Synthesis of a Metallopeptide− PNA Conjugate and Its Oxidative

Bioconjugate Chem. 2005, 16, 178−183

178

Synthesis of a Metallopeptide-PNA Conjugate and Its Oxidative Cross-Linking to a DNA Target Olga Kornyushyna, Ann J. Stemmler, Daina M. Graybosch, Ikuko Bergenthal, and Cynthia J. Burrows* Department of Chemistry, University of Utah, 315 S. 1400 East, Salt Lake City, Utah 84112-0850. Received October 8, 2004; Revised Manuscript Received November 18, 2004

A nickel(II)-PNA bioconjugate was prepared by formation of a salicylaldimine complex with the amino terminus of a peptide-PNA hybrid with the sequence Arg-His-Gly-[TACCTAGCAT]PNA-Arg-CONH2. Hybridization to complementary oligodeoxynucleotides was demonstrated, and covalent adduct formation was observed upon addition of KHSO5 as oxidant. In the absence of PNA, the reactivity of the phenolic radical generated as an intermediate was found to be G . T . C, A; by inclusion of the PNA delivery agent, cross-links between the two oligomers could be observed with T and C bases in the vicinity of the nickel complex, although G was still the most reactive site. The metal complex could be removed by treatment with EDTA following which the Schiff base linkage was readily hydrolyzed. The final result in this case is a salicylaldehyde moiety appended at the target site in DNA.

INTRODUCTION

Scheme 1. Design of the M(sal-XH) Motif

Both nucleic acids and proteins are rich in nucleophilic sites as well as containing heterocyclic components susceptible to oxidation. Oxidation of either of the two biopolymers generates an electron-deficient site that can lead to covalent bond formation with a nucleophilic partner. For example, oxidative adducts to DNA bases include the well characterized addition of tyrosine’s phenol group to both cytosine and thymine heterocycles as part of the molecular basis of DNA-protein crosslinking (1, 2). Additionally, our laboratory has shown that a phenolic radical generated as part of a metallosalicylaldehyde complex undergoes facile addition to guanine forming a covalent adduct to RNA or DNA (3-5). Such a radical can be effectively produced with a nickel(II) salen complex (Scheme 1, M ) Ni) whose one-electron oxidation is predominantly centered on the ligand rather than the metal (6). The N-terminal peptide sequence XXH (X ) any amino acid) is also a good binding site for nickel(II) producing a redox-active complex and allowing conjugation of the complex to any peptide or protein sequence via elaboration of the C-terminus of XXH to include a DNA-binding sequence. Indeed, both metallosalens and metallopeptides have served as reactive components of bioconjugates for site-selective modification of DNA (710). By combining these two motifs, the ability of nickel(II) salen to form oxidative adducts (11) and the peptide features of the NiXXH complex (12), we designed the Ni(sal-XH) complex. In this complex, the minimal peptide unit is a dipeptide in which the second amino acid is histidine to provide a strong coordination site for the metal, and the first amino acid is any primary aminecontaining R-amino acid (i.e. X * proline) such that the amino terminus can form a Schiff base with salicylaldehyde (Scheme 1). We previously reported the structure and properties of a prototype complex, Ni(sal-RH), the first tetradentate * To whom correspondence should be addressed. E-mail: [email protected].

metal complex of a peptide-salicylaldimine (13). Crystallographic analysis showed the formation of a highly planar nickel(II) complex with coordination to the phenolate oxygen, the imine nitrogen, one deprotonated amide nitrogen, and an imidazole nitrogen with no additional axial ligands. An arginine residue at the N-terminal position enhanced both water solubility of the complex and its electrostratic interaction with DNA. Oxidation of the nickel(II) complex could be triggered in either of two ways, using the strong oxidant KHSO5, or by adding a reducing agent, HSO3- to an aerobic aqueous solution of Ni(sal-RH). In the latter case, the nickel complex serves initially as a catalyst for in situ autoxidation of sulfite to HSO5- (14-16) and subsequently as a substrate for ligand-centered oxidation. In the initial study, we found guanine bases in oligodeoxynucleotides exhibited the highest intrinsic reactivity with the Ni(sal-RH) complex under oxidative conditions (13), as was the case for other nickel salentype complexes (3, 5, 17, 18). Our subsequent goal was to explore the utility of Ni(sal-XH) as a DNA modifying agent under conditions where the salicylaldimine radical could be delivered site specifically to bases other than guanine. Targeted delivery of DNA alkylating agents has

10.1021/bc0497564 CCC: $30.25 © 2005 American Chemical Society Published on Web 12/21/2004

Metallopeptide−PNA Cross-Linking to DNA Scheme 2. Structure of 1, Ni(sal-RH)G[TACCTAGCAT]PNA-RCONH2

been demonstrated for a variety of conjugates to distamycin analogues (19, 20), and metallopeptides have been used for site-specific cleavage of nucleic acids via conjugation to proteins (9), distamycin-like polyamides (21), and PNA (7). In the present work, we examine the ability of PNA conjugates of the Ni(sal-XH) motif to covalently attach the salicylaldehyde unit to various bases of an oligodeoxynucleotide in order to demonstrate the utility of the Ni(sal-XH) to ultimately modify DNA with a unique aldehyde moiety. Peptide nucleic acid (PNA) can serve as an ideal delivery agent to target specific sites in nucleic acids due to its exceptional properties for nucleic acid binding and ease of synthesis (22). PNAs have high binding affinity for DNA because of their neutral charge, and because PNA-DNA duplexes show higher melting temperatures than DNA-DNA duplexes, shorter sequences can be used to impart high stability to a duplex. PNA is less tolerant of base pair mismatches than is DNA; a single mismatch typically leads to an 8-20 °C lowering of the Tm in a PNA-DNA duplex ensuring higher sequence selectivity than for DNA-DNA duplexes (23-25). We therefore designed the Ni(sal-XH)-PNA conjugate, 1 (Scheme 2), that includes a PNA decamer conjugated to an Nterminal RHG peptide for formation of the appropriate redox-active nickel complex. Its reactivity toward complementary targets presenting any of the four natural bases in proximity to the metal complex was then examined. EXPERIMENTAL PROCEDURES

Synthesis of NH2-RHG-[TACCTAGCAT]-R-CONH2. The PNA-peptide conjugate was prepared at a 0.1 mmol scale using Rink Amide AM resin (0.145 g) to build the conjugate. Fmoc- and Bhoc-protected monomers for PNA synthesis were purchased from Perseptive Biosystems; other protected amino acids were purchased from NovaBiochem. The initial arginine at the C-terminus was coupled to the resin using standard peptide synthesis protocols. The solvent system was DMF, the activating agent was N-methylmorpholine in DMF, and the deprotecting agent was 20% piperidine in DMF. The peptide synthesizer was then switched to an NMP (N-methylpyrrolidine)-based solvent system (20% DMSO in NMP) (26) for addition of the Bhoc-protected PNA monomers. The activating agent was 10% DIPEA (N,N-diisopropylethylamine) and 10% lutidine in 20% DMSO/NMP, and the deprotecting agent was 30% piperidine in 20% DMSO/ NMP. Each PNA monomer (0.1 mmol) was weighed into vials along with HBTU (0.3 mmol). An endcapping

Bioconjugate Chem., Vol. 16, No. 1, 2005 179

reagent (3 mL) was used that consisted of 5% acetic anhydride and 6% lutidine in 20% DMSO/NMP. The mixing times and activating/deprotecting steps reflected published protocols (22). Next, the solvent system of the synthesizer was switched back to DMF, as described above, to add the three N-terminal amino acids. FmocGly-OH, Fmoc-His(trt)-OH, and Fmoc-Arg(pbf)-OH were added sequentially using standard protocols. Endcapping was achieved using acetic anhydride. The final step was deprotection of the last amino acid without endcapping. The PNA-peptide was cleaved from the resin using a cleavage cocktail of 95% trifluoroacetic acid, 4% m-cresol (necessary to cleave Bhoc protecting group) and 1% triisopropylsilane (a radical scavenger). The cocktail was shaken with the beads at room temperature for 1 h. The PNA-peptide conjugate was dried and washed three times with diethyl ether and then dried by evaporation. The sample was HPLC purified using an analytical C18 column (Viadac, 4.6 × 250 mm). The dried sample was dissolved in 35 mL of 0.1% TFA and filtered. A gradient from 100% solvent A (0.1% aqueous TFA) to 18% solvent B (acetonitrile) was used. The peak at 26 min. was analyzed by ESI-LC-MS (see Supporting Information) and showed a molecular mass at 3193.4 (calcd ) 3193.7). Synthesis and Purification of DNA Oligomers. Oligodeoxynucleotides were synthesized using standard automated solid-support chemistry protocols on an Applied Biosystems Model 392B synthesizer using the manufacturer’s protocols. Gel electrophoresis on 20% polyacrylamide gels with 7M urea at 40 W provided the purified oligodeoxynucleotides. The purity of synthesized oligodeoxynucleotides was confirmed by mass spectral analysis on a Micromass Quattro II instrument. Electrophoretic Mobility Shift Assays. The 14nt oligodeoxynucleotides were 5′-end labeled using T4 polynucleotide kinase and [(γ-32P] ATP. Unreacted [(γ-32P] ATP was separated from labeled oligonucleotides with MicroSpin G-25 columns (Amersham Pharmacia Biotech). DNA oligonucleotides were annealed to the PNApeptide strand in 10 µL solutions of 10 mM NaH2PO4. The DNA concentration was kept constant (2.2 µM) while the PNA-peptide concentration varied from 0 to 2.2 µM. The resultant duplexes were applied to a 15% nondenaturing polyacrylamide gel and subjected to electophoresis at 2 W for 4.5 h in the cold box (4 °C). Radiolabeled duplex formation was quantified by storage phosphor autoradiography (Molecular Dynamics Storm 860). Formation of the 1:DNA Duplexes. 5′-[32P]-Endlabeled oligodeoxynucleotides (3N14, N ) A, C, G, T) were annealed to the PNA-peptide strand in 200 µL solutions containing 100 mM NaCl plus10 mM NaH2PO4 (pH ) 8) to yield 1 µM solutions of DNA/PNA-peptide duplexes. Ni(sal)2 was prepared by mixing stoichiometric amounts of Ni(OAc)2 in 4:1 ethanol:water and salicylaldehyde in ethanol and collection of the precipitate. Approximately 1.5 mg of Ni(sal)2 was added to each duplex solution and shaken overnight in the cold box at 4 °C. Excess amounts of Ni(sal)2 were removed by filtration through SPIN-X columns, followed by washes with 100 µL of distilled H2O (final volume ) 300 µL). The solution volume was then reduced to 100 µL to yield 2 µM 1:DNA duplex. Cross-Link Formation. A 20-µL aliquot of 2 µM radiolabeled 1:DNA duplex was added to 4 µL of 1 mM KHSO5 in a 40-µL solution of 100 mM NaCl/10 mM NaH2PO4 (pH ) 7) and incubated for 15 min at room temperature. Reactions were quenched by adding 2 µL of 20 mM EDTA and 2 µL of 50 mM HEPES followed by dialysis

180 Bioconjugate Chem., Vol. 16, No. 1, 2005

Figure 1. Sequences of the peptide-PNA conjugate and oligodeoxynucleotides used in these studies.

overnight. Oxidized and unmodified 1:DNA duplex was then treated with 0.2 M piperidine for 30 min at 90 °C. Both unmodified and oxidized 1:DNA duplex not treated with piperidine were used as controls in these experiments. All reactions were lyophilized to dryness, resuspended in 4 µL of loading buffer, applied to a 15% polyacrylamide gel in the presence of 7M urea, and subjected to electrophoresis for 3 h at 40 W. Formation of the high molecular weight products was then monitored by storage phosphor autoradiography (Molecular Dynamics Storm 860). Adduct Formation of Ni(sal-RH) with Plasmid DNA. Ni(sal-RH) was prepared as previously described (13). Reactions with the single-stranded plasmid from M13mp18 phage were conducted using 100 µM Ni(salRH) and 50 µM Na2SO3 using the same reaction and buffer conditions as for oligonucleotide work. Primer extension and PCR were performed using 5′-[32P]-end labeled d(GTTTTCCCAGTCACGAC), the -40 primer relative to the Hind III restriction site, according to the procedure for the Thermo Sequenase cycle sequencing kit (Amersham). Analysis and imaging was conducted as described above. RESULTS AND DISCUSSION

The peptide bioconjugate 1 was designed to comprise a 10-mer PNA sequence for hybridization to its complementary sequence in DNA along with the N-terminal XH motif for formation of the redox-active Ni(sal-XH) complex. A glycine spacer was added between the metal ligating peptide and the PNA sequence, and arginines were included at both the N terminus and the C terminus for additional water solubility and electrostratic interactions with DNA. Although lysine residues could also have served this purpose, they may have interfered with Schiff base formation at the amino-terminal site. Finally, the C terminus of the oligomer was left as a neutral amide rather than a carboxylate group by synthesis on Rink Amide resin, again for facilitating binding to DNA. The synthesis of the peptide-PNA portion of 1 was achieved through a combination of standard protocols for peptide and for PNA synthesis on solid support. The nickel salicylaldimine complex of the peptide could be prepared either before or after hybridization to the DNA complement. The completed synthesis of 1 was analyzed by ESIMS (see Supporting Information) after HPLC purification of the peptide-PNA ligand. The PNA conjugate 1 was tested for hybridization to DNA complements by both thermal melting studies and gel mobility shift analysis using the four oligodeoxynucleotides shown in Figure 1. Each of the 14-mers contained the 10-mer complementary sequence to the PNA segment at its 5′ end, a one-nucleotide spacer of deoxyadenosine, thought to be the least reactive of the four bases, and a trinucleotide NNN sequence at the 3′ end that would be oriented in proximity to the Ni(sal-RH) group and would serve as the target sites for oxidative adduct formation. Tm values were above 55 °C, and a gel shift experiment indicated tight binding between the PNA-peptide con-

Kornyushyna et al.

Figure 2. PNA-peptide binding examined by the gel mobility shift assay. In a total volume of 10 µL, a radiolabled DNA (3T14) was incubated with various amounts of a complementary PNApeptide as described in the Experimental Procedures. The samples were analyzed on the 15% nondenaturing polyacrylamide gel. PNA concentrations in lanes 1-10 were 0, 0.011, 0.022, 0.034, 0.069, 0.14, 0.28, 0.55, 1.1, and 2.2 µM.

Figure 3. DNA-PNA cross-link formation after incubation DNA + bioconjugate 1 with KHSO5 as described in the Experimental Procedures. For each series, lane 1 contains unoxidized DNA‚1 in buffer; lane 2 contains unoxidized DNA‚1 treated with 0.2 M piperidine; lane 3 - DNA‚1 incubated with 100 µM KHSO5; lane 4 - DNA‚1 incubated with 100 µM KHSO5 and treated with 0.2 M piperidine. All reactions were subjected to electrophoresis on a denaturing 15% polyacrylamide gel. The positions of the high molecular weight products are indicated by the arrow.

jugate and each of the 3N14 complements. A representative assay is shown in Figure 2 for 3T14. To assess the ability of the PNA-Ni(sal-RH) bioconjugate to form covalent cross-links to target oligodeoxynucleotides, we analyzed both oxidation and adduct formation by denaturing gel electrophoresis (Figure 3). For each of the DNA 14-mers containing the complement to the PNA sequence plus a 3′-NNNA overhang near the Ni(sal-RH) complex, the 1:DNA duplex was treated with KHSO5 to trigger oxidation of the nickel(II) complex. Because nickel-salicylaldimine complexes typically undergo ligand-centered oxidation, the resulting phenolic radical is expected to couple to one of the 3′ overhanging nucleotides. For each set of experiments, the first two lanes represent control studies without oxidant and -/+ piperidine treatment. Piperidine treatment would be expected to result in strand scission at sites of base oxidation and/or adduct formation (27). Lanes 3 and 4 show adduct formation and strand scission -/+ piperidine treatment, respectively. It is clear from the results in Figure 3 that guanosine nucleotides retain the highest level of reactivity toward oxidative cross-link formation with thymidine also showing substantial adduct formation. The ability of dC to undergo adduct formation was apparent but small, while that of dA was almost nonexistent. Treatment of the

Bioconjugate Chem., Vol. 16, No. 1, 2005 181

Metallopeptide−PNA Cross-Linking to DNA Scheme 3. Proposed Mechanism of Cross-Link Formation and Hydrolysis

cross-linked duplexes with piperidine led to some loss of intensity presumably due to strand scission as was previously shown for the parent complex, Ni(sal-RH) (13). In all cases, the overhanging nucleotides appear also to be subject to oxidative damage leading to strand scission as evidenced by the bands observed below starting material on the gel. This is likely due to the rather high concentration of KHSO5 employed in the reaction (100 µM). Previously, we found that Na2SO3 could be used in place of KHSO5 wherein the nickel complex served to catalyze in situ oxidation of sulfite to monoperoxysulfate, avoiding the use of excess oxidant (13); however, trials with Na2SO3 in the present system led to lower yields of cross-link formation as well. On the basis of prior work in our laboratory and others, we propose the following mechanism of covalent adduct formation. First, the PNA strand hybridizes with DNA delivering the redox-active nickel complex to the vicinity of the 3′-overhang of the DNA target strand. Reaction with KHSO5 as oxidant (or nickel-catalyzed autoxidation of Na2SO3) leads to ligand-centered oxidation generating a phenol radical on the salicylaldimine moiety. Radical attack at C8 of guanine (Scheme 3) or the 5,6 double bond of T or C leads to covalent adduct formation. The adducts appear to be sensitive to piperidine treatment, giving rise to strand scission at the site of cross-linking under Maxam-Gilbert conditions (Figure 3, lanes 4). In the case of guanine C8 phenolic adducts, we previously showed that the adducts were highly sensitive to further oxidation by a pathway analogous to that of 8-oxoguanosine, leading to a guanidinohydantoin derivative. This further oxidation product likely accounts for the additional highmolecular weight band observed by PAGE (Figure 3) under conditions where relatively high concentrations of oxidant were used. This was well characterized for the parent compound Ni(sal-RH) by ESI-MS analysis of the various adducts isolated from the gel (13). Furthermore, it was demonstrated that the peptide delivery vehicle could be released from its target by treatment with EDTA. Removal of the coordinating nickel ion presumably sensitizes the salicylaldimine to hydrolysis. The final product after EDTA treatment is therefore the original strand with a salicylaldehyde moiety tagging the site of adduct formation. The extent of reaction at the various positions of the NNN overhang were analyzed after piperidine treatment. While all three positions were reactive, there was in each case a slight preference for reaction at the internal positions, N12 and N13, as opposed to the final nucleotide,

Figure 4. Primer extension analysis of single-stranded phage DNA (M13mp18) cross-linked to Ni(sal-RH). A portion of the sequence analyzed is shown at left with reactive sites in boldface. Lanes T, C, A, G are Sanger sequencing lanes of the single-stranded phage using ddATP, ddGTP, ddTTP, and ddCTP, respectively. Lanes 1-4 are reaction lanes. Cross-linking of Ni(sal-RH) with ss phage M13mp18 sterically inhibits Thermo Sequenase and hence primer extension stops before bases crosslinked to Ni(sal-RH). Control lane 5 demonstrates the effect of Na2SO3 in the absence of Ni(sal-RH). Control lane 6 is primer extension in the absence of Na2SO3 and Ni(sal-RH).

N14 (see Supporting Information), and the trend was the same for all bases except T, for which all three positions were equally reactive. An explanation consistent with these data is that bioconjugate 1 positions the reactive phenol radical nearest the bases that are 2-3 nucleotides beyond the complementary duplex region. To examine whether the observed reactivity of the nickel salicylaldimine group with T or C, in addition to G, could be found in a larger sample of DNA, we analyzed the single-stranded plasmid M13mp18 with the parent complex, Ni(sal-RH), lacking the PNA delivery unit.

182 Bioconjugate Chem., Vol. 16, No. 1, 2005

Kornyushyna et al.

Scheme 4. Utility of the Ni(sal-XH) Motif in Bioconjugates for Delivery of an Aldehyde Functional Group to Specific Sites in DNA

Conditions were chosen under which we previously observed a high degree of adduct formation as opposed to simple oxidative damage. These conditions involved the use of relatively low concentrations of sulfite such that the strong oxidant HSO5- was only formed via nickel-mediated autoxidation, and the concentration of HSO5- remained negligible. Oxidative adduct formation was analyzed using Thermo Sequenase, an exonucleasefree thermostable DNA polymerase. Primer extension was inhibited by the presence of an adduct, and thus the bands analyzed in lanes 1-4 of Figure 4 represent adduct sites that are one nucleotide shorter than the dideoxy sequencing lanes for which insertion opposite the template base has occurred. The data shown represent a small segment of a larger analysis. In general, reactivity still was seen only at guanines; however, a small number of thymines were also observed to form adducts. These data is therefore in keeping with the assessment that the overall reactivity preference of the phenolic radical generated from the Ni(sal-RH) complex is G > T > C . A. CONCLUSIONS

We have demonstrated that a Schiff base complex of nickel(II) can be readily appended to a synthetic peptidePNA conjugate for oxidative cross-linking to a complementary strand of DNA. Minimal requirements for the formation of the nickel complex are an N-terminal XH dipeptide motif in which X represents any R-amino acid with a primary amine available for imine formation with salicylaldehyde. The nickel(II) complex can then be prepared before or after conjugation of PNA to its DNA target. Oxidation with KHSO5, or in situ formation of this oxidant by nickel-catalyzed sulfite autoxidation, leads to oxidative coupling between the phenol ring of salicylaldehyde and a DNA base. Although the intrinsic reactivity of the phenol radical is greatest toward guanosine (G > T > C > A), the reactivity of T and C is enhanced by delivery of the redox-active metal complex in proximity to these bases by the PNA conjugate. Even with delivery, however, adenine remains low in reactivity. The ability to remove the metal complex and its PNA targeting agent by simple addition of EDTA is a further benefit of this design. The final result is a DNA strand whose targeted site bears a salicylaldehyde tag (Scheme 4). Such a unique aldehyde may be further functionalized with aldehyde reactive reagents leading to site-specific biotinylation, fluorescent labeling, or cross-linking to DNA binding proteins via reductive amination. Such features greatly expand the arsenal of metallo-PNA conjugates that has recently seen development in other laboratories as well (28-33).

ACKNOWLEDGMENT

We thank Dr. James G. Muller for assistance with mass spectrometry. This work was supported by a grant from the National Science Foundation (0137716). Supporting Information Available: ESI-MS characterization of 1 and quantitative analysis of PAGE results for site reactivity of 1 in oligo targets. This material is available free of charge via the Internet at http:// pubs.acs.org. LITERATURE CITED (1) Margolis, S. A., Coxon, B., Gajewski, E., and Dizdaroglu, M. (1988) Structure of a hydroxyl radical induced cross-link of thymine and tryosine. Biochemistry 27, 6353-6359. (2) Gajewski, E., and Dizdaroglu, M. (1990) Hydroxyl radical induced cross-linking of cytosine and tyrosine in nucleohistone. Biochemistry 29, 977-980. (3) Woodson, S. A., Muller, J. G., Burrows, C. J., and Rokita, S. E. (1993) A primer extension assay for modification of guanine N7 by Ni(II) complexes. Nucleic Acids Res. 21, 55245525. (4) Muller, J. G., Paikoff, S. J., Rokita, S. E., and Burrows, C. J. (1994) DNA modification promoted by water-soluble nickel(II) salen complexes: A switch to DNA alkylation. J. Inorg. Biochem. 54, 199-206. (5) Muller, J. G., Kayser, L. A., Paikoff, S. J., Duarte, V., Tang, N., Perez, R. J., Rokita, S. E., and Burrows, C. J. (1999) Formation of DNA adducts using nickel(II) complexes of redox-active ligands: A comparison of salen and peptide complexes. Coord. Chem. Rev. 185-186, 761-774. (6) Goldsby, K. A. (1988) Symmetric and unsymmetric nickel(II) Schiff base complexes: Metal-localized versus ligandlocalized oxidation. J. Coord. Chem. 19, 83-90. (7) Footer, M., Egholm, M., Kron, S., Coull, J. M., and Matsudaira, P. (1996) Biochemical evidence that a D-loop is part of a four-stranded PNA-DNA bundle. Nickel-mediated cleavage of duplex DNA by a Gly-Gly-His Bis-PNA. Biochemistry 35, 10673-10679. (8) Routier, S., Bernier, J.-L., Catteau, J.-P., and Bailly, C. (1997) Recognition and cleavage of DNA by a distamycinsalen-copper conjugate. Bioorg. Med. Chem. Lett. 7, 17291732. (9) Harford, C., Narindrasorasak, S., and Sarkar, B. (1996) The designed protein M(II)-Gly-Lys-His-Fos(138-211) specifically cleaves the AP-1 binding site containing DNA. Biochemistry 35, 4271-4278. (10) Long, E. C. (1999) Ni(II)-Xaa-Xaa-His metallopeptide- DNA/RNA interactions. Acc. Chem. Res. 32, 827-836. (11) Rokita, S. E., and Burrows, C. J. (2003) in Small Molecule DNA and RNA Binders (Demeunynck, M., Bailly, C., and Wilson, W. D., Eds.) pp 126-145, Wiley-VCH, Weinheim. (12) Harford, C., and Sarkar, B. (1997) Amino terminal Cu(II)and Ni(II)-binding (ATCUN) motif of proteins and peptides: Metal binding, DNA cleavage, and other properties. Acc. Chem. Res. 30, 123-130.

Bioconjugate Chem., Vol. 16, No. 1, 2005 183

Metallopeptide−PNA Cross-Linking to DNA (13) Stemmler, A. J., and Burrows, C. J. (1999) The Sal-XH mofif for metal-mediated oxidative DNA-peptide crosslinking. J. Am. Chem. Soc. 121, 6956-6957. (14) Muller, J. G., Hickerson, R. P., Perez, R. J., and Burrows, C. J. (1997) DNA damage from sulfite autoxidation catalyzed by a nickel(II) peptide. J. Am. Chem. Soc. 119, 1501-1506. (15) Lepentsiotis, V., Domagala, J., Grgic, I., van Eldik, R., Muller, J. G., and Burrows, C. J. (1999) Mechanistic information on the redox cycling of Ni(II/III) complexes in the presence of sulfur oxides and oxygen. Correlation with DNA damage experiments. Inorg. Chem. 38, 3500-3505. (16) Wietzerbin, K., Pratviel, G., Bernadou, J., Meunier, B., Muller, J. G., Jameton, R. A., and Burrows, C. J. (1999) Hydroxylation, epoxidation and DNA cleavage reactions mediated by the biomimetic MnTMPyP/O2/sulfite oxidation system. Inorg. Chem. 38, 4123-4127. (17) Zhou, X., Shearer, J. M., and Rokita, S. E. (2000) A Ni(salen)-biotin conjugate for rapid isolation of accessible DNA. J. Am. Chem. Soc. 122, 9046-9047. (18) Mandal, S. S., Varshney, U., and Bhattacharya, S. (1997) Role of the central metal ion and ligand charge in the DNA binding and modification by metallosalen complexes. Bioconjugate Chem. 8, 798-812. (19) Bailly, C., and Chaires, J. B. (1998) Sequence-specific DNA minor groove binders. Design and synthesis of netropsin and distamycin analogues. Bioconjugate Chem. 9, 513-538. (20) Kumar, D., Veldhuyzen, W. F., Zhou, Q., and Rokita, S. E. (2004) Conjugation of a hairpin pyrrole-imidazole polyamide to a quinone-methide for control of DNA cross-linking. Bioconjugate Chem. 15, 915-922. (21) Bailly, C., Sun, J.-S., Colson, P., Houssier, C., He´le`ne, C., Waring, M. J., and Henichart, J.-P. (1992) Design of a sequence-specific DNA-cleaving molecule which conjugates a copper-chelating peptide, a netropsin residue, and an acridine chromophore. Bioconjugate Chem. 3, 100-103. (22) Nielsen, P. E., and Egholm, M. (1999) Peptide Nucleic Acids: Protocols and Applications, Horizon Scientific Press, Wymondham, UK. (23) Ratilainen, T., Holmen, A., Tuite, E., Nielsen, P. E., and Norden, B. (2000) Thermodynamics of sequence-specific binding of PNA to DNA. Biochemistry 39, 7781-7791.

(24) Ratilainen, T., Holmen, A., Tuite, E., Haaima, G., Christensen, L., Nielsen, P. E., and Norden, B. (1998) Hybridization of peptide nucleic acid. Biochemistry 37, 12331-12342. (25) Jensen, K., Orum, H., Nielsen, P. E., and Norden, B. (1997) Kinetics for hybridization of peptide nucleic acids (PNA) with DNA and RNA studied with the BIAcore technique. Biochemistry 36, 5072-5077. (26) Thomson, S. A., Josey, J. A., Cadilla, r., Gaul, M. D., Hassman, C. F., Luzzio, M. J., Pipe, A. J., Reed, K. L., Ricca, D. J., Wiethe, R. W., and Noble, S. A. (1995) Fmoc mediated synthesis of peptide nucleic acids. Tetrahedron 51, 61796194. (27) Burrows, C. J., and Muller, J. G. (1998) Oxidative nucleobase modifications leading to strand scission. Chem. Rev. 98, 1109-1151. (28) Petersen, L., de Koning, M. C., van Kuik-Romeijn, P., Weterings, J., Pol, C. J., Platenburg, G., Overhand, M., van der Marel, G. A., and van Boom, J. H. (2004) Synthesis and in vitro evaluation of PNA-peptide-DETA conjugates as potential cell penetrating artificial ribonucleases. Bioconjugate Chem. 15, 576-582. (29) Mokhir, A., Kraemer, R., and Wolf, H. (2004) Zn2+Dependent peptide nucleic acids probes. J. Am. Chem. Soc. 126, 6208-6209. (30) Mokhir, A., Kraemer, R., Voloshin, Y. Z., and Varzatskii, O. A. (2004) Synthesis and DNA binding properties of dioxime-peptide nucleic acids. Bioorg. Med. Chem. Lett. 14, 2927-2930. (31) Mokhir, A. A., and Kraemer, R. (2003) Conjugates of PNA with naphthalenediimide derivatives having a broad range of DNA affinities. Bioconjugate Chem. 14, 877-883. (32) Verheijen, J. C., van der Marel, G. A., van Boom, J. H., and Metzler-Notle, N. (2000) Transition metal derivatives of peptide nucleic acid (PNA) oligomersssynthesis, characterization, and DNA binding. Bioconjugate Chem. 11, 741-743. (33) Bigey, P., Soennichsen, S. H., Meunier, B., and Nielsen, P. E. (1997) DNA Binding and cleavage by a cationic manganese porphyrin-peptide nucleic acid conjugate. Bioconjugate Chem. 8, 267-270.

BC0497564