DOI: 10.1002/chem.201301659

Spectroscopic Elucidation of the Inhibitory Mechanism of Cys2His2 ZincFinger Transcription Factors by Cobalt ACHTUNGTRENNUNG(III) Schiff Base Complexes

Marie C. Heffern,[a] Josh W. Kurutz,[b] and Thomas J. Meade*[a]

Introduction

Advances in the understanding of metal–protein binding in-teractions have stimulated the development of metal-basedprotein inhibitors to modulate biological processes and alterdisease progression.[1] CoIII Schiff base complexes bearinglabile axial ligands ([Co ACHTUNGTRENNUNG(acacen)(L)2]

+) inhibit histidine-con-taining proteins and enzymes including zinc finger (ZF)transcription factors (TFs) and metalloendopeptidases (Fig-ure 1 a).[1c,2] The mechanism of inhibition is believed todepend on the dissociative exchange of the labile axial li-gands of the complex, enabling the [Co ACHTUNGTRENNUNG(acacen)(L)2]

+ com-plex to coordinate the imidazole ring of histidine (His) resi-dues and disrupt protein function.[2d,3]

Abstract: Transcription factors are keyregulators in both normal and patho-logical cell processes. Affecting the ac-tivity of these proteins is a promisingstrategy for understanding gene regula-tion and developing effective therapeu-tics. CoIII Schiff base complexes ([Co-ACHTUNGTRENNUNG(acacen)(L)2]

+ where L= labile axial li-gands) have been shown to be potentinhibitors of a number of zinc metallo-proteins including Cys2His2 zinc fingertranscription factors. Inhibition by [Co-ACHTUNGTRENNUNG(acacen)(L)2]

+ of the target protein isbelieved to occur through a dissociativeexchange of the labile axial ligands forhistidine (His) residues essential forfunction. Here, we report a seriesof spectroscopic investigations withmodel peptides of zinc fingers that elu-cidate the interaction between [Co-

ACHTUNGTRENNUNG(acacen)(L)2]+ complexes and zinc

finger transcription factors. Observedchanges in NMR chemical shifts and2D 1H-1H NOESY NMR spectra dem-onstrate the preference of [Co-ACHTUNGTRENNUNG(acacen)(L)2]

+ complexes to coordi-nate His residues over other aminoacids. The conformation of [Co-ACHTUNGTRENNUNG(acacen)(L)2]

+ upon His coordinationwas characterized by 1H NMR spec-troscopy, near-UV CD, and electronicabsorption. These studies reveal thatthe resulting His-coordinated [Co-ACHTUNGTRENNUNG(acacen)(L)2]

+ complex possesses anoctahedral structure. The effects of

[Co ACHTUNGTRENNUNG(acacen)(L)2]+ complexes on the

zinc-finger structure were assessed bythe degree of hydrogen bonding(probed by 2D NMR spectroscopy)and secondary-structure profiles mea-sured by far-UV CD. These structuralstudies demonstrate the ability of [Co-ACHTUNGTRENNUNG(acacen)(L)2]

+ complexes to disruptthe bba structure of zinc fingers, result-ing in primarily random-coil conforma-tions. A mechanism is described where-in [Co ACHTUNGTRENNUNG(acacen)(L)2]

+ complexes inhibitzinc finger transcription factor activitythrough selectively coordinating Hisresidues in the zinc finger by dissocia-tive ligand exchange and disrupting thebba structural motif required for generegulation.

Keywords: cobalt · Schiff bases ·spectroscopy · transcription factors ·zinc fingers

[a] M. C. Heffern, Prof. T. J. MeadeDepartment of Chemistry, Molecular Biosciences, NeurobiologyBiomedical Engineering, and Radiology, Northwestern UniversityEvanston, Illinois 60208-3113 (USA)E-mail : tmeade@northwestern.edu

[b] Dr. J. W. KurutzDepartment of Chemistry, Northwestern UniversityEvanston, Illinois 60208-3113 (USA)

Supporting information for this article is available on the WWWunder http://dx.doi.org/10.1002/chem.201301659.

Figure 1. Structures and proposed mechanism of inhibition of histidine-containing proteins by CoIII Schiff base complexes. A) CoIII Schiff basecomplexes contain a CoIII metal center stabilized by a tetradentate acety-lacetonatoethylenediimine ligand (acacen). The axial positions bearlabile ligands that are believed to undergo dissociative ligand exchangeallowing coordination of imidazole side chains of His residues. B) CoIII

Schiff base complexes used in the present study: [CoACHTUNGTRENNUNG(acacen) ACHTUNGTRENNUNG(NH3)2]+

(1) and [Co ACHTUNGTRENNUNG(acacen)ACHTUNGTRENNUNG(4MeIm)2]+ (2). C) Naming scheme for histidine pro-

tons. Focus is placed on the methine protons of the imidazole referred toas H2 and H5.

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His-containing ZF TFs regulate changes in gene expres-sion through sequence-specific interactions with DNA.These proteins are known to mediate vital cell processes,such as embryonic development and apoptosis.[4] Small mol-ecule inhibitors of transcription are powerful chemicalprobes to study gene regulation, bypassing the difficultiesassociated with the current genetic methods of gene knock-down (RNAi) or knockout.[4] In addition to their role innormal physiology, aberrant activation of ZF TFs has beenstrongly implicated in pathological processes includingcancer metastasis and tumorigenesis.[4c,5] Oncogenic TFshave been shown to regulate the production of multiple on-cogenic proteins, and inhibition of one of these TFs may po-tentially disrupt several downstream protein targets of cur-rent chemotherapy.[4a,c,5,6] Consequently, small molecule TFinhibitors are promising agents for potent therapeutic inter-vention.[5a,6,7]

Recently, highly specific and potent inhibition of ZF TFsby [Co ACHTUNGTRENNUNG(acacen)(L)2]

+ complexes with labile ammine ligands([Co ACHTUNGTRENNUNG(acacen) ACHTUNGTRENNUNG(NH3)2]

+ =1, Figure 1 b) was achieved by at-tachment of targeting DNA oligonucleotides to the acacenequatorial ligand.[1c,2a–d] The 1–DNA conjugates selectivelydisrupt DNA-binding activity of the target TFs, namely theSnail[2a,c] and Gli[2b] families, which are both implicated indevelopment and cancer progression. Through inhibition ofTF DNA-binding activity, the conjugates were shown toalter biological processes associated with the TFs in cancercell lines both in vitro (such as transcriptional repression)and in vivo (e.g., neural crest formation in Xenopus laevisand denticle belt patterning in Drosophila embryo mod-ACHTUNGTRENNUNGels).[2a, b] The ability of these 1–DNA conjugates to affect sig-naling pathways through TF inhibition strongly suggests thatCoIII Schiff base complexes have potential as research toolsand ultimately new therapeutics.

[Co ACHTUNGTRENNUNG(acacen)(L)2]+ complexes with labile ligands like 1 are

thought to inhibit TFs by disrupting the structure ofCys2His2 ZFs in the DNA-binding domains.[2c,d] The properfolding of a Cys2His2 ZF into a functional structure is depen-dent on the presence of ZnII. Tetrahedral coordination ofZnII stabilizes the ZF into a bba motif comprising a Cys2-containing antiparallel b-sheet in the C terminus and His2-containing a-helix in the N terminus.[8] The a-helices oftandem ZFs in TFs cooperatively bind the major groove ofDNA in a sequence-specific manner to confer gene regula-tory function.[8b, 9] Displacement of ZnII and insertion of theoctahedral [Co ACHTUNGTRENNUNG(acacen)(L)2]

+ complex in the ZnII bindingsite is proposed to disrupt the bba structure, impairingDNA recognition and transcriptional activity.[2d] To date,a detailed characterization of the interaction between [Co-ACHTUNGTRENNUNG(acacen)(L)2]

+ complexes and Cys2His2 ZFs has not beenperformed. Elucidation of this interaction is necessary forthe further development of this class of CoIII complexes asTF inhibitors.

This study investigates the interaction of [Co-ACHTUNGTRENNUNG(acacen)(L)2]+ complexes with ZF motifs at the molecular

level using model peptides. Protein–ligand interactions areoften evaluated with 3D structures determined by solid-state

X-ray crystallography or distance restraints from solution-state NMR spectroscopy. However, the fluxional conforma-tion dynamics of the ZF peptides treated with [Co-ACHTUNGTRENNUNG(acacen)(L)2]

+ complexes (such as 1) made high-resolution3D structures of the model peptides prohibitively difficult toobtain. In order to overcome this challenge, the model pep-tides were investigated by a series of spectroscopic methodsto elucidate a comprehensive mechanism of inhibition. 2DNMR spectroscopy, circular dichroism (CD), and electronicabsorption spectroscopy experiments were performed, andthe results demonstrate that 1 selectively coordinates Hisresidues at its axial positions. Consequently, 1 disrupts thestructure of the bba ZF motif necessary for function. In ad-dition to providing valuable insight into the TF inhibitoryactivity of CoIII Schiff base complexes, the present workdemonstrates the unique employment of spectroscopicmethods to characterize interactions between metal coordi-nation complexes and target proteins.

Results and Discussion

Model peptides : The model peptides used in this study aresummarized in Table 1. Two 26-mer model peptides were

used to elucidate the interaction between 1 and the zinc-finger motif: ZF4 derived from the fourth ZF of the fiveZF-containing human Snail 1 (exon residue 207–233),[10] andCP1, a consensus zinc-finger peptide optimized for zincbinding.[11] The interaction between 1 and the His residuesin the zinc-binding region of ZFs was investigated usinga short His-rich (HR) peptide fragments: a 10-mer modelpeptide derived from the His-rich region of ZF4 (residues16–25) named HR1.

Selectivity of CoIII Schiff base coordination to histidines :Previous studies have suggested that protein inhibition of1 occurs by interactions between the metal center and Hisresidues that are crucial for function.[2a–d,3, 12] 2D 1H NMRspectroscopy experiments were conducted to determinewhether 1 is selective for binding His over other aminoacids.

Table 1. Sequences of the model peptides used to understand the interac-tion between [Co ACHTUNGTRENNUNG(acacen)(L)2)]+ and ZFs.[a]

Name Sequence

Peptides containing bba zinc-finger motifZF4 KSCPH CSRAF ADRSN LRAHL QTHSD VCP1 PYKCP ECGKS FSQKS DLVKH QRTHT G

Peptide containing His-rich region of Zn binding siteHR1 RAHLQ THSDV

[a] Two 26-mer peptides, ZF4 and CP1, were used to study effects on thewhole zinc-finger motif and a shorter His-rich (HR) peptide, HR1 fo-cused investigations on the His coordination. Residues involved in ZnII

binding are underlined and all histidines are italicized.

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NMR chemical shifts of His protons : Although structuralchanges may affect chemical shifts of peptide protons due toaltered hydrogen bonding, coordination of amino acids totransition metal complexes is expected to influence chemicalshifts to a greater extent.[2d, 13] The chemical shifts in the aro-matic regions of the 1H NMR and the 1H-1H TOCSY spectraof the model peptides listed in Table 1 were analyzed inthe presence (1–peptide) or absence (apo-peptide) of1 (Figure 2, and Figure S1 in the Supporting Information).

Correlations from 2D TOCSY experiments arise from mutu-ally spin–spin coupled nuclei and can resolve overlappingpeaks with different coupling environments, such as thebackbone NH and His imidazole protons.

Free ZF4 (apo-ZF4) is expected to adopt a random-coilstructure due to the absence of a ZnII ion to stabilize it.[8] Inthe spectra of free ZF4 (apo-ZF4; Figure 2 a, gray, and Fig-ure S1a in the Supporting Information), peaks correspond-ing to the side-chain methine protons H2 and H5 (seenaming scheme in Figure 1 c) of free His residues occur at7.84–7.96 ppm and 6.96–7.02 ppm, respectively. In the spec-tra of ZF4 treated with 1 (1–ZF4; Figure 2 a, red), the H2and H5 protons are shifted upfield to 7.25–7.28 ppm and6.37–6.42 ppm, respectively. Therefore, adding 1 to ZF4 in-duces changes in chemical shifts (Dd) of approximately0.48–0.60 ppm for H2 and H5. Similar phenomena were ob-served for the CP1 and HR1 peptides (Figure 2 b and c, andFigure S1b and S1c in the Supporting Information), indicat-

ing that 1 significantly shifts His proton resonances upfieldrelative to the free peptides. The upfield shift of the His imi-dazole resonances in the presence of 1 agrees with previous1D 1H NMR spectroscopy studies investigating the interac-tion of 1 with a Cys3His peptide derived from the HIV-1 nu-cleocapsid protein, NCp7.[2d] Further, the shifts in 1H reso-nances of the ZF peptides can be correlated to the forma-tion of a 1–peptide adduct observed by ESI-MS (Figure S2in the Supporting Information).

In order to assess the coordination preference of 1 forHis residues over other amino acids (especially the Cys resi-dues involved in ZnII coordination), the effects of the addi-tion of 1 on the resonances of the amino acid residueswithin the model peptides were compared. 1H NMR reso-nances of the ZF4 and HR1 peptides in the apo-peptidesand 1–peptides were assigned using 1H-1H 2D TOCSY and2D NOESY experiments (Tables S1–S3 in the SupportingInformation). Addition of 1 to the CP1 peptide (1–CP1) ledto a loss of several backbone NH 1H resonances relative tothe apo-CP1 complex, precluding complete assignment ofthe 1–CP1 spectra. This loss of signal is likely due to confor-mational heterogeneity resulting from multiple coordinationpossibilities between 1 and the His residues of CP1. The dif-ference in proton chemical shifts between the 1–peptidesand apo-peptides were determined for each assignable reso-nance (Dd= d(1-peptide)�d(apo-peptide)) and plottedagainst the peptide sequence (Figure 3, and Table S3, Figur-es S3, and S4 in the Supporting Information).

The Dd values of the model peptide protons can be usedto assess the coordination preference of 1 for His over otherresidues in the peptide sequence. Relative to the Dd valuesof non-His side-chain protons, significant negative Dd valuesof the three His side-chain protons, particularly at the imida-zole protons (jDd j >0.45 ppm, upfield shift), are observedfor ZF4 (Figure 3, and Figures S3 and S4 in the SupportingInformation). In addition, the Dd magnitudes of the Hisside-chain protons are significantly greater than the Dd mag-nitudes of the backbone NH and the CHa of the peptide.This indicates that the chemical-shift effects at the His sidechains are not solely due to a change in peptide structure.No notable differences are observed between the Dd valuesof the two His backbone protons in the Cys2His2 bindingsite (His19 and His23) and the third His of ZF4 (His5) atthe concentration of 1 tested (2.2 molar equivalents of 1 toZF4).

The pronounced shifts of the His side-chain resonances incomparison to the other amino acid residues demonstratethe preferential coordination of His to 1 (Figure 3). Theseshifts are specifically observed at the His imidazole protons,and are markedly larger than the His CHa and CHb pro-tons (jDd j =0.05–0.08 ppm and 0.05–0.18 ppm upfield shift,respectively), indicating that coordination of 1 occurs at theelectron-donating imidazole nitrogen atoms of the His andnot the peptide backbone (Figure 3, and Figures S3 and S4in the Supporting Information).

Two effects may contribute to the observed chemical-shiftchanges in ZF4 upon replacing ZnII with 1. One could be

Figure 2. Overlays of the downfield region of the 2D 1H-1H TOCSY spec-tra of ZF4 and CP1 at 15 8C and HR1 at 10 8C incubated with (red) andwithout 1 (gray). Boxes indicate cross-peaks between histidine side chainmethine protons H2 and H5 for all His residues in each peptide. Notableupfield shifts of His proton resonances are observed in all peptides in thepresence of 1 (red boxes) as compared to resonances of the free peptides(gray boxes).

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local electronic effects associated with coordination to theCoIII metal center. The other could be global structuralchanges that alter the electronic environments of the pep-tide protons. The latter may be observed if 1 stabilizesa structure that deviates from the expected random coil ofthe apo-ZF4. To determine the magnitude of the local elec-tronic effects induced by 1 on the His2 region of theCys2His2 ZnII binding site, the truncated 10-mer derivativeof ZF4, HR1, was evaluated in the absence (apo-HR1) orpresence (1–HR1) of 1. Chemical-shift changes observed inthe HR1 model peptide must be associated primarily withlocal electronic effects rather than structural changes, sincethe HR1 peptide is too short to adopt significant secondarystructure.

The Dd values for HR1 show significant upfield shifts ofthe His protons from coordination to 1, as observed withZF4 (Figure S5 in the Supporting Information). The Dd

magnitudes are especially strong for the imidazole methineH2 and H5 protons. Significant changes in the chemicalshifts of the arginine residue were not observed after bind-ing to 1, further confirming that 1 is selectively coordinatedby His residues. The consistency and degree of the coordina-tion demonstrated in the Dd plots of the ZF4 and HR1model peptides indicate that His residues are the selectiveligand target of 1, regardless of peptide structure and se-quence. Previous work has shown that addition of a targetingdomain to the acacen backbone of [CoACHTUNGTRENNUNG(acacen)(L)2)]+ com-plexes confers specificity.[2c,d,f] This study gives the firstdirect evidence to demonstrate that [Co ACHTUNGTRENNUNG(acacen)(L)2)]+

complexes alone (without targeting domains) possessa degree of specificity through selective binding to His resi-dues over other amino acids in a peptide sequence.

NOESY correlations to His protons : The interaction be-tween His residues and 1 was further analyzed with the1H-1H NOESY spectra of the His imidazole protons of the

ZF4, CP1 and HR1 model peptides (Figure 4). Protons ofmolecules partaking in intraresidue and intermolecular in-teractions within NOE proximity (~4 �) of His residues inthe model peptides can be identified through NOE correla-tions to the His protons. The protons of the nonlabile equa-torial acacen chelate can be used to probe intermolecular in-teractions involving 1 with 1H NMR experiments. For helpwith assignment, the resonances of correlations arising fromthe 1H-1H NOESY spectra of ZF4, CP1, and HR1 in thepresence of either ZnII (Zn–peptide) or 1 were compared tothe 1H spectrum of [CoACHTUNGTRENNUNG(acacen) ACHTUNGTRENNUNG(4MeIm)2] (2 in Figure 1 b,4MeIm =4-methylimidazole). Complex 2 was used asa small molecule model of [Co ACHTUNGTRENNUNG(acacen)(L)2]

+ with two Hisresidues at the axial positions.

As expected for both the Zn–peptide and 1–peptideNOESY spectra, intraresidue NOE correlations were ob-served between the His H5 and His Hb (Figure 4, and Fig-ure S6a in the Supporting Information). Weak intraresidueNOE correlations were observed between the H2 and H5 ofthe imidazoles, but cannot be seen at the contour levelsshown in Figure 4. In addition to the expected intraresidueHis NOE peaks, the spectra of the 1–peptides exhibit strongNOE correlations from the His H2 and H5 protons to reso-nances at 2.0, 2.2, and 3.5 ppm and weak NOE correlationsat 5.2 ppm. The chemical shifts of these resonances correlatewell to previous assignments of the protons of the acacenchelate[14] in the [CoACHTUNGTRENNUNG(acacen)(L2)]+ complex.

The chemical-shift correlations indicate that these NOEpeaks represent intermolecular NOE from close proximityof the acacen ligand of 1 to the His imidazole protons. TheNOE measurements support a model wherein His residuesof the peptides coordinate the CoIII center of 1 whereasCoIII coordination of the acacen equatorial chelate is re-tained (see scheme of NOE correlations in Figure S6b in theSupporting Information). Further, no detectable NOEs be-tween other amino acid protons and the protons of the

Figure 3. The Dd (d(1–peptide)�d(apo-peptide)) of amino acid side chain 1H chemical shifts [ppm] plotted against peptide sequence of ZF4 at 15 8C. Forclarity, only the individual protons of the His residues are labeled whereas the remaining residues are labeled by the residue name at the correspondingHb/Hb1. The remaining protons are plotted in order of closeness to the Hb (e.g., K1 is plotted along the x axis as Hb1, b2, Hg, Hd, and He but labeledas K1 at the Hb1 position). Fully labeled Dd plots are available in Supporting Information (Figure S5). The Dd of the His protons are highlighted withdark gray and hatched columns for emphasis and labeled according to Figure 1 b. Coordination selectivity was observed by the significant effects of 1 onthe His imidazole 1H resonances as compared to the other amino-acid protons.

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acacen chelate were observed in the full NOESY spectra ofthe 1-treated peptides validating the selectivity for interac-tions of 1 with the His imidazoles (Figure S7 in the Support-ing Information).

The chemical shifts of the observed NOE correlations tothe His H2 and H5 protons were compared to 2. 4MeIm canbe used as a simple model of the His side chain;[15] there-fore, 2 was studied as a model of [Co ACHTUNGTRENNUNG(acacen)(L)2]

+ axiallycoordinated to two His residues. The resonances of the HisH2 and H5 NOE cross-peaks exhibit strong resemblance tothe 1H spectrum of 2. The chemical shifts of the imidazole(protons e and f in Figure 4) and acacen protons (protons b,c, and d in Figure 4) of 2 correlate well to the chemicalshifts of the His H2 (labeled as H2-H5, H2-a, H2-b, H2-c inFigure 4) and H5 (labeled as H5-H2, H5-a, H5-b, H5-c inFigure 4) cross-peaks. The only exception to the chemical-shift correlation between 2 and the 1–peptide NOESYcross-peaks is the methyl resonance of the 4MeIm ligand at

2.2 ppm. This discrepancy is ex-pected since the 4-methyl groupis replaced by a downfield-shift-ed Hb in His residues. Theagreement in chemical shiftsbetween the His H2 and H5NOEs and 2 confirms the pres-ence of a CoIII-to-His coordina-tion interaction (Scheme S1 inthe Supporting Information).Together with the chemical-shift perturbations observed inthe Dd plots, the NOESY ex-periments demonstrate the se-lective intermolecular coordina-tion of 1 with His residueswithin ZF model peptides. Toour knowledge, these studiesare the first application of1H-1H NOESY experiments todetect selective binding ofmetal-based protein inhibitorsto amino acids within peptidesystems.

Conformation of His-coordinat-ed [Co ACHTUNGTRENNUNG(acacen)(L)2]

+ com-plexes : The changes in NMRchemical shifts and NOESYcorrelations demonstrate that1 interacts with His residues. Togain further insight into theconformation of the His-coordi-nated [Co ACHTUNGTRENNUNG(acacen)(L)2]

+ com-plex, the structure of the 1–HR1 adduct with respect to theCoIII metal center was investi-gated by CD spectroscopy andelectronic absorption studies.

HR1 was used as a peptide model to focus investigations onthe interactions of 1 with the His residues in the Cys2His2

ZnII binding site.

CD of the acacen ligand: CD spectroscopy was used to eval-uate the degree of chirality of [Co ACHTUNGTRENNUNG(acacen)(L)2]

+ in the 1–HR1 adduct. The near-UV CD of the acacen electronic ab-sorption band of 1–HR1 was measured and compared to theCD of the free HR1, 1, and the small molecule model, 2(Figure 5).

The CoIII-bound acacen ligand exhibits electronic absorp-tions in the near-UV region due to p–p* intra ACHTUNGTRENNUNGli ACHTUNGTRENNUNGgand transi-tions.[16] Monodentate ligands with free rotation (such asNH3) at the axial positions result in achiral, and thus optical-ly inactive [Co ACHTUNGTRENNUNG(acacen)(L)2]

+ complexes. Coordination ofa nonsymmetric His2 ligand that precludes free rotation inthe axial positions (such as a peptide with two coordinatingHis) is expected to introduce chirality to the resulting [Co-

Figure 4. Overlays of the 1H-1H NOESY spectra with 150 ms mixing time (blue) and 1H-1H TOCSY spectra(red) with 60 ms mixing time of His H2 (top panel) and H5 (bottom panel) protons of 1–ZF4 and 1–CP1 at15 8C, and 1–HR1 at 10 8C. For comparison, the 1D 1H NMR spectrum of 2 ([Co ACHTUNGTRENNUNG(acacen) ACHTUNGTRENNUNG(4MeIm)2]

+), a smallmolecule model of [Co ACHTUNGTRENNUNG(acacen)(L)2]

+ coordinated to two His at 10 8C is included along the right-hand edgesof both sections of the Figure. The intraresidue TOCSY and NOESY correlations of the His residues are la-beled and indicated by arrows. In addition to the intraresidue correlations, NOE correlations are present withresonances that correspond to the protons in the acacen ligand of [Co ACHTUNGTRENNUNG(acacen)(L)2]

+. These NOEs are indicat-ed by dashed boxes that include the homologous NMR peaks of 2. The presence of such NOEs demonstratesthe close proximity of the protons of the acacen chelate to the His imidazole protons. The homology of 1H res-onances to 2 validates the formation of a [Co ACHTUNGTRENNUNG(acacen)ACHTUNGTRENNUNG(His)x]

+ species upon adding 1 to His-containing pep-tides.

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ACHTUNGTRENNUNG(acacen)(L)2]+ species that can be detected by CD. In con-

trast to the NMR spectroscopy investigations, 2 was studiedas a negative control in these CD studies since free rotationaround the CoIII–4MeIm bond allowed by monodentate co-ordination would render the complex optically inactive. Al-though the HR1 peptide is optically active in the far-UVregion (190–250 nm) due to the chiral peptide bonds, thepeptide alone was found not to have any detectable CD inthe near-UV region (>250 nm).

As expected, no detectable CD signal was observed for1 or HR1. Upon treatment of HR1 with 1 to yield 1–HR1,negative CD signals were observed with minima at 280 and350 nm. The appearance of these signals indicates that HR1

and 1 interact to introduce chirality to the [Co-ACHTUNGTRENNUNG(acacen)(L)2]+ complex. No CD signal was observed in the

spectra of 2 in this region; thus, the observed optical activitycan be attributed to His coordination specifically withina model peptide. Plots of the CD signals at 350 nm (CDminimum) and 330 nm (lmax of acacen absorption band) nor-malized to the signal of free HR1 (signal of the complex di-vided by signal of free HR1; Figure 5 b) show the depend-ence of signal intensity on the concentration of 1. At thelowest concentration of 1 analyzed (1/HR1 =1:1), the ob-served signals at 350 and 330 nm are threefold greater thanthe CoIII complexes without peptide. The introduction of op-tical activity to 1 in the presence of HR1 suggests that thepeptide coordinates to 1 through ligands with restricted ro-tation. Such rotational restriction likely results from simulta-neous coordination of the two His residues of HR1 at theaxial positions of 1.

Electronic absorption of the acacen ligand : To characterizethe conformation and geometry of the 1–HR1 adduct, itselectronic absorption was evaluated and compared to 1 and2 (Figure 6). In contrast to the CD spectra, 1–HR1 exhibitsan electronic absorption spectrum that closely resembles 2,demonstrating the presence of an octahedral [CoACHTUNGTRENNUNG(acacen)-ACHTUNGTRENNUNG(His)2]

+ structure with the acacen in the equatorial planeand His2 coordination at the axial positions. The observedabsorption bands agree with the previously published p–p*intra ACHTUNGTRENNUNGli ACHTUNGTRENNUNGgand transition of CoIII-bound acacen, namely a maxi-mum between 330–340 nm and a shoulder approximately360 nm.[16]

In previous studies subtle differences in the p–p* transi-tion lmax based on axial ligand identity were observed.[16]

The lmax of the 1–HR1, 1 and 2 p–p* intra ACHTUNGTRENNUNGli ACHTUNGTRENNUNGgand transitionbands were determined with the first derivative plots of theelectronic absorption spectra (Figure 6 b). The lmax of the p–p* intra ACHTUNGTRENNUNGli ACHTUNGTRENNUNGgand transition absorption band of 1–HR1 occursat 337 nm, slightly red-shifted from the lmax of 1 at 335 nmand equal to the lmax of 2. These observations demonstratethe retention of the octahedral conformation of the CoIII

center upon axial coordination of His within the HR1 pep-tide.

The electronic absorption data suggest that the discrepan-cy between the 2 and 1–HR1 CD spectra results from thedifference in chiroptical properties not associated withchange in the octahedral geometry or ligand conformation.Rather, the optical activity of 1–HR1 likely results from re-striction in CoIII–imidazole bond rotation that may resultfrom His2 coordination as compared to the monodentate co-ordination of the 4MeIm.

The near-UV CD spectroscopy and electronic absorptionstudies presented here demonstrate the first use of thesemethods for both verification of His coordination and [Co-ACHTUNGTRENNUNG(acacen)ACHTUNGTRENNUNG(His)x]

+ octahedral conformation in the studies ofCoIII Schiff base complexes. Spectroscopic properties of [Co-ACHTUNGTRENNUNG(acacen)(L)2]

+ species were monitored in the near-UVwavelengths and were isolated from the far-UV signals ofthe peptide. These studies can be readily translated to the

Figure 5. CD of 1–HR1 (1/HR1= 1:1) and controls in the region of theelectronic absorption bands of acacen at 25 8C. A) The near-UV CD of1–HR1 were compared with [Co ACHTUNGTRENNUNG(acacen) ACHTUNGTRENNUNG(4MeIm)2]Br, free HR1, and 1,negative controls for optical activity and chirality in the acacen absorp-tion region. The presence of CD signal between 250 and 400 nm in the 1–HR1 validate His2 coordination that introduces chirality to 1. B) A plotof CD signal at 350 and 330 nm (the CD minimum and the lmax of theacacen absorption bands, respectively) normalized to the signal of HR1demonstrates the increase in optical activity of 1–HR1 with increasingconcentrations of 1.

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characterization of [Co ACHTUNGTRENNUNG(acacen)(L)2]+ species in the pres-

ence of larger and more complex protein systems.

Structural disruption of the zinc-finger motif by [Co-ACHTUNGTRENNUNG(acacen)(L)2)]+ complexes : The interaction of 1 with Hisresidues is hypothesized to inhibit TFs by displacement ofthe tetrahedral ZnII ion with 1. This displacement is thoughtto disrupt the bba structural motif required for sequence-specific DNA recognition.[2d] The structural effect of 1 coor-dinating to the full zinc-finger motif ZF4 was investigatedby chemical-shift dispersion, exchange correlation NMRspectroscopy and far-UV CD spectroscopy.

Structural evaluation by NMR spectroscopy : The effect of1 on a ZnII-stabilized structure of ZF4 was evaluated by1H NMR spectroscopy (Figure 7). Peptide backbone NHprotons form hydrogen bonds within a structured peptidesystem that cause shifts in backbone NH proton resonances,dispersing the chemical shifts.[17] As expected, such disper-sion is observed in the 1H NMR spectra of ZF4 in the pres-ence of 2.2 equivalents of ZnII (Zn–ZF4), since ZnII stabiliz-es ZF motifs (Figure 8 c). In contrast to Zn–ZF4, the spectraof 1–ZF4 exhibit a high degree of peak overlap and suggesta low degree of structure commonly observed in random-coil conformations (Figure 7 a).[17,18] Upon addition of 1 toa solution of Zn–ZF4, loss in both signal and chemical-shift

dispersion is observed, suggesting that 1 is perturbing theZnII-stabilized structure (Figure 7 b). The resulting spectrumresembles 1–ZF4 (apo-ZF4 treated with 1). This may indi-cate that 1 is effectively displacing ZnII from the structureand forming a less structured adduct with 1. Perturbations inthe well-defined spectra of Zn–CP1 are also observed uponaddition of 1 (Figure S8 in the Supporting Information). Incontrast to ZF4, the spectrum of Zn–CP1 treated with thesame equivalents of 1 bears less resemblance to 1–CP1 and

Figure 6. A) Electronic absorption spectrum of 1 +HR1 (1–HR1) near the acacen p–p* intraACHTUNGTRENNUNGli ACHTUNGTRENNUNGgand transition at 25 8C in comparison to 1 and 2. The ab-sorption band of 1–HR1 closely resembles 2, validating retention of the octahedral conformation upon His binding at the axial sites. B) First derivativeof the p–p* transition band unambiguously assigns the lmax of 1–HR1 to 337 nm, equal to that of 2 and slightly red-shifted of the lmax of 1 at 335 nm.

Figure 7. 1H NMR spectrum of the backbone NH of: A) 1–ZF4, B) Zn–ZF4 challenged with 1, and C) Zn–ZF4. Addition of 1 to Zn–ZF4 leadsto a loss in signal and chemical-shift dispersion and produces a spectrumthat resembles 1–ZF4 (apo-ZF4 treated with 1).

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more retention of the Zn–CP1 NMR peaks. This may resultfrom tighter binding of ZnII to CP1 than to ZF4. CP1 con-tains an N-terminal aromatic residue (Phe11) that is be-lieved to stabilize the bba motif and ZnII coordination.[11]

The absence of such an aromatic residue in ZF4 could desta-bilize ZnII coordination. The resulting higher ZnII bindingaffinity of CP1 could decrease the sensitivity of CP1 to per-turbations by 1. Ongoing work is quantifying thermodynam-ics of metal binding (of both CoIII Schiff base complexes andZnII) to various ZF model peptides to further elucidate theparameters (such as sequence) that influence binding affini-ties and stoichiometries. Nonetheless, these 1H NMR spec-troscopy studies suggest that 1 can compete with ZnII tobind to ZF peptides and the interaction is influenced by theremaining peptide sequence and possibly the ZnII-bindingpropensities.

NMR spectroscopy investigations of the ZF4 structurewere conducted with 1H-1H NOESY experiments to assessthe exchange rate of backbone NH protons with water(Figure 8, and Figure S9 in the Supporting Information).NOE correlations can be used to detect chemical exchangeoccurring at timescales shorter than the mixing time in thepulse sequence.[17,18] In an aqueous environment, backboneNH protons of a structured system involved in hydrogenbonding are expected to display low rates of chemical ex-change with water. Loss of hydrogen bonding and increaseddisorder increase the rate of chemical exchange of the back-bone NH protons with water protons. This high rate of ex-change produces detectable water 1H-NH exchange correla-tions in the NOESY spectra. No detectable water 1H-NHcorrelations (d 1H2O =4.9 ppm, 15 8C) were observed in theZn–ZF4 NOESY spectra with 150 ms mixing time (Fig-ure S9b in the Supporting Information), confirming the pres-ence of a well-ordered ZF4 structure stabilized by ZnII coor-dination. In contrast, strong water 1H-NH exchange correla-tions were observed in the 1–ZF4 NOESY spectra with thesame mixing time, suggesting the absence of backbone NH

hydrogen bonding due to a loss of structure (Figure 8). Simi-lar water 1H-NH exchange correlations were observed in theNOESY spectra of apo-ZF4, which is expected to adopta random-coil structure (Figure S9a in the Supporting Infor-mation). The NOESY data validate the hypothesis that the1–ZF4 adduct does not adopt significant stable structure.

Assessment of secondary structure by CD spectroscopy : CDspectroscopic analysis of peptides and proteins at far-UVwavelengths (190–250 nm) can reveal important secondary-structure features. CD experiments were conducted withZF4 to correlate the structural insight from the NMR spec-troscopy experiments to secondary-structure effects of 1 onthe bba motif of the peptide. The far-UV CD spectra ofapo-ZF4 or ZF4 in the presence of ZnII or 1 were monitoredfor signals characteristic of random coils (peak minimum at198 nm) and bba ZF motifs (minima at 208 and 220 nm anda maximum at 190 nm).[19]

ZnII was titrated into apo-ZF4 to evaluate the ability ofZF4 to adopt a ZnII-induced ZF structure (Figure S10 in theSupporting Information). In the absence of ZnII, apo-ZF4displays a minimum at 198 nm indicating that the free pep-tide is primarily random coil. Upon addition of ZnII, the198 nm signal of apo-ZF4 is reduced in a concentration-de-pendent manner to reveal a spectrum characteristic of thebba motif with minima at 208 and 220 nm and a maximumat 190 nm. The observed behavior of ZF4 is consistent withpreviously studied ZF peptides,[8a,b,11b, 20] validating the pep-tide as an appropriate model of a folded ZF motif. A maxi-mum structural effect was achieved at a 4:1 ZnII/ZF4 ratio.This ratio was employed in the subsequent titration experi-ment of 1 to ensure that the Zn–ZF4 model possesseda ZnII-stabilized bba structure.

In previous work, addition of 1 was shown to induce therelease of the ZnII ion in a ZnII-bound ZF peptide.[2d] CDsignals of Zn–ZF4 (ZnII/ZF4= 4:1) treated with 1 weremonitored to determine the structural consequences of 1-in-duced displacement of the ZnII ion. Addition of 1 to Zn–ZF4 induced a concentration-dependent reduction of the208 nm minimum and 190 nm maximum of the bba motifand rise of the minimum at 198 nm of a random-coil struc-ture (Figure 9, and Figure S11 in the Supporting Informa-tion). The resulting CD spectrum closely resembles that ofthe apo-ZF4. It is worth noting that a small feature, namelya minimum at 227 nm, is observed that deviates slightlyfrom the shoulder observed in the apo-ZF4 spectra. Thisfeature may result from the formation of disulfide bonds be-tween the free cysteines of 1–ZF4 (see the Supporting Infor-mation for discussion). The CD data support a mechanismof inhibition whereby His coordination to 1 induces a loss ofthe functional bba ZF structure by displacement of the tet-rahedral ZnII and binding of the octahedral [Co-ACHTUNGTRENNUNG(acacen)(L)2]

+ complex.Previous enzyme studies have shown that replacement of

the labile axial ligands of [Co ACHTUNGTRENNUNG(acacen)(L)2]+ with N-hetero-

cycles (such as the 4-methylimidazole ligands of 2) signifi-cantly reduces inhibitory activity.[3,12] Recent studies have at-

Figure 8. 1H-1H NOESY with 150 ms mixing time at 15 8C in the back-bone NH�CHa region of 1–ZF4. Rate of chemical exchange of backboneNH protons with water protons (d 1H2O=4.9 ppm, 15 8C) was used toevaluate the degree of hydrogen bonding and consequently, structure.The position of the water peak is indicated by the dotted line. Peaksfrom the backbone NH of 1–ZF4 (as with apo-ZF4) exhibit detectablechemical exchange with water in comparison to those of Zn–ZF4 (Fig-ure S9 in the Supporting Information). This indicates a low degree of hy-drogen bonding from a disordered structure adopted by 1–ZF4.

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T. J. Meade et al.

tributed the decreased activity to tighter axial coordinationof such N-heterocycles in contrast to the labile ammines of1, strongly suggesting that His coordination occurs via disso-ciative ligand exchange at the axial positions.[14] In contrastto 1, [Co ACHTUNGTRENNUNG(acacen)(L)2]

+ complexes, like 2, with less labile li-gands exhibited incomplete dissociative substitution at bothaxial positions in the presence of competing N-heterocycles,such as free imidazole (Im), resulting in mixed ligand com-plexes (e.g., [Co ACHTUNGTRENNUNG(acacen) ACHTUNGTRENNUNG(4MeIm)(Im)]+ rather than [Co-ACHTUNGTRENNUNG(acacen)(Im)2]

+).CD analysis of Zn–ZF4 treated with 2 was conducted to

determine the dependence of ZF4 structural perturbation by1 on reactivity of the complex to axial ligand dissociation.Although the lability of the ammine ligands of 1 allows forHis coordination at both axial sites, the previous studies sug-gest that the more substitutionally inert 2 would be likely toonly coordinate one His residue. In contrast to effects ob-served with 1, addition of 2 to Zn–ZF4 yields a CD spec-trum slightly shifted from the bba signals but resemblingZn–ZF4 more than apo-ZF4 (Figure S12 in the SupportingInformation). No further structural changes detectable byCD were observed beyond the addition 0.25 equivalents of2. The discrepancy between the CD spectra of ZF4 with1 and 2 suggests that His coordination at both axial posi-tions is required to elicit complete disruption of the bba

motif and supports a dissociative ligand exchange mecha-nism for potent inhibitory activity.

The evaluations of backbone NH hydrogen bonding byNMR spectroscopy and secondary structure by far-UV CDexperiments indicate that 1 disrupts the ZnII-dependent bba

structure of ZF4 in favor of a less ordered random-coil con-formation closely resembling apo-ZF4. The structural effectscorrelate to axial ligand lability by the reduced effect of 2on ZF4 secondary structure. In conjunction with the ob-served His-coordination behavior of 1, these studies validate

the proposed mechanism of inhibition. The CoIII Schiff basecomplex inhibits ZF proteins by coordinating His residuesin the ZnII-binding site through dissociative axial ligand ex-change thereby disrupting the bba structural motif requiredfor function.

Conclusion

The present work provides a comprehensive molecular un-derstanding of the interactions between [Co ACHTUNGTRENNUNG(acacen)(L)2]

+

complexes and ZF motifs that confer potent inhibition oftranscription factors. Due to the disorder of the model pep-tides treated with 1, alternative techniques to traditional 3Dstructure elucidation were required to determine the bindingsite, selectivity, and mode of inhibition. NMR spectroscopy,CD and electronic absorption spectroscopy studies demon-strated that His residues coordinate to 1 at both axial ligandpositions while retaining octahedral geometry. The coordina-tion of His to 1 results in a loss of the structure stabilized byZnII tetrahedral coordination. Consequently, protein func-tion dependent on the zinc-finger motif (such as sequence-specific DNA binding of TFs) is inhibited. These investiga-tions illustrate the remarkable specificity of [Co-ACHTUNGTRENNUNG(acacen)(L)2]

+ complexes for His over other amino acids re-vealing a degree of selectivity at the binding site even in theabsence of a targeting molecule on the acacen backbone.

The knowledge gained in these studies has provided theanalytical foundation to move from a qualitative under-standing of the peptide–complex interaction to quantitativeinformation by extension of the techniques employed. Al-though the model peptides in these investigations were de-rived from ZF TFs, the implications for the mechanism ofaction of [Co ACHTUNGTRENNUNG(acacen)(L)2)]+ complexes translate to otherHis-containing targets. Additionally, with the growing fieldof medicinal inorganic chemistry, new methods for under-standing protein–metal interactions are required. The ana-lytical approaches demonstrated in this work can be readilytranslated to other bioactive coordination complexes andpromote the development and design of transition metal-based therapeutics.

Experimental Section

Materials : Two 26-mer ZF model peptides, ZF4 (KSCPHCSRAFADRSNLRAHLQTHSDV) and CP1 (PYKCPECGKSFSQKSDLVKHQRTHTG), were purchased from GenScript USA, Inc.(Piscataway, NJ, USA). Two His-rich (HR) peptide fragments, a 10-mernamed HR1 (RAHLQ THSDV) and a 7-mer (GHIRTHG) named HR2,were synthesized by standard solid-phase peptide synthesis techniques.Fmoc chemistry and HBTU/DIPEA activation on Wang resin were used.Following TFA cleavage, the peptides were purified by preparativeHPLC using acetonitrile/water gradients in the presence of 0.05 % TFAand 230 nm detection wavelength on an Atlantis T3 column (Waters).The identities and purities (>95%) of the products were confirmed byESI-MS and analytical HPLC.

The two CoIII Schiff base complexes used in these studies, [Co ACHTUNGTRENNUNG(acacen)-ACHTUNGTRENNUNG(NH3)2]+ (1) and [Co ACHTUNGTRENNUNG(acacen) ACHTUNGTRENNUNG(4MeIm)2]

+ (2 ; Figure 1) were synthesized

Figure 9. CD of apo-ZF4 (free), Zn–ZF4 and Zn–ZF4+1 at 25 8C. Theapo-ZF4 is characterized by a minimum at 198 nm expected of a randomcoil. Zn–ZF4 exhibits a maximum at 190 nm, a minimum at 208 nm, anda shoulder at 224 nm, characteristic of a bba motif. The addition of 1 toZn–ZF4 disrupts the bba motif as illustrated by a loss of the 208 nm min-imum of the bba motif and a shift towards the random coil 198 nm mini-mum.

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as previously described.[14, 16] Materials for synthesis of the complexes,ZnSO4·7 H2O, ZnCl2·6H2O and D2O were purchased from Sigma–Aldrich(St. Louis, MO, USA). Perdeuterated [D11]Tris was purchased from Cam-bridge Isotope Laboratories (Andover, MA, USA). Purchased materialswere used without further purification. Deionized water was obtainedfrom a Millipore Q-Gard system equipped with a Quantum EX car-tridge.

NMR spectroscopy : NMR experiments for ZF4 and CP1 were performedon a Bruker Avance II 900 MHz spectrometer equipped with a sensitivi-ty-enhanced triple-resonance 5 mm inverse TCI cryoprobe. Samples ofZF4 and CP1 were prepared in peptide concentrations of 1.5 mm and dis-solved in 90:10 (v/v) H2O/D2O solution buffered at pH 7.0 with 20 mm

deuterated [D11]Tris for solutions containing peptide alone or peptideand 1. ZnII-treated peptides (Zn–peptide) were prepared differently so asto minimize ZnII coordination by Tris buffer. Zn–peptide samples werefirst prepared without buffer, then buffered to pH 7.0 with a maximumcapacity of 10 mm deuterated [D11]Tris. The model peptides were co-incu-bated with 2.2 equiv of either ZnII (ZnSO4·7 H2O) or 1 for at least 1 h at37 8C and degassed for at least 2 h prior to data acquisition. All NMRspectroscopy experiments for ZF4 and CP1 were performed at 15 8C in5 mm NMR tubes. The chemical shifts [ppm] were measured downfieldfrom an internal standard (4,4-dimethyl-4-silapentane-1-sulfonic acid(DSS)). Solvent suppression for 1D, TOCSY and NOESY experimentswas achieved using excitation sculpting using 1808 water-selective pulseswith gradients.[21] 1D experiments were accumulated using 32 k datapoints with 256 scans. Gradient-based phase-sensitive 1H-1H 2D experi-ments were performed using mixing times of 20 or 60 ms for TOCSYsand 150 or 300 ms for NOESYs. 2D experiments were accumulated using2048 data points in t2 for 64 t1 values with 16 scans. 1D NMR spectrosco-py data were processed with TopSpin 2.1.6 software (Bruker Instruments)and analyzed by MestReNova 7.0.3 program (MestreLab Research S.L.).2D NMR spectroscopy data were processed with NMRPipe[22] and ana-lyzed with NMRViewJ (One Moon Scientific).

A combination of 1D, TOCSY and NOESY experiments was employedto assign the spectra of peptides treated with either ZnII or 1. To assignthe chemical shifts of the amino acids, protons in the peptides were firstclassified into spin systems using 1H-1H TOCSY spectra acquired with20 ms and 60 ms mixing times. Subsequently, sequence-specific assign-ments were determined using backbone NHi-CHai and CHai-NHi+1 cor-relations of the 1H-1H NOESY spectra with 150 and 300 ms mixing times.

NMR spectroscopy experiments for HR1 were performed on a BrukerAvance III 600 MHz spectrometer equipped with a broadband multinu-clear 5 mm inverse BBI probe optimized for 1H applications. Sampleswere 5 mm in 2 or peptide concentration and dissolved in 90:10 (v/v)H2O/D2O solution buffered at pH 7.0 with 100 mm deuterated [D11]Tris.For chemical-shift determination experiments, peptides were co-incubat-ed with 2.2 equiv of either ZnSO4·7H2O or 1 for at least 1 h at 37 8C anddegassed for at least 2 h prior to data acquisition. Peptide 1D, TOCSYand NOESY experiments for chemical-shift evaluation were performedsimilarly to ZF4 and CP1 but at 10 8C in 5 mm NMR tubes. Data wereprocessed and analyzed similarly to ZF4 and CP1. Similar NMR spec-troscopy experiments were performed on 2, a substitutionally inert [Co-ACHTUNGTRENNUNG(acacen)(L)2]

+ complex. Since the 4-methylimidazole ligand of 2 is in-tended to mimic the His imidazole side chain, 2 was implemented asa small molecule model of [CoACHTUNGTRENNUNG(acacen) ACHTUNGTRENNUNG(His)2]

+ and the chemical shiftswere compared to those of the 1–peptide adducts.

CD spectroscopy: CD experiments were performed on a Jasco J-815 CDspectrometer at 25 8C. For experiments evaluating the [Co ACHTUNGTRENNUNG(acacen)(L)2]

+

intra ACHTUNGTRENNUNGli ACHTUNGTRENNUNGgand transfer band, samples were either 125 mm in HR1 concentra-tion (with or without varying concentrations of 1) or 62.5 mm [Co-ACHTUNGTRENNUNG(acacen)(L)2]

+ in the absence of peptide. The aqueous solutions weremaintained at pH 7.0 with 10 mm Tris buffer. For 1–HR1 samples, peptideand 1 were co-incubated at varying molar equivalents (1/peptide =1.0,2.0, or 4.0) for at least 1 h at 37 8C. The spectra of 10 mm Tris at pH 7.0was subtracted from all the sample spectra. Experiments were performedat 25 8C in 1 mm Hellma cuvettes. CD signal was measured from 190 to400 nm with five accumulations at a scan-rate of 1 nm min�1.

For CD experiments evaluating zinc-finger structure, samples were33.6 mm in ZF4 concentration. The aqueous solutions were maintained atpH 7.0 with 5 mm phosphate buffer instead of Tris buffer to minimizebackground signal in the far-UV region. For Zn–ZF4 samples, ZF4 wasco-incubated with varying molar equivalents of ZnII (ZnSO4·7H2O) andincubated at least 1 h at 37 8C. For samples treated with 1, Zn–ZF4 at4.0 molar equivalents of ZnII-to-peptide was co-incubated with varyingmolar equivalents of 1 for at least 1 h at 37 8C. For comparison, the sameexperiments were performed with 2 as with 1. Since 2 is less substitution-ally labile than 1, its effects were tested to correlate structural perturba-tions to reactivity by dissociative ligand exchange. Experiments were per-formed at 25 8C in 1 mm Hellma cuvettes. CD signal was measured from190 to 260 nm with five accumulations at a scan-rate of 1 nm min�1. AllCD data were processed and analyzed with CDPro software (ColoradoState University).

Electronic absorption spectroscopy : 1, 2 and 1–HR1 samples for elec-tronic absorption spectroscopy were prepared in the same way as for CDspectroscopy experiments evaluating the [Co ACHTUNGTRENNUNG(acacen)(L)2]

+ intra ACHTUNGTRENNUNGli ACHTUNGTRENNUNGgandtransfer band. Electronic absorption data were recorded on a Hewlett–Packard HP 8453 diode array spectrophotometer at 25 8C. First derivativeplots were produced using GraphPad Prism 6 program (GraphPad Soft-ware, Inc.).

Additional experimental information of concentration-dependent1H NMR spectroscopy, ESI-MS analysis, and free thiol analysis with Ell-man�s reagent can be found in the Supporting Information.

Acknowledgements

The authors gratefully acknowledge Natsuho Yamamoto, Allison S.Harney, Robert J. Holbrook, Daniel J. Feld, and Lauren M. Matosziukfor helpful discussions. The authors gratefully acknowledge JosephCoomes, Viktorie Reichova, and Emily Testa for technical support.M.C.H. would like to acknowledge the National Science FoundationGraduate Research Fellowship. Portions of this work were completed atthe Integrated Molecular Structure Education and Research Center,Keck Biophysics Facility, and Biomolecular NMR Center at Northwest-ern University; and the Center of Structural Biology at the University ofIllinois, Chicago through the Chicago Biomedical Consortium. This workwas supported by funding from the Center of Cancer NanotechnologyExcellence (CCNE) initiative of the National Institutes of Health Nation-al Cancer Institute under award U54A119341.

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Biomol. NMR 1995, 6, 277.Received: April 30, 2013Revised: October 5, 2013

Published online: November 6, 2013

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