Optimizing the Targeted Chemical Nuclease Activity of 1,10

James Gallagher, Chi-hong B. Chen, Clark Q. Pan, David M. Perrin, Young-Moon Cho, and. David S. Sigman*. Department of Biological Chemistry, School of...
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Bioconjugate Chem. 1996, 7, 413−420

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Optimizing the Targeted Chemical Nuclease Activity of 1,10-Phenanthroline-Copper by Ligand Modification James Gallagher, Chi-hong B. Chen, Clark Q. Pan, David M. Perrin, Young-Moon Cho, and David S. Sigman* Department of Biological Chemistry, School of Medicine, and Department of Chemistry and Biochemistry, Molecular Biology Institute, University of California, Los Angeles, California 90095-1570. Received January 26, 1996X

Our interest in improving the efficiency of targeted scission reagents has prompted us to study the influence of ring substituents on the nuclease activity of 1,10-phenanthroline-copper conjugated to oligonucleotides and DNA-binding proteins. Since methyl substitution at all but the 2 and 9 positions enhances the copper-dependent chemical nuclease activity of 1,10-phenanthroline, we have compared the reactivity of conjugates prepared from 5-(aminomethyl)-1,10-phenanthroline (MOP) to those of conjugates prepared from 5-amino-1,10-phenanthroline (amino-OP). Tethering MOP derivatives to the Escherichia coli Fis protein enhances DNA scission several-fold at the weaker cleavage sites initially observed with conjugates prepared from amino-OP. However, scission efficiency is not increased at the stronger cleavage sites, or when scission is targeted to single-stranded DNA by a complementary oligonucleotide. These results are consistent with a change in the rate-determining step for cleavage associated with the differential accessibility of the DNA-bound coordination complex to solvent and reductant. Although the free bis cuprous complex of 2,9-dimethyl-1,10-phenanthroline (neocuproine) is redox-inactive, an oligonucleotide tethered to neocuproine through C5 of the phenanthroline ring efficiently cleaves a complementary DNA sequence. These results establish that the nucleolytic species in targeted scission is the 1:1 cuprous complex and suggest that the oxidative reaction proceeds through a copper-oxo intermediate rather than a metal-coordinated peroxy species. However, substituents at the 2 and 9 positions of the ligand will often hinder close approach of the phenanthroline-copper moiety to the oxidatively sensitive ribose as shown by the preference of the oligonucleotide-targeted chimera for cleavage of single-stranded regions and the failure of neocuproine-DNA-binding protein chimeras and a C2-tethered chimera to cleave DNA.

INTRODUCTION

The generation of new functional biomolecules has been the focus of current research with catalytic antibodies (1), ribozymes (2), in vitro selection schemes (3), and site-directed mutagenesis (4-6). Functionalization of biomolecules by conjugation with the cleavage reagent 1,10-phenanthroline-copper(I) constitutes another direction in the development of reactive molecules with designed specificities (7-9). This redox-active coordination complex has proven especially useful in the development of highly specific chemical nucleases and proteases (9) since it does not generate diffusible hydroxyl radicals but rather reacts via a copper-oxo species (10). Several factors contribute to the specificity of chimeric reagents prepared with 1,10-phenanthroline-copper. Preeminent among these are the orientation and proximity of the redox-active copper ion to the oxidatively sensitive site (11). However, the intrinsic reactivity of the copper-oxo intermediate is also likely to play a role in the specificity and efficiency of the chimeric scission reagents. Since substituents on the phenanthroline (OP) ring modulate the redox activity of the chelates (12), we initiated a study to determine if the cleavage efficiency and selectivity can be improved by derivatization of the 1,10-phenanthroline ligand. In the first series of experiments, our goal was to synthesize more efficient cleavage reagents by inserting a methylene group between the 1,10-phenanthroline ring system and the iodoacetamido * Address correspondence to this author at the Molecular Biology Institute. Phone: 310-825-8903. Fax: 310-206-7286. E-mail: [email protected]. X Abstract published in Advance ACS Abstracts, June 1, 1996.

S1043-1802(96)00028-6 CCC: $12.00

substituent used for covalent attachment of OP to the targeting biomolecule. Our prior observation that the 1:2 cuprous complexes of 5-methyl- and 5,6-dimethyl-1,10phenanthroline were more active as catalysts in the oxidation of thiols and in the untargeted cleavage of DNA than (OP)2Cu+ motivated this modification of the ligand (12). We similarly reasoned that methyl substituents at phenanthroline carbons 2 and 9 might enhance the reactivity of the 1:1 cuprous complex of phenanthrolineDNA-binding ligand chimeras. Therefore, oligonucleotides were alkylated with 2,9-dimethyl-5-(iodoacetamido)1,10-phenanthroline and then used to cleave complementary DNA sequences. Although 2,9-dimethyl-1,10phenanthroline does not form a redox-active 2:1 complex because steric clash between the ortho methyl groups inhibits the redox cycling through the square-planar cupric complex, this kinetic barrier is not relevant in targeted scission where only a single ligand is linked to a carrier (13-15). Our results indicate that insulation of the phenanthroline ring system from the amido group by a methylene spacer does not increase the nucleolytic activity when oligonucleotides are the targeting vehicles. However, substantial rate enhancements are observed in proteintargeted scission at certain cleavage sites, particularly those at which only weak cleavage is observed without the methylene spacer. In contrast, at sites at which strong cleavage is observed without the insulating methylene group, insertion of the methylene group does not enhance scission. The observation of rate enhancement at a specific site may depend on whether reduction or oxidation of the copper is the rate-determining step in © 1996 American Chemical Society

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Table 1

X-OP

2:1 copper complex

1:1 copper complex

nuclease activity of the 2:1 complex:b concentration at 50% cleavage (µM)

1,10-phenanthroline (OP) 5-CH3-OP 5-CH3CONH-OP 5-CH3CONHCH2-OP

12 2.6 21 7.8

57 30 80 44

19 7.4 57 25

chemical redoxy activity:a half-life (min)

a Half-lives for the oxidation of mercaptopropionic acid (MPA) by the 2:1 and 1:1 copper complexes of X-OP (8 µM:2 µM or 2 µM:2 µM X-OP:CuSO4). b Cleavage of a 141 bp long Lac UV-5 promoter (-96 to +45) by (X-OP)2Cu+.

reaction at that site. Our results further indicate that chimeras prepared from 2,9-dimethyl-1,10-phenanthroline [neocuproine (NC)] and oligonucleotides are competent site-directed cleavage reagents. Therefore, the steric factors that inhibit the redox chemistry of the 2:1 neocuproine-cuprous complex [(NC)2Cu+] are not present in the 1:1 complex linked to a targeting ligand. RESULTS

Insulation of the Amido Moiety from 1,10-Phenanthroline. (a) Untargeted Bis Complexes. The methylene group of 5-(aminomethyl)-1,10-phenanthroline (MOP) (16) inhibits any electronic interaction of an amido moiety, prepared from MOP, with the 1,10-phenanthroline ring system. Before preparation of phenanthrolinelinked chimeras for site-directed scission, the catalytic oxidation of thiols and the oxidative cleavage of duplex DNA by the 2:1 and 1:1 complexes of 5-(acetamidomethyl)-1,10-phenanthroline (AMOP) and copper(I) was studied to confirm the anticipated enhanced reactivity of these coordination complexes compared to those of 5-acetamido-1,10-phenanthroline. The disappearance of the free, titratable sulfhydryl group of 3-mercaptopropionic acid (MPA) according to the method of Ellman (17) provides a convenient assay of the redox activity of the copper chelates. Both the 1:1 and 2:1 cuprous chelates prepared from 5-AcNHCH2OP (AMOP) have half-lives for the oxidation of MPA to the disulfide that are 2-3-fold shorter than those of the corresponding chelates prepared from 5-acetamido-1,10-phenanthroline (5-AcNHOP) (Table 1).

This enhanced redox activity is also reflected in the nuclease activity of the untargeted 2:1 1,10-phenanthroline-cuprous complex toward a 141-base pair (bp) 32P 5′end-labeled DNA probe containing the lac UV-5 promoter. Quantification of the disappearance of the DNA parent band as a function of concentration of the cleaving agent (Table 1) indicates that (AMOP)2Cu+ is a more potent nuclease than (5-AcNHOP)2Cu+ and approaches the nucleolytic activity of (OP)2Cu+. The scission patterns of the different chelates are the same for this DNA segment. These results indicate that the binding specificity of the tetrahedral cuprous chelates does not depend on substituents at the C5 position of the ligand. (b) Oligonucleotide-Targeted Scission. The simplest test system to assay the intrinsic reactivity of a tethered coordination complex involves the targeted scission of a single-stranded DNA by a complementary OP-linked deoxyoligonucleotide (18). In the first series of experiments, insertion of the methylene group insulated the

Figure 1. Targeted scission of a single-stranded DNA by an oligonucleotide derivatized with a series of 5′-substituted 1,10-phenanthrolines: (top) oligonucleotide sequences; (bottom) lane a, 80-mer single-stranded DNA probe; lane b, MaxamGilbert G+A digest; lanes c-g, probe treated with CuSO4 and MPA and hybridized to no OP-oligonucleotide (lane c), AOPoligonucleotide (lane d), AMOP-oligonucleotide (lane e), AAOPoligonucleotide (lane f), or AGMOP-oligonucleotide (lane g).

1,10-phenanthroline nuclease from the tethering amido group. The sequences of the relevant oligonucleotides are presented in the top panel of Figure 1. No significant effect of insulating the 1,10-phenanthroline from the amido group is apparent from the cleavage data (bottom panel of Figure 1). For example, the cleavage efficiency of the 50-mer oligonucleotides derivatized with IAMOP was virtually identical to that observed with IAOP. The reactivities of oligonucleotides derivatized with IAAOP

Optimizing the Activity of 1,10-Phenanthroline−Copper

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Figure 2. (Top) Ribbon diagram of Fis dimer showing the positions of the two amino acids modified with derivatives of 1,10-phenanthroline. (Bottom) Schematic representation showing the binding of Fis dimer to DNA.

and IAGMOP were also compared. Chimeras prepared from these two 1,10-phenanthroline derivatives have isomeric tethers of the same length. The patterns of reactivity were virtually identical, indicating that there is no advantage in oligonucleotide-targeted scission either by lengthening the tether to the phenanthroline ring system or by inserting the methylene spacer.

(c) Protein-Targeted Scission. Since the oxidative chemical nuclease activity of 1,10-phenanthroline-copper proceeds by a multiple step pathway that includes the reduction of the cupric complex ion, followed by the reductive cleavage of hydrogen peroxide to generate the copper-oxo species directly responsible for scission (19), it is possible that the limiting step in the cleavage chemistry of the targeted and untargeted reactions differs or depends on the carrier used for directing the cleavage. Since proteins are effective targeting ligands of the chemical nuclease activity, we investigated the influence of the intervening methylene group in this method of sitespecific scission. This question was addressed with the Escherichia coli Fis protein (top panel of Figure 2), a 98-amino acid homodimer with four R-helices per monomer that functions in a number of biological systems, including recombination, transcription, and DNA replication, yet lacks a well-defined consensus sequence (20, 21). The helixturn-helix C and D subunits of each monomer comprise the DNA binding domain, and binding induces a consid-

Figure 3. Single-stranded cleavage of the proline permease promoter by bound Fis-OP-Cu conjugates. The numbers on the right refer to the positions of the flanking DNA. Fis binding sites are defined by a 15 bp core with the sequences denoted as -1L to -10L for the left flank and -1R to -10R for the right flank. The parent bands are not shown. AOP ) CH2C(O)NHOP. AMOP ) CH2C(O)NHCH2-OP. AAOP ) CH2C(O)NHCH2CH2C(O)NH-OP. AGMOP ) CH2C(O)NHCH2C(O)NHCH2-OP.

erable degree of DNA bending (22). Four phenanthroline derivatives, IAMOP, IAOP, IAMGOP, and IAAOP, were linked to the Fis mutants R71C and N73C. In the FisDNA model, the guanidino group of the Arg-71 side chain points toward, but does not directly contact, the DNA flanking the 15-base pair binding site. The Asn-73 side chain contacts the phosphates on the edge of the binding site (Figure 2B). Since the difference in the length of the tethers in the Fis-AMOP and Fis-AOP conjugates is one methylene unit, the change in the scission efficiency may not be due to differences in the intrinsic reactivity of the coordination complex. However, the isomeric Fis-AMGOP and Fis-AAOP chimeras have tethers of the same length and chemical composition. As a result, any change in reactivity will reflect insulation of the carboxamide from the aromatic system of the 1,10-phenanthroline ligand. Figure 3 shows single-stranded cleavage by the FisOP-Cu conjugates at two known binding sites on a 266base pair DNA from the promoter region (-157 to +109) of the E. coli proline permease. A dramatic enhancement of the scission upon insertion of the methylene group is evident upon comparison of lane 2 to lane 3 and lane 6 to lane 7. For example, conjugate 73-AOP cleaves weakly only in the right flanking sequence of the Fis binding site I, while 73-AMOP shows strong cleavage in both flanking regions of binding sites I (-48 to -34) and II (-88 to -74). Since Fis-OP-Cu conjugates 71-AAOP, 71-AMGOP, 73-AAOP, and 73-AMGOP have isomeric tethers of identical length, the insulating effect of the methylene group (compare lane 4 to lane 5 and lane 8 to lane 9) is responsible for the observed enhancements. Scission is enhanced by a factor of 1.7-3.2 at most sites (Table 2). However, scission efficiency is not always enhanced. For sites that are efficiently cleaved by chimeras prepared from 5-(iodoacetamido)-1,10-phenanthroline, in-

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Table 2. Ratios of DNA Scission by Fis-OP Conjugates with Isomeric Tethers scission state 71-AGMOP:71-AAOP 73-AGMOP:73-AAOP

-6R

-4L

-5R

-3L

2.4 2.7

1.8 0.8

3.2 1.7

1.7 0.7

sulation of the ring system by a methylene derivative does not yield increased scission. Indeed, a slight decrease is observed at the two sites most efficiently cleaved by the chimera prepared with N73C and IAAOP (Table 2). The insulated OP derivatives only enhance scission at the weaker scission sites of these conjugates. Targeted Scission by Chimeras Prepared with a Neocuproine Analog. Substituents at phenanthroline carbons 2 and 9 inhibit the oxidative activity of the copper chelates with stoichiometry of two ligands per metal ion. However, targeted scission with chimeric nucleases incorporating 1,10-phenanthroline-copper reflects the reactivity of the 1:1 complex. The lack of reactivity of the free 2:1 complex of 2,9-dimethyl-1,10-phenanthroline [neocuproine (NC)], therefore, does not necessarily mean the 1:1 complex is inactive. Given the enhanced reactivity of copper complexes composed of methyl-substituted phenanthrolines (12), chimeric nucleases prepared with the neocuproine derivative (BrANC) could be more reactive than the parent phenanthroline. To test this idea, the same oligonucleotide-targeting system described in the top panel of Figure 1 was used to assay the cleavage competence of NC-linked oligonucleotides.

The results (Figure 4) clearly indicate that oligonucleotides linked to phenanthroline (lanes a and b) and neocuproine (lane e) are directly comparable in scission yield, although the patterns of scission are not identical. Although NC-oligonucleotides cleave within the doublestranded region, they cleave more efficiently in the less sterically constrained single-stranded regions than do the AOP-linked conjugates. Central to this comparison is that chimeras prepared with NC-linked oligonucleotides cleave by the same reaction mechanism as OP-linked oligonucleotides. The principal criterion for similarity (or identity) of mechanism is that the cleavage reaction is dependent on the cuprous ion and hydrogen peroxide. The absolute requirement for a reducing agent (e.g., 3-mercaptopropionic acid or ascorbic acid) indicates the importance of cuprous ion, and the inhibition of scission by catalase reflects the importance of hydrogen peroxide (23). In all the OP conjugates described thus far, the carrier was linked through phenanthroline C5. The cleavage efficiency of 1,10-phenanthroline derivatives in which the tether was placed at positions 2 or 4 was also explored. An oligonucleotide linked to OP by a 2-aminomethyl substituent (using the phenanthroline derivative 2-IAMOP) did not lead to measurable cleavage in the target DNA even in the presence of added hydrogen peroxide (Figure 4, lane f). Since this analog should be comparable in intrinsic reactivity to the 5-aminomethyl derivative, linkage through the 2 position must inhibit the close approach of the copper-oxo species to the C1′H of the

Figure 4. Targeted scission of a single-stranded DNA by oligonucleotides tethered to neocuproine via C5 and to 1,10phenanthroline via C2, C4, and C5: lane a, AOP-oligonucleotide; lane b, AAOP-oligonucleotide; lane c, ASOP-oligonucleotide; lane d, 5-glycylneocuproine-oligonucleotide (see Experimental Section); lane e, ANC-oligonucleotide; lane f, 2-AMOPoligonucleotide.

deoxyribose, the presumed oxidative target of the nuclease activity. Because of its inactivity, further chimeras were not prepared with this analog. Modified oligonucleotides were also prepared with the derivative IASOP. As in the case of the C2-substituted conjugates, no cleavage was observed (Figure 4, lane c).

Targeting the Template Strand of the E. coli RNA Polymerase-Lac UV-5 Open Complex. The most novel reactivity of the chemical nuclease bis(1,10phenanthroline)-copper(I) is its cleavage of the singlestranded template strand of catalytically competent open transcription complexes formed between promoters and RNA polymerases (24-27). Although hyperreactivity of the nuclease at several prokaryotic and eukaryotic transcription start sites has been reported (28, 29), we have studied the E. coli lac UV-5 promoter and E. coli RNA polymerase most extensively. The single-stranded template strand is also accessible to sequence-specific

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Optimizing the Activity of 1,10-Phenanthroline−Copper

hybridization to complementary oligonucleotides, and we have demonstrated that OP-linked 5′-UGGAA-3′ specifically cleaves the template strand of the E. coli RNA polymerase-lac UV-5 open complex at positions -4 to -6 (30). Since NC exhibited preferential cleavage of single-stranded regions of DNA in the experiments summarized in Figure 4, we examined the targeted scission reaction by NC-linked UGGAA. The NCUGGAA conjugate caused little detectable scission of the template strand. This result suggests that the 2,9dimethyl groups block the steric approach to the singlestranded template strand of the open complex. This potential steric interference, albeit different in origin from that responsible for the lack of oxidative activity of the 2:1 coordination complex, (NC)2Cu+, suggests that the neocuproine derivative will not be as generally useful as other OP derivatives in targeted scission. Moreover, neocuproine linked to Fis N71C did not cleave Fis binding sites (data not shown), again indicating that the methyl groups at the 2 and 9 positions may block the access of the copper-oxo group to the oxidatively sensitive C1′H of the deoxyribose in bent DNA. While these experiments suggest the limitations of NC in the preparation of targeted scission reagents, they confirm the view that diffusible reactive species are not being generated by this redox-active coordination complex. DISCUSSION

The chimeras prepared by tethering a series of 1,10phenanthroline derivatives to oligonucleotides and proteins have provided valuable insights into the mechanism of DNA scission by 1,10-phenanthroline-copper(I) linked to a targeting ligand. The studies with 2,9-dimethyl-1,10phenanthroline (neocuproine) have been instructive in two contexts. First, they have provided definitive, confirmatory evidence that the nucleolytic species formed upon tethering 1,10-phenanthroline to a ligand is active as the 1:1 complex. If the cleavage reaction always required assistance from a second ligand, these conjugates would be cleavage-inactive. The preferential reactivity of neocuproine-oligonucleotide conjugates toward single-stranded DNA, however, indicates that steric constraints continue to influence the reactivity but arise from the intereference of the ortho methyl groups with the target rather than with a second NC ligand. The second context in which the studies with NC are informative is that their competence as scission reagents suggests that the oxidative reaction proceeds through a copper-oxo intermediate rather than through a metalcoordinated peroxy species. Methyl substituents at phenanthroline carbons 2 and 9 would disfavor a metalcoordinated peroxy species. Yet there are no differences in the extent of DNA cleavage by the NC- and OPoligonucleotide chimeras. As a result, this mechanism is excluded. Despite the enhanced reactivity in the untargeted scission of DNA by (5-AcNHCH2OP)2Cu+ as compared to that of (5-AcNHOP)2Cu+, site-specific scission reagents incorporating the 5-(carboxamidomethyl)-1,10-phenanthroline unit are not routinely more efficient than chimeras prepared with the carboxamido group directly attached to the phenanthroline ring system. The explanation for this dilemma may reside in the multistep reaction pathway of the chemical nuclease. The first step in the kinetic sequence is the reduction of the cupric complex to the corresponding cuprous complex, which then binds to the DNA and reductively cleaves hydrogen peroxide to generate the oxo species directly responsible for DNA cleavage (31). Previous work has suggested that there is an obligatory order in the reaction sequence (12).

Since the 2:1 complex (OP)2Cu2+ intercalates into DNA and is not accessible to reduction (32), the only kinetically viable pathway for the formation of the cuprous complex is the reduction of the freely diffusible cupric complex.

(OP)2Cu2+ + 1e- f (OP)2Cu+

(1a)

(OP)2Cu+ + DNA h (OP)2Cu+-DNA

(1b)

H2O2

(OP)2Cu+-DNA 98 (OP)2Cu3+dO-DNA f products (1c) In untargeted scission, the reductive cleavage of hydrogen peroxide may be the slow step at the low concentrations of in situ-generated hydrogen peroxide. The immeasurably fast rate of DNA cleavage observed when millimolar concentrations of hydrogen peroxide are added to the reaction mixture supports this observation (14). The aminomethyl derivatives would react more quickly because the electron-releasing methylene group stabilizes the cupric complex and therefore facilitates the formation of the higher valent cupric ion formed by hydrogen peroxide reduction. In untargeted scission, scission is more efficient because the slow step of the reaction (i.e., reduction of hydrogen peroxide, eq 1c) is enhanced. In targeted scission, on the other hand, the 1:1 OPCu complex may be bound tightly to DNA, in which case the cupric complex must be reduced at its specific site even though the negatively charged DNA may hinder the approach of the anionic 3-mercaptopropionate. The failure to see an enhanced rate of cleavage in targeted scission may result from a difference in the ratedetermining step in untargeted and targeted scission. In targeted scission, the slow step of the cleavage reaction may be reduction of the DNA-bound cupric complex (e.g., k2, RSH). This first and rate-limiting step would not be enhanced by the 5-methylene substituent. Chimeras prepared with the 5-methylene derivatives will only show rate enhancement when the metal ion can be easily reduced and the reductive cleavage of peroxide (e.g., k3, H2O2) limits the overall rate of scission. This is likely to occur at weak sites of scission because in these cases the copper ion does not approach as closely to the oxidatively sensitive bonds of the deoxyribose and therefore is more accessible to solvent and reductant. L-OP–Cu2+ + DNA

k1 k–1

L = targeting ligand or OP

L-OP–Cu2+

RSH k2

L-OP–Cu+

H2O2

k3

DNA

DNA

L-OP–Cu+

O

k4

products

DNA

In summary, these studies indicate that the efficiency of targeted scission is primarily a function of the orientation of the OP-Cu to the oxidatively sensitive C1′H of the deoxyribose (33). Modification of the phenanthroline leads to changes in the reactivity of these conjugates but not always in a predictable manner. These studies demonstrate two important mechanistic features of these chimeras. (a) Only a single phenanthroline ligand in the coordination complex is essential for scission activity, and (b) a metal-coordinated peroxy intermediate is not involved in the scission. The important contribution of orientation to scission efficiency underscores the fact that the oxidative species responsible for scission is not freely diffusible. The variable reactivity of the 5-aminomethyl OP derivatives in the Fis-targeted scission indicates that

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the rate of formation of the cuprous complex may be ratelimiting in the overall reaction especially at strong scission sites where the metal ion may not be freely accessible to diffusible reductants such as 3-mercaptopropionic acid or ascorbic acid. EXPERIMENTAL SECTION

Synthesis of Neocuproine Derivatives. 5-Nitroneocuproine. Neocuproine (GFS Chemicals) (2.0 g) in a mixture of concentrated H2SO4 (15 mL) and fuming HNO3 (9 mL) was heated to reflux for 3-5 h. The reaction mixture was then added to ice and enough solid NaOH to give an alkaline pH. The product was extracted twice with equal volumes of CHCl3, the CHCl3 was removed on a rotary evaporator, and the product was recrystallized from a mixture of CHCl3 and hexanes, thereby yielding pure 5-nitroneocuproine (0.55 g, 27%): 1H NMR (CDCl , 200 MHz) δ 2.98 (s, 3H), 3.00 (s, 3H), 3 7.63 (d, J ) 8.4 Hz, 1H), 7.67 (d, J ) 8.8 Hz, 1H), 8.28 (d, J ) 8.2 Hz, 1H), 8.61 (s, 1H), 8.92 (d, J ) 8.8 Hz, 1H). 5-Aminoneocuproine. 5-Nitroneocuproine (0.5 g) was suspended in ethanol (40 mL), (NH4)2S (80 mL, 11% w/v in ethanol) was added, and the mixture was heated to reflux for 3-5 h. The solvent was removed on a rotary evaporator, water was added, and the pH was adjusted to 14 with aqueous KOH. The product was extracted three times with equal volumes of CHCl3. After removal of the CHCl3, the product was recrystallized from a mixture of CHCl3 and hexanes, thereby yielding 5-aminoneocuproine (0.3 g, 60%): 1H NMR (CDCl3, 200 MHz) δ 2.85 (s, 3H), 2.92 (s, 3H), 6.89 (s, 1H), 7.36 (d, J ) 8.4 Hz, 1H), 7.49 (d, J ) 8.4 Hz, 1H), 7.98 (d, J ) 8.2 Hz, 1H), 8.16 (d, J ) 8.6 Hz, 1H). 5-(Bromoacetamido)neocuproine (BrANC). 5-Aminoneocuproine (0.3 g) was dissolved in CHCl3 (10 mL), and a layer of aqueous KOH (0.5 M, 10 mL) was added on top. Bromoacetyl bromide (150 mL, 1.3 molar equiv, Aldrich Chemical Co.) was added via a Pasteur pipette to the organic layer on the bottom. The reaction mixture was allowed to stir overnight at 25 °C. The reaction was then analyzed by TLC, and additional bromoacetyl bromide was added if necessary until complete conversion to the acylated product was observed. Next, the solution was shaken vigorously, the clear organic phase containing the product was removed, the CHCl3 was removed on a rotary evaporator, and the product was recrystallized from hexanes: 1H NMR (CDCl3, 200 MHz) δ 2.82 (s, 3H), 2.90 (s, 3H), 4.28 (s, 2H), 7.42 (d, J ) 10 Hz, 1H), 7.47 (d, J ) 8.6 Hz, 1H), 8.10 (d, J ) 8.2 Hz, 1H), 8.19 (s, 1H), 8.40 (d, J ) 12 Hz, 1H). Synthesis of 5-(Aminomethyl)-1,10-phenanthroline and Its Derivatives. 5-(Bromomethyl)-1,10-phenanthroline. 5-Methyl-1,10-phenanthroline (367.1 mg, 1.892 mmol, GFS Chemicals), benzoyl peroxide (50.2 mg, 0.11 molar equiv), and N-bromosuccinimide (355.5 mg, 1.056 molar equiv) were added to dry benzene (19 mL) in a round-bottomed flask, and the mixture was illuminated, under an atmosphere of argon, with a 300 W incandescent light bulb placed about 5 in. from the flask. The temperature within the flask was maintained at 50-60 °C, and the reaction was allowed to proceed for 2 h. TLC and 1H NMR analyses of the supernatent at the end of the 2 h period indicated that no unreacted 5-methyl-1,10phenanthroline remained and that the major product was the desired bromide. The reaction mixture was cooled on ice for at least 1 h and then filtered to remove a reddish tar and the succinimide which precipitated during cooling. The solids were rinsed twice with benzene. The benzene was removed from the filtrate on a

Gallagher et al.

rotovap, and the resulting bromide was used without further purification: 1H NMR (CDCl3, 200 MHz) δ 9.24 (dd, J ) 4.7, 1.6 Hz, 1H), 9.20 (dd, J ) 4.4, 1.6 Hz, 1H), 8.54 (dd, J ) 8.4, 1.5 Hz, 1H), 8.19 (dd, J ) 8.0, 1.6 Hz, 1H), 7.84 (s, 1H), 7.72 (dd, J ) 8.4, 4.4 Hz, 1H), 7.62 (dd, J ) 8.2, 4.4 Hz, 1H), 4.94 (s, 2H). 5-(Aminomethyl)-1,10-phenanthroline (AMOP). Ethanol (16 mL) and concentrated aqueous ammonia (4 mL) were added to the crude 5-(bromomethyl)-1,10-phenanthroline, and the mixture was stirred at 22 °C for 2 h. Next, the ethanol and excess ammonia were removed on a rotovap. Dichloromethane was added to the residue, and then 0.1 N HCl was added until the aqueous layer had a pH of about 4. The aqueous layer containing the desired product was extracted several times with dichloromethane, then the pH was adjusted to about 12 with 1 N NaOH, and the product was extracted seven to ten times with dichloromethane. The dichloromethane extract was dried over Na2SO4, and the volatiles were removed on a rotovap, thereby giving the crude product as a yellow oil. The product was purified by flash chromatography on silica gel (2 cm inside diameter × 13 cm, 98:2:0.5 CHCl3:CH3OH:isopropylamine) to afford 5-(aminomethyl)-1,10-phenanthroline as a light yellow oil (138.9 mg, 35% from 5-methyl-1,10-phenanthroline): 1H NMR (CD3,OD, 200 MHz) δ 9.06 (dd, J ) 4.4, 1.4 Hz, 1H), 9.02 (dd, J ) 4.4, 1.5 Hz, 1H), 8.59 (dd, J ) 8.4, 1.3 Hz, 1H), 8.37 (dd, J ) 8.1, 1.4 Hz, 1H), 7.87 (s, 1H), 7.76 (dd, J ) 8.5, 4.4 Hz, 1H), 7.71 (dd, J ) 8.3, 4.4 Hz, 1H), 4.35 (s, 2H); 13C NMR (CD3OD, 50 MHz) δ 152.9, 152.8, 149.0, 148.5, 140.5, 139.9, 135.8, 132.1, 130.8, 127.3, 127.2, 126.8, 45.7; HRMS (EI) m/e 209.0958 (M+, calculated for C13H11N3 209.0953). 5-(Iodoacetamidomethyl)-1,10-phenanthroline (IAMOP). Iodoacetic acid (8.6 mg, 1.1 molar equiv) and 1,3dicyclohexylcarbodiimide (DCC, 9.0 mg, 1.05 molar equiv) were added to 5-(aminomethyl)-1,10-phenanthroline (8.7 mg, 0.042 mmol) in a round-bottomed flask. Chloroform (1.5-1.6 mL) was added, and the reaction flask was degassed and filled with Ar. The reaction mixture was allowed to stir at 22 °C overnight (14-20 h). The solid which had precipitated was removed by filtration. The clear, pale yellow filtrate was extracted three times with 0.01 N HCl. Next, the aqueous extract was washed once with deuteriochloroform. Then, the pH of the aqueous layer was raised to about 9 or 10 by adding saturated aqueous NaHCO3 dropwise. The product was then extracted into deuteriochloroform (4 × 2 mL). The clear, pale yellow extract was dried over anhydrous Na2SO4, which was removed by filtration. The volume of the solution was measured, and the purity and yield of the 5-(iodoacetamidomethyl)-1,10-phenanthroline were determined by 1H NMR using an internal standard (e.g., benzene). The yield is approximately 80%, and the purity is >95%. The product is stable for a few days in chloroform solution if stored at 4 °C but eventually decomposes to give a chloroform-insoluble tan- to purplecolored solid. For derivatization of nucleic acid oligomers or proteins, the CDCl3 solution of 5-(iodoacetamidomethyl)-1,10-phenanthroline was sufficiently concentrated to be added directly to a solution of the ligand to be derivatized in buffer-dimethylformamide: 1H NMR (CD3OD, 200 MHz) δ 9.11 (dd, J ) 4.4, 1.5 Hz, 1H), 9.08 (dd, J ) 4.4, 1.6 Hz, 1H), 8.61 (dd, J ) 8.4, 1.5 Hz, 1H), 8.43 (dd, J ) 8.1, 1.6 Hz, 1H), 7.95 (s, 1H), 7.81 (dd, J ) 8.4, 4.3 Hz, 1H), 7.76 (dd, J ) 8.0, 4.4 Hz, 1H), 4.95 (s, 2H), 3.79 (s, 2H); 13C NMR (CDCl3, 90 MHz) δ 167.5, 150.2, 150.0, 145.8, 145.7, 135.9, 132.2, 131.9, 127.6, 126.9, 126.1, 123.2, 123.1, 42.0, -0.66; HRMS (EI) m/e 377.0026 (M+, calculated for C15H12N3OI 377.0025).

Optimizing the Activity of 1,10-Phenanthroline−Copper

5-[N-(Phthaloyl)glycylamidomethyl]-1,10-phenanthroline. N-Phthaloylglycine (88.1 mg, 1.1 molar equiv) and DCC (89 mg, 1.1 molar equiv) were added to 5-(aminomethyl)-1,10-phenanthroline (82 mg, 0.39 mmol) in a 10 mL round-bottomed flask equipped with a septum and a magnetic stirring bar. Chloroform (3.9 mL) was added, and the reaction mixture was allowed to stir under an inert atmosphere at 22 °C overnight (16 h). The chloroform was removed on a rotovap, and NMR analysis of the CD3OD soluble portion of the resulting residue indicated that all the amine had reacted. The crude 5-[N(phthaloyl)glycylamidomethyl]-1,10-phenanthroline was used without purification. 5-(Glycylamidomethyl)-1,10-phenanthroline. Ethanol (8 mL) and hydrazine (36.7 mL, 97%, 3.0 molar equiv) were added to the crude 5-[N-(phthaloyl)glycylamidomethyl]-1,10-phenanthroline, and the reaction mixture was allowed to reflux under an atmosphere of argon for 30 min, at which time TLC analysis indicated all the 5-[N-(phthaloyl)glycylamidomethyl]-1,10-phenanthroline had reacted to give a more polar product. Next, the volatiles were removed on a rotovap, and the resulting off-white solid was triturated with 0.01 N HCl. The white solid was removed from the aqueous layer containing the product by filtration and was washed well with 0.01 N HCl and water. Then, the pH of the combined aqueous washes was adjusted to about 12 with 1 N NaOH, and the water was removed on a rotovap from a 30 °C water bath. The resulting solid was washed thoroughly with methanol to dissolve the product. A small amount of silica gel was added to the methanol extract, and then the methanol was removed on a rotovap. The resulting powder (the crude product adsorbed onto silica gel) was poured onto a prepacked flash column (2 cm inside diameter × 4 cm), and the product was eluted with 150 mL of 100:3:1 CHCl3:CH3OH: isopropylamine, followed by 100 mL of 100:6:2 CHCl3: CH3OH:isopropylamine. 5-(Glycylamidomethyl)-1,10phenanthroline was obtained as a light yellow foam in 56% overall yield (58.3 mg): 1H NMR (CD3OD, 200 MHz) δ 9.04 (m, 2H), 8.58 (dd, J ) 8.4, 1.5 Hz, 1H), 8.36 (dd, J ) 8.2, 1.7 Hz, 1H), 7.82 (s, 1H), 7.74 (m, 2H), 4.93 (s, 2H), 3.41 (s, 2H); 13C NMR (CD3OD, 90 MHz) δ 171.6, 151.0, 150.8, 146.9, 146.5, 137.7, 134.2, 133.9, 129.7, 128.7, 127.1, 124.9, 124.6, 43.6, 41.6: HRMS (EI) m/e 266.1168 (M+, calculated for C15H14N4O 266.1168). 5-[N-(2-Iodoacetyl)glycylamidomethyl]-1,10-phenanthroline (IAGMOP). Iodoacetic acid (6.8 mg, 1.1 molar equiv) and DCC (7.0 mg, 1.05 molar equiv) were added to 5-(aminomethyl)-1,10-phenanthroline (8.6 mg, 0.032 mmol) in chloroform (1.5-1.6 mL), and the reaction mixture was allowed to stir under argon at 22 °C overnight (14-20 h). The solid which had precipitated (DCU) was removed by filtration, and the clear, pale yellow filtrate was extracted three times with 0.01 N HCl (1-2 mL per extraction). Next, the aqueous extract was washed once with deuteriochloroform. Then, the pH of the aqueous layer was raised to about 9 or 10 by adding saturated aqueous NaHCO3 dropwise. The product was then extracted into deuteriochloroform (4 × 2 mL). The clear, pale yellow extract was dried over anhydrous Na2SO4, which was removed by filtration. The volume of the solution was measured, and the purity and yield of the 5-[N-(2-iodoacetyl)glycylamidomethyl]-1,10-phenanthroline were determined by 1H NMR using an internal standard (e.g., benzene). The yield is approximately 73%, and the purity is >95%. The product is stable for a few days in chloroform solution if stored at 4 °C but eventually decomposes. For derivatization of nucleic acid oligomers or proteins, the CDCl3 solution of 5-[N-(2-

Bioconjugate Chem., Vol. 7, No. 4, 1996 419

iodoacetyl)glycylamidomethyl]-1,10-phenanthroline was sufficiently concentrated to be added directly to a solution of the ligand to be derivatized in buffer-dimethylformamide: 1H NMR (CD3OD, 200 MHz) δ 9.08 (dd, J ) 4.4, 1.4 Hz, 1H), 9.05 (dd, J ) 4.4, 1.6 Hz, 1H), 8.59 (dd, J ) 8.4, 1.6 Hz, 1H), 8.42 (dd, J ) 8.1, 1.6 Hz, 1H), 7.88 (s, 1H), 7.78 (dd, J ) 8.4, 4.3 Hz, 1H), 7.75 (dd, J ) 7.9, 4.4 Hz, 1H), 4.95 (s, 2H), 3.96 (s, 2H), 3.79 (s, 2H). Modification of Oligonucleotides with OP Derivatives. The oligonucleotide was first 5′-labeled with thiophosphate by allowing the nucleotide (∼5 nmol) to react with [γ-35S] ATP (11.5 nmol, specific activity of 6.6 Ci/mmol) and T4 polynucleotide kinase in 100 µL of kinase buffer at 37 °C for 2 h. After phenol/chloroform extraction, the labeled product was desalted by passage through a Sephadex G-50 spin column. An aliquot (5 µL) was saved as a reference, while the remainder was immediately derivatized. Typically, a 30-fold excess of an iodoacetamido derivative of OP in aqueous DMF was used, and the reaction was carried out at 37 °C for 2 h. The modified product was electrophoresed on a 15% denaturing polyacrylamide gel along with the γ-35Slabeled but underivatized reference oligonucleotide. The OP-derivatized product, which moved more slowly than the reference, was excised, eluted, and precipitated with ethanol. The neocuproine-derivatized oligonucleotide was also prepared from 5-glycylneocuproine. The 5′-phosphorylated oligonucleotide was first converted into its imidazolide by reaction with [3-(dimethylamino)propyl]carbodiimide (0.12 M) in imidazole hydrochloride buffer (100 µL, 0.1 M, pH 6.1) at room temperature for 2 h as reported previously (18, 34). 2,6-Lutidine (6 µL) was then added, followed by 5-glycylneocuproine (6 mg). This mixture was heated at 50 °C for 2 h. The product was purified as described above. Catalytic Oxidation of 3-Mercaptopropionic Acid. Each redox reaction mixture contained 1 mM MPA in 50 mM phosphate (pH 8.0) at 22 °C. At each time point, a 30 µL aliquot was diluted with 970 µL of a solution containing 0.25 mM dithiobis(2-nitrobenzoic acid) and 5 mM EDTA in 50 mM phosphate (pH 8.0). The absorbance of the 4-mercapto-2-nitrobenzoic acid produced was measured at 412 nm. Kinetics of DNA Scission by PhenanthrolineCopper Complexes. Cleavage reactions were carried out in 40 mM Tris/50 mM KCl/10 mM MgCl2, 5.8 mM MPA, and pH 7.9 buffer for 4.0 min at 37 °C. The reactions were quenched with EDTA, and the DNA was precipitated, redissolved in loading buffer, and electrophoresed in a 12% denaturing polyacrylamide gel. The relative amounts of uncut parent band were measured using a Molecular Dynamics Phospho-Imager 445SI. Cleavage of Single-Stranded DNA by Oligonucleotide-1,10-Phenanthroline-Copper(I) Chimeras. The single-stranded substrate was synthesized on a Pharmacia Gene Assembler, 5′-labeled with γ-32P and T4 kinase, and purified using a 10% denaturing polyacrylamide gel. In a typical cleavage experiment, a solution of substrate (0.1 pmol) and OP-derivatized oligonucleotide (1-5 pmol) in 20 µL of buffer (50 mM, pH 7.5 Tris/50 mM NaCl/5 mM MgCl2) was denatured by heating to 90 °C for 5 min and then allowed to reanneal at room temperature for 1 h. The cleavage reaction was initiated by addition of aqueous CuSO4 (2 µL, 200 µM) and MPA (2 µL, 58 mM). After 2 h at 37 °C, the reaction was quenched with neocuproine. Products were analyzed on a 8% denaturing polyacrylamide gel. Cleavage of the E. coli Proline Permease Promoter by Fis Protein-1,10-Phenanthroline-Cop-

420 Bioconjugate Chem., Vol. 7, No. 4, 1996

per(I) Chimeras. The Fis-1,10-phenanthroline conjugates were incubated with 32P end-labeled DNA probe in 20 mM Tris (pH 7.5)/80 mM NaCl/50 mM CuSO4 for 10 min at 22 °C. MPA was added to a concentration of 3 mM to initiate cleavage. The reaction was quenched with neocuproine after 1 h, loading buffer was added, and the cleavage products were separated by denaturing PAGE (12%). The relative intensities of the bands were determined using a Molecular Dynamics Phospho-Imager 445SI. ACKNOWLEDGMENT

This work was supported by National Institutes of Health Grant USPHS GM 21199. C.P. and D.M.P. were trainees of USPHS National Research Award GM 07185. LITERATURE CITED (1) Lerner, R. A., Benkovic, S. J., and Schultz, P. G. (1991) At the Crossroads of Chemistry and Immunology: Catalytic Antibodies. Science 252, 659-667. (2) Thorn, S. N.; Daniels, R. G., Auditor, M.-T. M., and Hilvert, D. (1995) Large rate accelerations in antibody catalysis by strategic use of haptenic charge. Nature 373, 228-230. (3) Szostak, J. W. (1992) In vitro genetics. TIBS 17, 89-93. (4) Malcolm, B. A., Rosenberg, S., Corey, M. J., Allen, J. S., Baetselier, A. d., and Kirsch, J. D. (1989) Site-Directed Mutagenesis of the Catalytic Residues Asp-52 and Glu-35 of Chicken Egg White Lysozyme. Proc. Natl. Acad. Sci. U.S.A. 86, 133-137. (5) Sprang, S., Standing, T., Fletterick, R. J., Stroud, R. M., Finer-Moore, J., Xuong, N. H., Hamlin, R., Rutter, W. J., and Craik, C. S. (1987) Three-dimensional Structure of Asn102 Mutant of Trypsin: Role of Asp102 in Serine Protease Catalysis. Science 237, 905-909. (6) Wagner, C., and Benkovic, S. (1990) Site directed mutagenesis: a tool for enzyme mechanism dissection. Trends Biotechnol. 8, 263-270. (7) Sigman, D. S., Bruice, T. W., Mazumder, A., and Sutton, C. L. (1993) Targeted Chemical Nucleases. Acc. Chem. Res. 26, 98-104. (8) Pan, C. Q., Landgraf, R., and Sigman, D. S. (1994) DNA Binding Proteins as Site-Specific Nucleases. Mol. Microbiol. 12, 335-342. (9) Wu, J., Perrin, D. M., Sigman, D. S., and Kaback, H. R. (1995) Helix packing of lactose permease in Escherichia coli studied by site-directed chemical cleavage. Proc. Natl. Acad. Sci. U.S.A. 92, 9186-9190. (10) Sigman, D. S., Mazumder, A., and Perrin, D. M. (1993) Chemical Nucleases. Chem. Rev. 93, 2295-2316. (11) Bruice, T. W., Wise, J., Rosser, D. S. E., and Sigman, D. S. (1991) Conversion of Lambda Phage Cro into an OperatorSpecific Nuclease. J. Am. Chem. Soc. 113, 5446-5447. (12) Thederahn, T. B., Kuwabara, M. D., Larsen, T. A., and Sigman, D. S. (1989) Nuclease Activity of 1,10-Phenanthroline-copper: Kinetic Mechanism. J. Am. Chem. Soc. 111, 4941-4946. (13) Graham, D. R., Marshall, L. E., Reich, K. A., and Sigman, D. S. (1980) Cleavage of DNA by Coordination Complexes. Superoxide Formation in the Oxidation by 1,10-Phenanthroline-Cuprous Complexes by OxygensRelevance to DNAcleavage Reaction. J. Am. Chem. Soc. 102, 5419-5421. (14) Marshall, L. E.; Graham, D. R., Reich, K. A., and Sigman, D. S. (1981) Cleavage of Deoxyribonucleic Acid by the 1,10Phenanthroline-Cuprous Complex. Hydrogen Peroxide Requirement and Primary and Secondary Structure Specificity. Biochemistry 20, 244-250. (15) Reich, K. A., Marshall, L. E., Graham, D. R., and Sigman, D. S. (1981) Cleavage of DNA by the 1,10-PhenanthrolineCopper Ion Complex. Superoxide Mediates the Reaction Dependent on NADH and Hydrogen Peroxide. J. Am. Chem. Soc. 103, 3582-3584. (16) Kimura, E., Bu, X., Shionoya, M., Wada, S., and Maruyama, S. (1992) New Nickel(II) Cyclam (Cyclam ) 1,4,8,11Tetraazacyclotetradecane) Complex Covalently Attached to Ru(phen)32+ (phen ) 1,10-Phenanthroline). A New Candidate

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