Synthetic analogues and biosynthetic intermediates of bleomycin. Metal-binding, dioxygen interaction, and implication for the role of functional groups in bleomycin action mechanism.

In order to clarify the role of bleomycin functional groups in action mechanism, the metal-binding, dioxygen activation, and DNA cleavage of several synthetic analogues and biosynthetic intermediates of bleomycin have been investigated. The present results support that 1) the beta-aminoalaninepyrimidine-beta-hydroxyhistidine portion of the bleomycin molecule substantially participates in the Fe(II) and dioxygen interactions, 2) the transposition of the pyrimidine (or pyridine) and imidazole groups in the Fe(II)-coordination is essential for the effective binding and activation of molecular oxygen by the bleomycin ligands, and 3) the gulose-mannose moiety plays an important role as an environmental factor for the efficient dioxygen reduction and DNA cleavage, although the sugar portion does not contribute significantly to the nucleotide specificity in the DNA strand scission. Certain oligopeptides are able to mimic the metal-binding and dioxygen activation by bleomycin, but not induce the effective DNA cleavage. Probably, the bithiazole DNA interaction site of bleomycin delivers the iron/dioxygen chemistry to particularly the DNA (formula, see text) nucleotide sequences.

DNA (3). Although the P-aminoalaninepyrimidine-/3-hydroxyhistidine portion appears to be capable of dioxygen activation by the chelation with ferrous ion (4), some possible transition metal-binding sites of BLM have been proposed on the basis of theoretical and spectroscopic investigations (5-7). Among them, the x-ray crystallographic analysis of biosynthetic intermediate P-3A-Cu(II) complex isolated from a culture broth of BLM demonstrated the most direct evidence for the metal coordination sites (8), and also an acid hydrolysis product of the BLM-Co(II1) complex was recently shown to be analogous to the structure of the P-3A-Cu(II) complex (9). However, these metal complexes are biologically inactive and do not activate molecular oxygen. The studies using depyruvamide BLM and N-acetyl BLM indicated that the a-amino group of the p-aminoalanine residue has an important effect on metal coordination, dioxygen activation, and DNA cleavage activity (10, 11). However, the rote of the gulose-mannose sugar portion for the BLM action is uncertain. Herein, we wish to report the metal-binding, dioxygen interaction, and DNA strand scission of the synthetic analogues and biosynthetic intermediates of BLM, and to implicate the role of the respective functional groups in the BLM action mechanism. The BLM-related compounds as shown in Chart 1 are used in this paper. 25 "C for 12 h. The resulting Schiff base (3) was hydrogenated over 5% Pd-C in MeOH, affording yellow foam (4) upon workup and chromatography on silica gel (eluted with 9 1 CHzCIz-MeOH) (4: 60% yield from 1, [a]? +36.2' (C, 1, CHCL)). Then, the secondary amino group of 4 was protected with benzyloxycarbonyl (2) group to effect a smooth peptide formation with histidine methyl ester. Treatment of 4 with a little excess of benzyl chloroformate in the presence of 0.1 N NaOH in CHzC12 (3 h, 25 "C) afforded colorless foam ( 5 ) upon workup and chromatography on silica gel (eluted with 9 1 CHZCIZ-MeOH) in 88% yield (5: [a]? +77.7" (C, 1, CHCL)). Condensation of 5 with histidine methyl ester (19) was carried out smoothly. Methyl pyridine-2-carboxylate derivative 5 was hydrolyzed (0.1 N LiOH, 30 min at 0 "C and 1 h a t 25 "C) and neutralization with 0.1 N HC1 and usual workup afforded free acid (6). Treatment of 6 with N,N'carbonyldiimidazole (tetrahydrofuran, 0 "C, 1 h) followed by conden-sation with histidine methyl ester (tetrahydrofuran, 25 "C, 12 h) afforded white powder (7) upon workup and chromatography on silica gel (eluted with 9:l CHzC12-MeOH) (7: 83% yield from 5, m.p. 168-170 "C, [a]? +60.1° (C, 0.1, CHCb)). The protective groups of 7 were removed with 30% HBr-AcOH (2.5 h at 25 "C), affording methyl ester (PYML-2) as a solid residue after workup. The methyl ester was hydrolyzed with 1 N NaOH at pH 9-10, and the solution was neutralized with 1 N HC1. After removal of the solvent, the residue was purified by Amberlite CG-50 (H' form, eluted with 1% aqueous NH,). Thus, yellow solid PYML-1 (8) was obtained in 87% yield upon usual workup (m.p. 120-122 "C, [a]? +2.85" (C, 1, H20)).

Role of Functional Groups
PEML was synthesized as follows (see Scheme 2). Methyl 6-formylpyridine-2-carboxylate (1) was treated with a large excess of ethylenediamine in EtOH in the presence of an activated molecular sieve a t 25 "C for 12 h. After filtration of the molecular sieve, the solution of the resulting Schiff base 9 was hydrogenated over 5% Pd-C. The crude hydrogenated product obtained by usual workup was treated with benzyl chloroformate (4 eq) in the presence of 1 N NaOH in CHzCh. Usual workup and chromatography on silica gel (eluted with 4 1 AcOEt-hexane) afforded crystalline tris(benzyloxycarbony1) derivative 10 (59% from 1, m.p. 106-107 "C). The protective groups of 10 were removed with 30% HBr-AcOH. After evaporation of the solvent, the residue was purified by Diaion WK-10 (H' form, eluted with 1% aqueous NHs), affording hydroscopic yellow solid PEML (11) in quantitative yield. All new compounds were chromatographically homogeneous and gave satisfactory analytical and spectral data.
Preparation of Metal Complexes-The metal complexes of the synthetic analogues and the biosynthetic intermediates were prepared according to previously reported procedures for the BLM-metal complexes (20)(21)(22). For the electronic spectral measurements, the Fe(I1) complexes with CO, CZH~NC, and NO were obtained anaerobically at pH 7.2 in a Thunberg cuvette equipped with a sidearm stoppered with a rubber septum. For the determinations of ESR, 'H NMR, and Mossbauer spectra, the Co(II), Fe(II), and Fe(I1)-CO complexes were formed under the fully anaerobic condition which was achieved by using a vacuum line. The Fe(I1)-NO complexes were prepared by addition of a few milligrams of sodium nitrite and sodium borohydride (or sodium dithionite) to the solution of the corresponding Fe(I1) CHO 1 SCHEME 2 complexes. Spin-trapping experiments using BPN (Aldrich) and DMPO (Aldrich) were carried out according to the previously reported procedure (23). DMPO was used after purification by filtration with charcoal.
Fe-Mossbauer spectra in the zero magnetic field were obtained at 110 K with a conventional constant-acceleration type. The radiation was detected by a proportional counter and multichannel analyzer system. All the given isomer shifts are relative to the iron metal at 300 K and all workups were carried out in an oxygen-free environment. The electrochemistry was performed at p = 0.1 (NaC104) and pH 7.2 on a hanging mercury drop electrode with a 100-mV/s scan rate by a Yanagimoto P-1000H cyclic voltammeter.
DNA Cleavage Experiments-Plasmid pBR 322 DNA was digested with HcnfI (Takara), and the restriction fragment (396 base pairs) was isolated from a 5% polyacrylamide gel (24). Terminal phosphates were removed by treatment with bacterial alkaline phosphatase (Bethesda Research Laboratories), and the 5' ends were labeled with ,'"P by using T4 polynucleotide kinase (Bethesda Research Laboratories) and [y-,"P]ATP (Amersham). This doubly end-labeled molecule was digested with Hue111 (Takara) and the singly end-labeled 327-base pair fragment was isolated by electrophoresis on a 6% polyacrylamide gel. The restriction fragments of DNA were incubated with the BLMrelated compounds, under the conditions described in the legends to the figure. Nucleotide sequences of the restriction fragments were determined as reported by Maxam and Gilbert (25). The present restriction fragment contains 327 bases and the 5' terminus corresponds to position 2845 in the pBR 322 DNA map.

RESULTS
Visible, Circular Dichroism, a n d Redox Characteristics of Copper(I0 Complexes- Fig. 1 shows the visible absorption and CD spectra of the PYML-l-Cu(II) complex at pH 7.2. These electronic and CD features closely resemble those of naturally occurring BLM-Cu(I1) complex which reveal an absorption maximum at 595 nm and CD extrema at 555 and 665 nm. The X, , , values of 597 and 595 nm indicate similar copper ligand fields for PYML-1 and BLM, because the magnitude of the ligand field around the central Cu(I1) is reflected in the d~z y z -d~~" y~ transition (26). The cyclic voltammogram of the PYML-1-Cu(I1) complex exhibited a quasi-reversible 1electron oxidation-reduction wave with an E112 value of -319 mV uersus the NHE. This redox potential also corresponds well to that (-327 mV uersus NHE) of the BLM-Cu(I1) complex (27). Table I summarizes the visible, CD spectral constants, and redox potentials for the Cu(I1) complexes of PYML-1, PEML, P-3A, deglyco BLM, and BLM. The red shift of the vd.d for the P-3A-and the deglyco BLM-Cu(I1) complexes suggests a distortion from square-planar configuration of the Cu(I1) site. Their Cu(II)/Cu(I) redox potentials are clearly higher than those of the Cu(I1) complexes of PYML-1 and BLM. Indeed, the x-ray crystallographic result of the P-3A-Cu(II) complex demonstrated that the Cu(I1) site is a distorted square-pyramidal structure with four chelate rings of 5-5-5-6 ring members coordinated by the a-amino, secondary amine, pyrimidine ring, deprotonated peptide of histidine residue, and histidine imidazole nitrogens, and that the Cu(I1) ion is displaced about 0.20 A from the basal plane in the direction of the axial a-amino nitrogen ligand (8). The longer wavelength shift of the d-d band and the positive shift of the El/? value for the deglyco BLM-Cu(I1) complex indicate that the ligand field splitting is somewhat weakened in comparison with that in the BLM-Cu(I1) complex. The high reduction potential of the PEML-Cu(I1) complex is presumably attributed to the structural strain of the 5-5-5-5 chelate ring members.
Electron Spin Resonance Features of Divalent Metal Complexes- Fig. 2 displays the ESR spectral comparison for the Cu(II), Co(II), Co(I1)-02, and Fe(II)-I4NO complexes between PYML-1 and BLM. Table I1 summarizes the ESR parameters for the divalent metal complexes of PYML, PEML, P-3A, deglyco BLM, and BLM. As seen in Fig. 2 A , the ESR spectra for the Cu(I1) complexes of PYML and BLM were characterized by axially symmetric gand A-tensor components, and both Cu(I1) complexes evidently presented comparable ESR   parameters. A similar axially symmetric ESR feature was also observed for the PEML-Cu(I1) complex, although the increasing gl, value and the decreasing All value are noted in this case. However, the ESR spectra for the Cu(I1) complexes of P-3A and deglyco BLM showed the copper hyperfine structures with lower symmetric g-anisotropies, suggesting a rhombic distortion of the Cu(I1) chromophore.
PYML, P-3A, and deglyco BLM formed the Co(1I) complexes and their dioxygen adducts similar to those of BLM (21). These ESR features are typical of a low spin squarepyramidal Co(I1) complex with the electronic configuration [(dxy,y,,z,)6(d,2)1] and of monoxygenated low spin Co(II)-O2 adduct complex. The PEML-Co(I1) complex was detected only under the fully anaerobic condition which was achieved by using a vacuum line, and its Allco value (84.3 G) was smaller than those (92-95 G ) of the Co(I1) complexes with PYML, P-3A, deglyco BLM, and BLM. This is due to the higher pK, of the axial a-amino base in PEML ligand, which clearly lacks the electron-withdrawing CONH:! group. Repeated and careful experiments demonstrated no formation of monoxygenated adduct for the PEML-Co(I1) complex.
Under anaerobic conditions, the present six ligands formed the high spin ferrous ( S = 2) complexes (see the following section). However, such high spin Fe(I1) ESR spectra are difficult to detect at 77 K because of their short iattice times. The nitric oxide adduct complexes were easily obtained by addition of a few milligrams of NaI4NO2 (or Na1'N02) and sodium dithionite (or sodium borohydride) to the Fe(I1) com- a The complex was detected only under the fully anaerobic condition which was achieved by using a vacuum line. plexes of PYML, P-3A, deglyco BLM, and BLM. In contrast, the PEML-Fe(I1)-NO complex was not detected even under the fully anaerobic condition. These ESR features exhibited rhombic symmetry with a triplet 14N (or doublet 15N) hyperfine splitting in the central g, signal, and are typical of the sixcoordination type (4). The changes of the nitrogen-hyperfine splitting lines and the AN values by the substitution of I4NO by 15N0 were in accord with the nuclear spin and magnetogyric ratio of I4N (I = 1 and YN = 1.934) and 15N (I = % and YN = -2.712) nuclei.
Visible, Proton Magnetic Resonance, a n d Mossbauer Spectra of Iron(II) Complexes-Except for the PEML-Fe(I1) complex, the Fe(I1) complexes of PYML-1, P-3A, deglyco BLM, and BLM reacted with carbon monoxide, ethyl isocyanide, and nitric oxide to form these adduct complexes. As shown in Table 111, the visible absorption spectra of the Fe(I1) complexes with CO, C2HSNC, and NO differ markedly from that of the original Fe(1I) complex. These dioxygen analogous adducts were characterized by their large extinction coefficients which are due to iron-ligand charge transfer transition, and the absorption maxima were shifted to a longer wavelength in the order CzHsNC > NO > CO. Upon carbon monoxide binding to the PYML-1-Fe(I1) complex, these paramagnetic shifted protons disappeared. As summarized in Table IV, the present 'H NMR results indicate that 1) high spin Fe(I1) ion ( S = 2) and diamagnetic Fe(I1) ion (S = 0) are present in the PYML-1-Fe(I1) complex and its CO adduct, respectively; 2) 'H NMR behavior between the Fe(I1) complexes of PYML-1 and BLM is remarkably similar; and 3) the gulose-mannose sugar protons are involved in the numerous paramagnetic resonances of the BLM-Fe(I1) complex. Indeed, we observed that the magnitude of the chemical shifts in the deglyco BLM-Fe(I1) complex is comparable to that in the BLM-Fe(I1) complex, but the resonance lines of the former are fewer than those of the latter (28). Therefore, it is reasonably supposed that the sugar moiety of BLM is located near  Values in parentheses are e. 'The formation of these dioxygen analogous adducts was not observed even under the fully anaerobic condition.  the Fe(I1)-coordination site spatially and that the sugar protons experience the paramagnetic effect of the central Fe(I1) ion. Fig. 4 shows the 57Fe-Mossbauer spectra of the PYML-1-Fe(I1) complex and its CO adduct at 110 K in zero magnetic field, which are characterized by a single quadrupole doublet. The quadrupole splitting (AEQ = 3.00 mm/s) and the isomer shift (SF, = +1.05 mm/s) of the PYML-1-Fe(I1) complex are remarkably close to those of the BLM-Fe(I1) complex (see Table IV) (22). The Mossbauer parameters are typical of a high spin ferrous ion. The PYML-1-Fe(I1)-CO complex has the Mossbauer characteristics (AEQ = 0.51 and Spe = +0.18 mm/s) which are similar to the BLM-Fe(I1)-CO complex (22) and consistent with a S = 0 ferrous assignment.

TABLE IV Proton chemical shifts and Mossbauerparameters for Fe(II) and Fe(II)-CO complexes of PYML-1 and BLM
Dioxygen Activation by Iron(II) Complexes (Spin Trapping)-From the viewpoint of dioxygen activation, the PYML-1 ligand was investigated and compared with BLM (4). The ESR spin trapping experiments using BPN and DMPO at pH 6.9 evidently revealed that hydroxyl radicals are generated from the PYML-1-Fe(II)-02 complex system. The ESR pattern and the parameters were as follows: BPN spin adduct (triplet of doublet, g = 2.0057, and a N = 15.3 G ) and DMPO spin adduct (quartet, g = 2.0058, and a N = upH = 15.2 G). In contrast with the corresponding PYML-1-Fe(I1)-O2 complex system, the CO introduction strongly interfered with dioxygen activation by the PYML-1-Fe(I1) complex (see Fig. 5). Carbon monoxide is in competition with dioxygen for interaction with the PYML-1-Fe(I1) complex and is a typical O2 antagonist, just as with the BLM-Fe(I1) complex. Table V summarizes the relative spin concentration of hydroxyl radical BPN spin adduct in the Fe(I1) complex systems of BLM, deglyco BLM, P-3A, PYML-1, and PEML. Here, it is of special importance to note that 1) the radical spin concentrations of the PYML-1 (or P-3A)-Fe(II) and the deglyco BLM-Fe(I1) complex systems were estimated to be approximately 20 and 40% of that of the corresponding BLM-Fe(I1) complex system, respectively, and 2) the dioxygen activation ability of the PEML-Fe(I1) complex system was negligibly small in comparison with that of the corresponding BLM system. The result corresponds well to the recent observation (29) in which the deglyco BLM-Fe(I1) complex system gave about half as much ["Hlthymine release from PM-2 DNA as the BLM-Fe(I1) complex system. Therefore, it is inferred that the gulose-mannose sugar portion and the bithiazole-containing tripeptide S moiety contribute to the more effective dioxygen activation by the BLM ligand.

DNA Cleavage Reaction of Iron(Ig Complex Systems-In
the DNA strand scission, the nucleotide sequence specificity of the deglyco BLM-Fe(I1) complex system was determined and compared with that of the corresponding BLM system. Cleavage of double-stranded restriction fragments of plasmid pBR 322 DNA, 327 nucleotides in length, is illustrated in Fig.  6. Although the BLM-Fe(I1) complex system preferentially Of special interest is the fact that the deglyco BLM-Fe(I1) and the BLM-Fe(I1) complex systems revealed almost identical patterns of the cleavage in the DNA f r a h e n t s labeled at the 5' terminus, although the DNA cleavage ability of the deglyco BLM system was somewhat less active than that of the BLM system (see Figs. 6 and 7). The present result strongly demonstrates that the gulosemannose sugar moiety does not give an alteration for the nucleotide sequence specificity in the DNA cleavage reaction by the BLM antibiotics. Under the same experimental condition, on the other hand, the PYML-1-Fe(I1) complex system showed significantly less activity.

DISCUSSION
Although the role of metal ions in the BLM action mechanism has been actively studied, the controversial information for the transition metal-binding sites of BLM has been obtained. The coordination of the sugar carbamoyl group for Cu(I1) (31) and Fe(I1) (6) complexes of BLM, and the bindings of the diaminopropionamide and the P-aminopropionamide groups for Co(II1) complex (32) have been proposed. Recent 'H nuclear relaxation study of the BLM-Mn(I1) complex also suggested the bithiazole group as a metal ligand (7). The present study provides the most reliable evidence for the proposed metal-binding sites (4,5) in which 1) the p-aminoalanine-pyrimidine-P-hydroxyhistidine region of the BLM molecule is substantially important for the Fe(II), COW), and Cu(I1) interactions and 2) the gulose-mannose and the methylvalerate moieties in BLM are not necessarily participating as direct ligands toward the metal coordination. This result is consistent with the structural assignment by x-ray analyses for the P-3A-Cu(II) complex (8) and the Co(II1) complex of pseudo-tetrapeptide A of BLM (33). The synthetic ligand PYML-1 includes 1) simplification of the pyrimidine nucleus of BLM to pyridine nucleus, 2) use of a simplified side chain, ([(S)-2-amino-2-carbamoylethyl]amino} methyl group, and 3) use of histidine for /3-hydroxyhistidine of BLM. Nevertheless, the physicochemical properties of the PYML-1-divalent metal complexes are remarkably similar to those of the corresonding BLM metal complexes. Certainly, PYML-1 is a simplified and valuable analogue which corresponds to the amine-pyrimidine-imidazole portion of BLM.
As seen between PYML and PEML, the substitution of the imidazole group by the amino group gives significant influence on dioxygen-binding and -activation by the Fe(I1) complexes. In contrast with PYML, P-3A, deglyco BLM, and BLM ligands, the Co(I1)-Op, Fe(I1)-CO, Fe(II)-C?HSNC, and Fe(I1)-NO adduct complexes of PEML were not observed by all means. Therefore, the presence of the imidazole group, in particular the transposition of pyrimidine (or pyridine) and imidazole groups in the Fe(I1)-coordination, appears to be essential for the effective binding and activation of molecular oxygen by the Fe(I1) complexes of BLM-related ligands. We previously reported that 1) the spectroscopic and crystal field parameters of the BLM-iron complexes with CO, C2H5NC, NO, N:t-, OH-, and CH:,NH:! are similar to those of the corresponding hemoprotein complexes, except for the CNadducts and 2) the iron ligand donors in BLM are arranged in a rigid square-pyramidal configuration with a 5-5-5-6 ring member, as seen in the case of heme (22). Probably, the aromatic nitrogen-containing and electron-rich structure formed by the coordination of pyrimidine (or pyridine) and imidazole nitrogen ligands truns to each other, contributes to the same consequence on iron electronic state as found in the hemoproteins.
The remarkable similarity of the divalent metal complexes between deglyco BLM and BLM definitely indicates that the ' I carbamoyl group of the sugar portion in BLM does not directly coordinate to the metal ions. It is well known that the addition of HzOz induces the conversion from the inactive BLM-Fe(II1)-OH-complex ( gl = 1.893, g 2 = 2.185, and g: 3 = 2.431) to the active BLM-Fe(III)-02H-species (gl = 1.937, gz = 2.171, and g , = 2.254) for the DNA cleavage (34)(35)(36). When hydrogen peroxide (1-10 mM) was added to the deglyco BLM-Fe(II1)-OH-complex (1 mM) which shows the typical low spin ferric ESR signals at gl = 1.887, gl = 2.180, and g a = 2.432, it quite similarly changed to the ESR spectrum with g, = 1.937, g2 = 2.171, and g3 = 2.254, whereas, the decreasing dioxygenactivation ability of the deglyco BLM-Fe(I1) complex system suggests that the gulose-mannose moiety is situated near the Fe(I1)-coordination site and plays an important role as the environmental factor in the efficient dioxygen activation, just as pivalamidophenyl groups in picket-fence porphyrins (37,38). The previous ESR studies demonstrated that the environment of in-plane ligands with the pyrimidine and imidazole groups in the BLM-Fe(I1)-NO complex is altered by the binding of DNA (39,40). Antholine et al. (41) observed by 'H NMR study that the binding to poly(dA-dT).poly(dA-dT) changes the environment of the imidazole and methylpyrimidine rings of the BLM-Fe(I1)-CO complex but does not have a similar effect on these rings when the iron is absent. As shown in the BLM-Fe(I1)-NO complex, the binding of DNA to the deglyco BLM-Fe(I1)-NO complex also induced a greater separation of the g, and g, absorptions in comparison with the original ESR spectrum of the deglyco BLM-Fe(I1)-NO complex (see Table 11). Herein, it is of interest that the difference g, (2.060-2.041) and g, (1.976-1.962) values of the BLM complexes are larger than those (g, = 2.046-2.038 and g, = 1.976-1.963) of the deglyco BLM complexes. A weak interaction such as hydrogen bond between the sugar group of BLM and DNA phosphate group may be responsible for the larger g separations in the BLM complex. As clearly demonstrated in the DNA cleavage by the deglyco BLM-Fe(I1) complex system, however, the absence of the gulosemannose portion in BLM ligand gives no noticeable effect on the nucleotide sequence specificity. Therefore, the sugar moiety does not contribute significantly to the specificity of DNA binding, which is mainly due to the intercalation with the bithiazole group (42). Recently, the binding specificity of the four BLM analogues, BLM-BI', BLM-A?, BLM-Bs, and peplomycin, was compared, and the result also shows no significant contribution of the terminal amine side chains to the DNA nucleotide specificity (43). On the other hand, it has been noted that the somewhat altered specificity of tallysomycin must be attributed to the presence of the additional amino sugar (43). The present guanine-pyrimidine (5' + 3') specificity of BLM represented by sequences is consistent with the observations by D'Andere and Haseltine (44) and Takeshita et al. (30,43) who used lactose operon pL 53 and bacteriophage 4x174 DNA fragments, respectively. The synthetic analog PYML-1-Fe(I1) complex showed the effective dioxygen activation, but was remarkably less active than the corresponding BLM complex in the DNA cleavage reaction. The DNA binding molecule to deliver a metal ion to the site of the DNA helix where activated molecular oxygen attacks the DNA is required for the efficient DNA strand scission. The examples involve the 1,lO-phenanthroline-cuprous complex (45,46) and the methidiumpropyl-EDTA-ferrous complex (47) which result in the DNA cleavage under the presence of oxygen. On the other hand, the BLM-Cu(1) complex has been reported to produce oxygen radicals under aerobic conditions (48) and to form a carbon monoxide adduct (49). However, the BLM-Cu(1) complex did not cleave the present pBR 322 DNA fragment in the presence of 2-mercaptoethanol (or sodium dithionite). This phenomenon is probably attributed to the low redox potential of the BLM-copper complex. Indeed, the BLM-iron complex has the much higher redox potential (Ell2 = -+0.15 V uersus NHE) (50).
In summary, 1) the P-aminoalanine-pyrimidine-P-hydroxyhistidine portion of BLM molecule is substantially important for the Fe(I1) and dioxygen interactions, and indeed the synthetic analog PYML is able to mimic the metal binding and dioxygen reduction by BLM ligand, 2) the pyrimidine group of BLM can be probably replaced by the pyridine group without loss of its function of this group, but the substitution of the imidazole by the amino groups shows the significant decrease of the effective O2 binding and activation, 3) although the gulose-mannose group does not contribute essentially to the nucleotide specificity in the DNA cleavage, the sugar portion plays an important role as the environmental factor in the efficient oxygen activation and DNA cleavage, and 4) the gulose-mannose, methylvalerate, and bithiazole moieties in BLM are not clearly participating as direct ligands toward the Fe(I1) binding. We believe that the present results promise good hope for the molecular design of a synthetic compound with the function of selective DNA base cleavage.