation in the antibiotic nonactin (Fig. 22A).75m Since these O-rich moieties are preferred ligands for alkali and alkaline earth metal ions, the preference in metal binding is thus not because of the ligands but controlled by different mechanisms Figure 22. Schematic structures of nactins (A): Nonactin (R1, R2, R3, and R4 ¼ CH3), monactin (R1 ¼ C2H5; R2, R3, and R4 ¼ CH3),trinactin (R1and R3 ¼ C2H5; R2 and R4 ¼ CH3), and tetranactin (R1, R2, R3, and R4 ¼ C2H5); valinomycin, which has a cyclic struc-ture with three repeating units of (L-Val L-Lactate D-Val D-hydroxylisovalerate) (B); lasalocid (C), and monensin (D).
such as the different molecular structures, the different sizes of the metal-binding site, the differentionic radii of metal ions (i.e., 0.66, 0.95, 1.33, 1.48, and 1.69 A ˚ for Liþ , Naþ , Kþ , Rbþ , and Csþ , respectively), the use of different moieties for metal binding, and/or the different degrees of hydrationenergy of cations. For instance: (i) while lasalocid with a linear structure has a larger apparent affinityfor its Ca2þ binding than its Kþ binding in the presence of vesicle membrane,323 the cyclic nactinsexhibit higher preference toward monovalent cation binding and best in the binding with NH þ The larger binding cavity in nonactin than in tetranactin (Fig. 22A) results in a binding affinity ofCsþ > Naþ for nonactin, but Naþ > Csþ for tetranactin.324 (iii) The large ‘‘metal binding cavity'' invalinomycin (Fig. 22B) allows its binding with one (e.g., K þ ) or two (e.g., Ba2þ ) metal ions.312 (iv)X-ray crystal structures show that Kþ is bound to valinomycin at the ‘‘internal binding site,'' whereasNa þ at an ‘‘external binding site.''322 (v) While the apparent formation constants for Kþ and Naþbinding to nonactin in dry acetone are in the same order, that for Na2þ is significantly decreased in thepresence of water which indicates the importance of hydration in cation binding to nactins.325 2. Carboxylic Ionophores This family of ionophores are comprised of a linear polycycloether backbone and a carboxylate group,as represented by lasalocid and monensin (structures C and D, Fig. 22). Most of these ionophores bindmonovalent cations with a one-to-one ratio and bind divalent cations with a metal:ligand ratio of 1:2,in which the cyclic ether-O atoms serve as metal-binding ligand.326 Recent FT-IR and 7Li- and 23Na-NMR studies suggest that lasalocid forms a fluxional 1:1 complex with Li þ and 2:2 complexes withKþ , Rbþ , and Csþ ions in solid and in chloroform,327 and 1:1 and 2:2 complexes with Naþ inchloroform.328 The neutral and acid complexes of lasalocid with alkali metals, Tlþ , Agþ , andalkaline earth metal ions have been studied with 1H- and 13C-NMR, and have been suggested topossess similar structures.329 Lanthanide(III) (Ln3þ ) complexes of lasalocid with different metal-to-drug ratios have also been reported.330 Extraction of water-soluble Ln3þ–acetylacetonato (acac)complexes into organic solvent by lasalocid was observed, in which ternary lasalocid–Ln3þ–acaccomplexes are proposed to form with high preference toward smaller Ln3þ ions.331 Metal com-plexation of lasalocid has recently been reviewed.332 3. Gramicidin Family Gramicidins are a family of peptide ionophores3,333 produced by Bacillus brevis as a mixture of a fewcongeners with different amino acid compositions, of which Gramicidin A is the major componentwhose primary structure is shown below.3 Formyl-L-Val1– Gly2– L-Ala3– D-Leu4– L-Ala5– D-Val6– L-Val7– D-Val8– L-Trp9– D-Leu10– L-Trp11– D-Leu12– L-Trp13– D-Leu14– L-Trp15– ethanolamine.
L-Trp11 in gramicidin A is replaced by L-Phe in gramicidin B, and by L-Tyr in gramicidin C. This family of ion channeling antibiotics exhibit a different mechanism for their action from the cation-carrying ionophores described in the above section. Gramicidins can insert into the lipid bilayer as adimer and span across the membrane, forming a unique b-double-stranded helix which creates achannel of 4 A ˚ wide for cations to permeate (Fig. 23).334 This channel exhibits an cation selectivity of H þ > NHþ > Csþ > Rbþ > Kþ > Naþ > Liþ > N(CH3)4 in 0.1 M salt,335 yet does not show permeability to divalent metal ions Mg2þ , Ca2þ , Ba2þ , and Zn2þ . The divalent metal ions canbind to the b-double-stranded helix structure at the entrance of the channel which prevents thetransport of monovalent cations.336 The trend in the alkali metal binding affinity follows the sameorder as the relative ionic mobility, ionic radius, and hydration energy of the ions.3 The structures of several gramicidin congeners and their metal-bound forms have been de- termined with X-ray crystallography337 and NMR spectroscopy in both micelles338 and solidstates333,339,340 (Fig. 23). Similar configurations are observed for the peptide backbone in bothsolution and solid states.337–341 However, deviations are observed among side chains, particularly Figure 23. Structures of gramicidin A determined with NMR spectroscopy (top, Protein Data Bank ID: 1MIC.pdb) and X-ray crystal-lography (middle, top view; bottom, side view, Protein Data Bank ID: 1AV2).Three Cs2þ ions located in the channel are found in thecrystal structure.
Trp-9. The structures in solution determined with NMR spectroscopy exhibit a higher degree ofirregularity than the structures in solid state determined with crystallography, such as the shape of thechannel (Fig. 23, top). The binding of monovalent metal ions does not seem to cause significantstructural change of gramicidins. The structure similarity of this antibiotic with and without boundcations is quite reasonable since the insertion of gramicidins into membranes does not rely on thebinding of metal ions as in the case of other ionophores described above. In addition, the similaritydoes not create further energy barrier for metal binding and transportation. According to thesestructures, the hydrophobic amino acid side chains are located outside the channel. This moleculararrangement allows gramicidins to exhibit extensive hydrophobic interaction with membrane uponinsertion into the membrane, whereas the carbonyl groups are positioned inside the channel forinteraction with and transportation of cations.
The structures of gramicidins A, B, and C in micelles obtained by means of 2D-NMR techniques are very similar, with background atom RMSD 90.5 A ˚ ascribed to similar structures.338 The side chains in these three congeners also have similar configurations. Despite their very similar structuresand monovalent cation specificity, gramicidins A, B, and C exhibit different cation binding andtransporting properties, such as the conductance and activation energy for ion transport. The minorvariations in the structures of the three congeners cannot explain these significantly differentproperties. Change in dipole moment of the side chains (i.e., dipole moments of 2.08, 0, and 1.54 D forTrp, Phe, and Tyr, respectively) was suggested to cause the difference. The decrease in the singlechannel conductance of Naþ for several natural and synthetic gramicidin congeners was found tocorrelate with the replacement of a Trp by Phe, which decreases the dipole moment.338a B. Iron Sequestering and Antibiotic Activities The extremely small solubility product Ksp of 1038 for Fe(OH)3 makes soluble Fe3þ in aqueoussolutions very scarce. To overcome this low availability of iron under aerobic conditions,microorganisms excrete Fe3þ -specific siderophores (Fig. 24) which bind Fe3þ with extraordinarilyhigh affinity constants in the range of 1030–1052 M1 and transport Fe3þ into cells via specificreceptors.317,342 Once the Fe3þ complexes of siderophores are transported into the cells, the iron canbe released upon reduction. In addition to the iron transport activity of siderophores, the Cu2þ , Co3þ ,and Ni2þ complexes of the siderophore desferal were found to exhibit an interesting ‘‘alternativebioactivity'' toward the cleavage of plasmid DNA and oligonucleotides343 which points a newdirection for design of new ‘‘chemical nucleases.'' 1. Mechanism and Structure of Siderophores The biosynthesis of siderophores is regulated by cellular concentration of Fe2þ .344 When theconcentration is low, Fe2þ is dissociated from an ferric uptake regulatory (Fur) protein, resulting inthe binding of Fur to the operon that initiates the synthesis and excretion of siderophores for Fe3þsequestering.342 The Fe3þ–siderophore complexes can be recognized by species-specific receptorsand transported into the cells. For example, although ferrichrome A serves as an iron carrier for fungi,it does not serve that purpose in bacteria; whereas ferrichrome does.345 Thus, a rational approach tochemotherapy becomes possible when it is based on iron transport mechanism in microorganisms317 Figure 24. Two prototypical families of Fe-free siderophores: (A) hydroxamate-based ferrioxamines B (R ¼ NHþ (R ¼ NHCOCH3), and E (R ¼ NHCOCH2CH2 thatclosesthe chainto give a 3-fold symmetric ringstructure) and (B) catechol-basedenterobactin.
by (a) controlling iron transport via iron chelating, (b) transport of an antibiotic substance viaconjugating with siderophores (e.g., albomycin discussed below), and/or (c) inactivation of sidero-phore receptors via inhibitor binding.
The crystal structures of several hydroxamate-containing Fe3þ -bound siderophores have been resolved which allow a detailed comparison of their structures and correlation of their structures withfunction. While the crystal structures show that the iron complexes of ferrioxamines B,346 D1,347 andE348 and desferrioxamine E349 are mixture of L- and D-cis isomers, ferrichrome complexes350 areshown to be exclusively the L-cis isomers. This cis configuration seems to be important for therecognition of these siderophores by membrane receptors (see next Section), wherein the ‘‘carbonylface'' is considered to play an important role since the ‘‘oxime face'' is relatively more shielded thanthe carbonyl face in the structures of these siderophores.
2. Albomycin Structure and Receptor Binding Albomycin is a prototypical siderophore antibiotic produced by Streptomyces. It is a broad-spectrumantibiotic active against several Gram-positive and -negative bacteria with low minimum inhibitionconcentrations, and is even active toward those bacteria resistant to penicillin, streptomycin, TC, anderythromycin.317,351 This antibiotic is a natural conjugate comprised of an iron-binding tri-d-N-hydroxy-d-N-acetyl-L-ornithine site analogous to that of ferrichromes and an antibiotic moiety ofthioribosyl pyrimidine (Fig. 25). Albomycin is recognized by the ferrichrome receptor in the outercell membrane of E. coli,352 and is activated after cleavage by peptidase N to release the antibioticmoiety. Thus, albomycin-resistant bacteria are found to lack either the receptor353 or the peptidase.354 Iron-depleted siderophores are expected to have quite flexible and extended structures. Upon Fe3þ binding, the metal binding ligands are held together by the metal and exhibits a compact metal-binding configuration which is recognized by the ferrichrome receptors (cf. Fig. 25). A few structuresof the membrane transporter protein FhuA (ferric hydroxamate uptake A protein) with and without abound Fe3þ–sederophore have been determined (Fig. 25).355 The Fe3þ binding moiety of the Fe3þcomplexes of phenylferricrocin, albomycin, ferrichrome, and ferricrocin have quite similar coordina-tion chemistry upon their binding to FhuA, and are bound to the receptor in a similar orientation andinteracts with the same amino acid side chains, including Tyr and Arg side chains. The cis con-figurations of the Fe3þ–siderophore complexes bound to the uptake proteins are found to be the sameas those of Fe3þ–siderophores without bound to the protein.346–350 Similar interactions and confi-guration are also observed in the crystal structure of gallichrome–FhuD complex.356 The binding ofFe3þ–ferichrome to FhuA induces a significant conformational change through the N-terminaldomain, including an unwinding of helix 2 near the binding site and a 11-A ˚ translation of the loop next to it, as well as a 17-A ˚ moving of Trp22 on the oppose side from the binding site. How these changes result in the uptake of the Fe3þ–siderophore complexes and transport through cell membranecould not be revealed in the crystallographic studies.
Recently, a semi-synthetic rifamycin analogue CGP4832 was found to bind to and actively transported by FhuA protein, despite its distinctly different structure from those of albomycin andferrichrome.357 In addition to FhuA, the membrane transporters FepA (ferric enterobactin transportprotein)358 and FecA (ferric citrate uptake protein which recognizes and transports the dinuclearFe(III)2–citrate2 complex)359 are also characterized to contain the 22-stranded b-barrel structure asfound in FhuA,360 with a cross section of 35–45 A ˚ . The above studies have provided the molecular basis for rational design of antibiotic siderophores that target bacteria through specific siderophorereceptors of the bacteria.
C. Perspectives of Metal Ions in Medicine In addition to the metalloantibiotics discussed in this review, a number of drugs and potentialpharmaceutical agents also contain metal-binding or metal-recognition sites, which can bind or


Figure 25. Top: Schematic structure of albomycin. Middle: The crystal structure of the Fe3þ albomycin FhuA complex (ProteinData Bank ID1QKC).The Fe3þ drug complex is shown in red color (Fe3þ in green), and the protein as gray ribbons.The binding siteof the drug complex is located inside the FhuA‘pocket.' Bottom: The structure of Fe3þ albomycin seen in the crystal structures ofthe Fe3þ albomycin FhuA complex, in which the residues involved in H-bonding with the Fe33þ albomycin are shown inblue color.
interact with metal ions and potentially influence their bioactivities and might also cause damages ontheir target biomolecules. Numerous examples these ‘‘metallodrugs'' and ‘‘metallopharmaceuticals''and their actions can be found in the literature, for instance: (a) several anti-inflammatory drugs, suchas aspirin and its metabolite salicylglycine,361 ibuprofen,362 the indole derivative indomethacin,363bioflavonoid rutin,364 diclofenac,365 suprofen,366 and others367 are known to bind metal ions andaffect their antioxidant and anti-inflammatory activities; (b) the potent histamine-H2-receptorantagonist cimetidine368 can form complexes with Cu2þ and Fe3þ , and the histidine H2 blocker antiulcer drug famotidine can also form stable complex with Cu2þ ;369 (c) the anthelmintic andfungistatic agent thiabendazole, which is used for the treatment of several parasitic diseases, forms aCo2þ complex with metal:drug ratio of 1:2;370 (d) the Ru2þ complex of the anti-malaria agentchloroquine exhibits an activity two to five times higher than the parent drug against drug-resistantstrains of Plasmodium faciparum;371 (e) a number of Ru2þ /3þ and Rh2þ /3þ complexes are found tobind DNA and exhibit anti-tumor activities;256,372 (f) the antiviral trifluoperazine forms complexeswith VO2þ , Ni2þ , Cu2þ , Pd2þ , and Sn4 þ which exhibit higher inhibition activities than the metal-free drug when tested on Moloney murine leukemia virus reverse transcriptase;373 (g) the clinicallyuseful b-lactamase inhibitor sulbactam can form complexes with Ni2þ , Cu2þ , and Fe3þ ;374 (h) a fewhormone-anchored metallodrugs have been prepared which show enhanced receptor binding andhigher activities against cancer cells;241,375 (i) the thiosemicarbazone-conjugated isatin (which showsa broad-spectrum bioactivity376) can bind late first-row transition metal ions and exhibit activitytoward human leukemia cell lines, however, without inducing cell apoptosis;377 and (j) metalcomplexes (including Be2þ , Mg2þ , Mn2þ , Co2þ , Ni2þ , Cu2þ , Zn2þ , Cd2þ , Pb2þ , Fe3þ , Al3þ , andLa3þ ) of several carbonic anhydrase inhibiting sulfonamides378 have been investigated for theirtopical intraocular pressure lowering properties and as potential agents against gastric acidimbalances.
There are also a number of metallodrugs and metallopharmaceuticals which have been utilized for the treatment of diseases and disorders or as diagnostic agents,233e,379,380 such as gold antiarthriticdrugs, bismuth antiulcer drugs, gadolinium MRI contrast agents, technetium radiopharmaceuticals,metal-based X-ray contrast agents, and photo- and radio-sensitizers, vanadium as insulin mimics, andlithium psychiatric drugs. The metal ion Liþ can be considered the smallest effective metallodrugwhose carbonate and citrate salts exhibit significant therapeutic benefit in the treatment of manicdepression (bipolar mood disorder).381 Some recent studies by means of 3D-MRI techniques indicatethat the volume of the brain gray matter is increased in bipolar disorder patients treated with Liþ .382The status of Liþ in cells have been extensively studied and recently reviewed.381,383 It is alsointeresting to point out that the metal ion Sb3þ may be regarded as the simplest ‘‘metalloantibiotic''from a broader viewpoint of the term, whose salts (including N-methylglucamine antimonite and Na-stibogluconate) have been utilized for the treatment of leishmaniasis against the protozoan parasiteLeishmania.384 The antiprotozoal mechanism of Sb3þ is thought to be attributed to its binding totrypanothione that is essential for the growth of the parasite.
This review has summarized the structure, function, and activity of several different families of metalloantibiotics, and has also pointed out the design and potential utilization of metal complexes forbattling pathogenic microorganisms. Because of the increase in bacterial resistance toward manycurrently used antibiotics in recent years, further development of new antibiotics has become anurgent mission. Better understanding of the structure, function, and mechanism of existingmetalloantibiotics and the mechanism of antibiotic resistance will lead to better design of metalcomplexes for this mission. As the chemical properties of metal ions can vary significantly and canalso be further fine-tuned by proper design of drug ligands for targeting different biomolecules andbiocomponents, new generations of various ‘‘metalloantibiotics'' isolated from natural resources orobtained via chemical syntheses and/or modifications that exhibit more effective antiparasitic,antibacterial, antiviral, and anti-tumor activities can be foreseen.
The NMR studies of bleomycin, anthracyclines, streptonigrin, aureolic acids, and bacitracin carriedout in the author's laboratory have been supported by the start-up funds, Research and CreativeScholarship Grants and the PYF Award of the University of South Florida, and by the Edward L.
Cole Research Grant (F94USF-3) of the American Cancer Society—Florida Division. The author's co-workers Dr. Xiangdong Wei, Dr. Jon Epperson, and Dr. Jason Palcic have made significantcontributions in better understanding of structure–function relationship of several metalloantibioticsdiscussed in this review.
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384. Yan S, Ding K, Zhang L, Sun H. Complexation of antimony(III) by trypanothione. Angew Chem Int Ed Professor Li-June Ming completed his undergraduate education at the Chinese Culture University in Taiwan,earned his M.S. degree on fast kinetic study of configurational change of metal complexes with Professor Chung-Sun Chung at the National Tsing Hua University in Taiwan, and obtained his Ph.D. degree on nuclear magneticresonance studies of Cu,Zn-superoxide dismutase with Professor Joan Valentine at the University of California atLos Angeles in 1988. After 3 years of postdoctoral research on several Fe enzymes and synthetic complexes withProfessor Lawrence Que, Jr., at the University of Minnesota, he joined the Chemistry Department and theInstitute for Biomolecular Science at the University of South Florida in 1991. His research interest is onhydrolytic metalloenzymes and chemistry, metalloantibiotics, and metallopolymers. Nuclear magnetic reso-nance techniques have been extensively used in his research for the investigation of paramagneticmetallobiomolecules and synthetic metal complexes.

Source: http://chuma.cas.usf.edu/~ming/Publications/abstracts/metalloantibiotics.pdf

Microsoft word - technical specs chapt 7 pass process

PASS CR (MICRO Type II) CAPE SEAL and PASS QB Rejuvenating Seal for Residential Roads SECTION 700 - PASS CR Scrub Seal The work shall consist of furnishing all necessary labor, materials and equipment for the transporting, application of the polymer modified asphaltic emulsion PASS or equal, ¼" by No. 10 premium aggregate to conform to the Provisions of Section 37-2, of the Standard Specifications, Plans and these Special Provisions. The work shall be done in the following order: preparing the pavement surface; applying the emulsion; scrubbing the applied emulsion with an emulsion broom; applying premium aggregate; rolling the ¼" by No. 10 premium aggregate; and sweeping up excess aggregate and more fully described below.

Clasificacin terminolgica

CLASIFICACIÓN TERMINOLÓGICA Y CODIFICACIÓN DE ACTOS Y TÉCNICAS MÉDICAS ORGANIZACIÓN MÉDICA COLEGIAL CODIFICACIÓN DE ESPECIALIDADES OMC La codificación de especialidades en el marco de la OMC se realiza mediante un código numérico de 2 dígitos, empezando por Medicina General como 01 y Pediatría como 02, para seguir con las sucesivas especialidades por orden alfabético desde 03 correspondiente a Alergología hasta el 41 para Urología.