Nickel(II)—bleomycin: spectral investigation of the metal binding site

Nickel(U)-Bleomycin: Spectral Investigation of the Metal Binding Site Andrea Dei and Dante Gatteschi lstiruto di Chimica Florence,

Generale

ed Inorganica.

Facolta di Farmacia.

Universita di Fire&-e.

italy

ABSTRACT Bleomycin (Mm) solurions conraining the nickel(M) ion have been investigated rhmugh *H m-or. &and field and circulardichroism spectroscopies. It has been foundtheblmbinds the metal ion in a pH dependent fashion. The spectral data are consistent with the presence of at least two species. it is suggested that in the low pH region blm binds to nickel(U) through the /3-aminoalanino residue. whereas inthehighpH region. the&unino-pyrimidine. imidazole. and amidogroupoffl-h\rdroxy-

hisridine arealso involvedincoordination.

INTRODUCTION in view of the relevant important of transition metal ions in determining the biological activity of bleomycins (blm), several studies have been carried out in order to investigate the coordination chemistry of these molecules [ I-31. To date the bulk part of all research work has been focused on iron [4-IO], cobalt [ 1l-141. copper [2, E-191, and zinc [15, 16.201 metal-blm derivat&es. On the bases of the electronic spectra 121.blm is believed to form pseudooctahedral complexes with nickei(I1). Since these complexes are fully paramagnetic, we have attempted a ‘H nmr investigation, the isotropic shifts being expected to provide a powerful tool for elucidating the nature of the chromophore and for obtaining conformational information [2 I].

EXPERIMENTAL Bleomycin sulfate (Blenoxane) was a generous gift of Bristol Laboratories, (Syracuse, NY). It consists of approximately 70% bleomycin A,and 30% bleomycin B,. A molecular weight of 1550 was assumed. Solutions containing Ni-blm derivatives were obtained by mixing blm solutions in Dp (Merck 99.8%) with an appropriate volume of Address requests for reprints to Prof. D. Gatteschi, Istituto di Chimica Generale ed Inorganica,Facolta di Farmacia,Universitadi Firenze,Via G. Capponi 7,50121 Florence, Italy. lo-1 OfI~ -cBiochem&fq 17, 131-137 (1982) @ Ekvier Scie- Publishing Co.. Inc. 1982

52 VanderbiltAve.. New York. NY 10017

131 0162-0134/82/060131-0752.75

132

A. Dei and D. Gatteschi

nickel(H) nitrate solution in the same solvent. The solvent was then evaporated to dryness under vacuum and an appropriate volume of DIO was added. The final concentration of blm was - 10 -‘M_ The pH values of the solutions were then adjusted by addition of NaOD. The electronic spectra were recorded in the range of 10.000-30.000 cm--’ using a Car-y 17 D spectrophotometer. The circular dichroism (CD) spectra were carried out using a Jasco J-500 C spectropolarimeter in the range of 10.000-25.000 cm-‘. The ‘H nmr spectra were run with an instrument based on a Bruker CXP consolie and a low resolution Varian DA 60 1.41 T electromagnet, equipped with an external lock circuit affording I Hz long-texm stability. The spectra were recorded in the 30.000 Hz range as referenced to DSS 4.4dimethyl-4-silerpentaneI-sulfonate. The spectra were obtained using a modified DEFT pulse sequence in order to suppress the solvent signal. A Varian CFT 20 spectrometer operating at 80 MHz was used to record spectra in the O-l -000 Hz range. RESULTS The clinically used bleomycin is a mixture of essentially two derivatives: blm AZ (ca. 70%) and blm Bz (ca. 30%). Since it has been shown that the two blm congeners exhibit identical ligand properties towards metal ions [ 1. 10.22.23]. in the present investigation we have used the clinical mixture of the two congeners. The electronic spectra show two main peaks, in the range lO.OOO-20,000 cm-‘, whose position is pH dependent. At high pH a band is present at 13.000 cm --I (E = 23) and another at 18,800 cm-t (E = 18). These data compare satisfactorily with those reported by Dabrowiak [2]. On lowering the pH the two bands show a red shift. Eventually, at pH = 2.5, the bands move to 10,7OOcm-’ (E = 13) and 17,5OOcm-* (E= 8), respectively, with a shoulder at 12,800 cm-r_ No isobestic points are detected when the electronic spectra of the same solution at various pH values are compared. A similar behavior is observed in the CD spectra (Fig. 1). Also the *H nn; spectra of D1O solutions containing both nickel(B) and blm in -0.9 molar ratio show a marked dependence on the pH_ For pH < 3, neglecting the lines falling in the range - 10/O ppm (DSS reference). where the resonance signals of protons not interacting with the paramagnetic metal ion are expected_ at least five lines can be detected, four downfield and on upfield from DSS, as shown in Figure 2a. Although accurate determination of the areas of the peaks could not be made because of their broadness, it appears that the upfield signal is twice as intense as the four downfield signals. The intensities of all these signals decrease as the pH increases, while at the same time a new pattern of signals appears. A pH > 4.5, only this new set of signals is present in the spectrum (Fig. 2b). At intermediate PH. both sets of signals are simultaneouslv detected_ The high pH spectrum shows seven resonance lines, six downfield and one upfield from DSS _However, because of the broadness and the overlap of the signals in the range -6O/10 ppm, an accurate estimation of the area of the signals is not possible and therefore the number of resonating protons experiencing significant isotropic shifts is uncertain_ As the pH is varied, significant changes are observed in the diamagnetic range of the spectrum as well. At low pH the signals attributable to the imidazole and methyl group of the 4aminopyrimidine show no significant isotropic shift or broadening. However they become less intense as the pH increases, until they finally disappear at pH > 4.5. Protons

Nickel(II)-Bleomycin

133

I

I

I

500

4

8

700

600

nm

FIGURE

FIGURE

LCDspectra

2. Schematic

of Ni(II)-blm

1H nmr spectra

at pH = 2.9 (-)

and at pH = 5.7 (---).

at 34OCof D,O solutionsof Ni(II)-blm

at pH = 2.7 (a) and

ph = 7.4 (b).

+14 -233

(a)

-43

-151

I

-24

I -200

-2DD

I

(DSS)

-100

0

-100

0 (DSS)

v

ppm

134

A. Dei and D. Gatteschi

attributable effects.

to the dibenzothiazole

moiety

never experience

any significant

paramagnetic

DISCUSSION The electronic and CD spectra of the Ni-blm system show the existence of an equilibrium involving at least two species. In every case the coordination environment of the nickel(II) ion is octahedral_ The red shift of the bands shows that the donor atoms of the species predominant in the low pIi region exert a smaller ligand field strength as compared to the high pH ones. The energy of 3A,+3T,, transition (13,UOO cm -I) of the high pH species is extremely high for an octahedral complex and is in agreement with the low-spin behavior of the cobah complex [ 1 l] and with the high energy of the transitions in the copper(U) complex [ 16,241. That blm is actually bound to the metal, either at low or high pH, is shown by the CD spectra in which optical activity can be induced only by the interaction of the metal ion with the optically active blm. The ’ H nrnr spectra also show the existence of two paramagnetic species in solution whose relative abundance depends on the pH of the solution_ Although in principle the existence of more than two species cannot be excluded. the fact that the decrease of the low pH signal intensities is paralleled by au increase in the intensities of the high pH signals leads us to suggest that the observed pattern of resonances should be attributed to only two predominant species. Furthermore. the simultaneous detection of signaIs of both the species indicates that the molecular processes involved in the equilibrium are slow on the I H nmr time scale. This behavior is usually observed in nickel(M) complexes when reactions involving the substitution of two or more donor atoms occur. as for instance in chelate ring formations [21,25]_ Similar considerations hold when one considers that the spectrum of the Ni-blm species at low pH does not exhibit any change upon variation of the molar ratio Ni(II)/blm_ It can be concluded, therefore, that in the predominant species in this pH range more than one donor atom of th e _glycopeptide must be involved in coordination towards nickel( II)_ The fact that for pH < 3 the resonance signals of the pyrimidine methyl group and imidazole do not experience any appreciable shift or significant broadening upon addition of nickel(M) salts indicates that neither 4-amino-pyrimidine and fl-hydroxyhistidine moieties are involved in coordination towards nickel(I1). The values of the isouopic shifts of the signals at -223 and - 15 1 ppm are large and the only reasonable candidates for the assignment are protons lying on o-carbons with respect to nitrogen donor atoms. Values of this order of magnitude are anticipated for protons having pseudoequatorial character in five- or six-membered chelate rings [2 I, 26-28J. The corresponding pseudoaxial protons are expected to exhibit smaller downfield shifts. Therefore it is suggested that the observed resonances can be explained by assuming the existence of one chelate ring in the Ni-blm species that predominates at low pH values- Since the most reasonable candidate seems to us the p-aminoalanine residue, the simplest structural hypothesis that can account for the experimental spectra is that shown in Figure 3. The X conformation should be stabilized in energy with respect to the 6 one by the presence of bulky equatorial substituents on the secondary amino groups. In this respect the signals with large downfleid shifts could be attributed to the two equatorial H, and H, protons, whereas the signal at c-20 ppm should be assigned to

135

Nickel(H)-Bleomycin

D2NOC FIGURE

3. Proposed structure of the Ni(II)-blm species existing as predominant at low pH.

the H, proton. The other signah at 43 and + 14 ppm should be attributed to the H, proton and to the /3-methylenic protons of the propionamide residue. The upfield shift of the Iatter signal is accounted for on the basis of spin polarization mechanism considerations. 129). Support is given to this binding model hypothesis by the results of i3C relaxation [7] and ESR studies [3, 171 on iron(R)- and copper(R)-blm systems, respectively. The conclusions of an iH nmr investigation on the o, &diaminopropionate nickel(R) complexes can he also usefully be referred to for comparison DO]_ The ligand field and CD spectra of the high pH Ni-blm species show that an octahedral complex is formed in which new donor atoms are involved in coordination towards nickel(U). The high ligand field strength exerted suggests that other nitrogen atoms bind to the metal acceptor. This is continned by the ’ H nmr spectra, which show a signiticant interaction of the metal ion with the 4-amino-pyrimidine and imidazole groups. It is apparent that the binding of these groups to the metal ion occurs only when the blm releases the fourth acidic proton [ 1,231. According to the results of the x-ray structural analysis of the Cu(P-3A) complex (241. it appears that the driving force for the above groups is the coordination of the deprotonated P-hydroxyhistidine function. Although this fact does not constitute an uncommon event in the chemisuy of metal-polypeptide derivatives, 13 I, 321, it is not easy to show experimentally that this occurs in the present Ni-blm

case. On the other hand, if this was not true, the 4-amino-pyrimidine

group

should be expected to be coordinated also in the species existing as predominant at lower pH values, since this group does not experience relevant basicity properties. Therefore it seems reasonable to propose the existence of tive nitrogen donors in the coordination sphere of the nickel(R) ion. The assignment of the *H nmr resonance signais is quite problematic. The signals at -225 and - 199 ppm may be attributed either to the two pseudoequatorial protons of the p-aminoalanino residue or to one of them and to the methinic protons adjacent to this amido group. The resonances of the pyrimidine methyl group and the methyiene of the propionamide residue are attributed on the basis of intensity considerations to the signals at -33 and +8 ppm. respectively. Imidazole protons and aliphatic pseudo axial proton signds are expected to fall in the range -6O/-20 ppm_ As mentioned above. the broadness and the overiap of these signals preclude any detailed assignment at this stage. In order to obtain some usefui conformational information it should be appropriate to compare these spectra with those of some related bleomycins as, e-g.. deamido

bleomycin

tdea bhn) or depyruvamide

bieomycin (dep blm).

136

A. Dei and D. Gatteschi

In conclusion, these results indicate that him in its nickeI(II) derivatives exhibits similar binding properties to those found in copper and cobalt(W) complexes. The isotropic shift data show that in solution an equilibrium involving two different pammagnetic chromophores occurs. This has been commonly explained by assuming the existence of two pH dependent complex formation equilibria [23]. However. it cannot be excluded that a more complicated pH dependent coordination processes occurs. Some support to this hypothesis may come from the fact that_no isobestic points were detected in the spectrophotometric titrations. Analysis of our data suggests that at low pH. metal coordination occurs through the P-aminoalanino residue. whereas at higher pH. the pyrimidine. imidazole. and amido groups of p-hydroxyhistidine are also coordinated_ These data give additional support to the current model of metal coordination of bleomycin. i7re authors are indebted to Bristol Labomtories for the generous gift of Blenoxane. expressed to Dr. Roberto Monnaxni and to Mr. Antonio Canetta for assistance.

Thanks are

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Nickel(II)-Bleomycti

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E. J. Evans, J. E. Grice, C. J. Hawkins, and M. R. Heard,Inorg

Received Novem her 20. I98I: accepted Febnrnly I4 I982

Chem 19,3496

(1980).