J. Am. Chem. SOC.1984, 106, 6905-6909
6905
The Binding of Yb(II1) to Adriamycin. A 'H NMR Relaxation Study I. J. McLennan and R. E. Lenkinski* Contribution from the Guelph- Waterloo Centre for Graduate Work in Chemistry, Guelph Campus, Guelph, Ontario, Canada NIG 2W1. Received March 21, 1984
Abstract: The binding of Yb(II1) to adriamycin in methanol was monitored by UV-vis absorption spectroscopy. A Job ratio plot based on UV-vis binding data indicated that the complex had a stoichiometry of 1:l. The formation of the complex was found to be dependent on the hydrogen ion concentration present in methanol. The 'H NMR spectrum of the Yb(II1)-adriamycin complex was found to contain two sets of resonances arising from the complex and the free adriamycin indicating the rate of exchange of adriamycin between its free and complexed state was slow on the 'H NMR chemical shift time scale. Transfers of magnetization experiments were carried out in order to assign the resonances arising from the paramagnetic complex. The spin-lattice relaxation times of the assigned resonances were determined. These relaxation times were analyzed in terms of the distances from the Yb(II1) ion to various protons in the drug. With use of these distances and the coordinates of the hydrogen atoms obtained from an X-ray structure, the site of metal complexation was found to be at the Cl1and CI2oxygens of the antibiotic characterized by an oxygen-Yb(II1) bond length of 2.5 A.
Adriamycin (Adm (I)) is an anthracycline aminoglycoside antibiotic. Adriamycin differs from daunomycin in that the CHzOH at position 14 is replaced by CH3. The pharmokinetic properties of Adm have been the subject of a large number of studies over the last 20 years.3 The first reported observation of metal binding to anthracyclines was by Calendi et al. in 1965.4 These authors found that large spectral changes occured in the visible spectrum of daunomycin upon the addition of an excess of Al(III), Fe(II), Fe(III), and Mg(I1) in aqueous solution. Myers OH
0
0
I
C"
P? NH
OH
I
et aL5have also observed perturbations in the absorption spectrum of Adm upon binding of Fe(II1) characterized by a shift from 479 to 608 nm with an isobestic point at 535 nm upon aeration in aqueous solution. From their studies the authors concluded that the species observed was probably the Fe(I1)-Adm complex. Using visible absorption, CD, and ESR spectroscopy, Greenaway and Dabrowiak6 monitored the binding of Cu(I1) to daunomycin, adriamycin, and N-(trifluoroacety1)daunomycin in water and have proposed that a bis Cu(I1) complex of the drugs exists from pH 6.0 to 8.5. It was suggested that Adm forms a complex with Cu(I1) at the quinone, hydroquinone ligands6 In addition, Spinelli and Dabrowiak' have reported the existence of a Cu(I1)-daunomycin-DNA ternary complex in which the Cu(I1) is bound either to the C,, c6 or the C,,, C I 2and is not intercalated with calf thymus DNA. Fritzsche et a1.* have suggested that iremycin, (1) Supported by the Natural Sciences and Engineering Research Council of Canada (R.E.L.).
(2) McLennan, I. J.; Lenkinski, R. E.; Yanuka, Y. J . A m . Chem. SOC., submitted for publication. (3) For a review see: Acamone, F. 'Anticancer Antibiotics"; Academic Press: New York, 1981; Vol. 17. (4) Calendi, E.; Di Marco, A,; Reggiani, M.; Scarpinato, B.; Valentina, L. Biochim. Biophys. Acta 1965, 103, 25-49. (5) Myers, C. E.; Gianni, L.; Simone, C. B.; Klecker, R.; Greene, R. Biochemistry 1982, 21, 1707-1713. (6) Greenaway, F. T.; Dabrowiak, J. C. J . Inorg. Biochem. 1982, 16, 91-107. (7) Spinelli, M.; Dabrowiak, J. C. Biochemistry 1982, 21, 5862-5870.
0002-7863/84/1506-6905$01.50/0
which is similar to daunomycin, forms a Cu(I1) complex which also binds to DNA. However, there is very little specific geometric information concerning the metal ion binding site@) in any of these reports. We have chosen to focus on the Yb(II1) complex of Adm for a variety of reasons. We have found evidence for the existence of a ternary complex of Adm-DNA-lantha~~ide(III).~ There is a large body of literature dealing with lanthanides as isomorphic replacements for Ca(I1) in biological Of the paramagnetic lanthanides Yb(II1) has been found to produce perturbations in the N M R spectra of ligands which are most amenable to structural interpretations (vide infra). In a separate study using UV, visible, and fluorescence spectroscopy, we have determined the stoichiometry and the equilibrium constants for various lanthanide complexes of Adm in aqueous solution.I2 In the course of this study we found, as was previously reported by others,l3-I5 that Adm undergoes self-association in aqueous solution. In the present study we present an analysis of the perturbations observed in the IH NMR spectrum of Adm induced by Yb(II1). The approach used in the analysis is similar to the one employed by Sykes and co-workers in the analysis of the ' H N M R spectral perturbations observed in the Yb(II1)-parvalbumin As is the case with Yb(111)-parvalbumin, the rate of dissociation of the Yb(II1)-Adm complex was found to be slow on the IH N M R chemical shift time scale at 400 MHz. This manifests itself in the appearance of distinct resonances of the paramagnetic complex (vide infra). These resonances were assigned by transfer of saturation techniques. The relaxation rates of these assigned resonances were analyzed in terms of the distances between the various protons and the Yb(II1) center. In the following sections we present the details of these experiments as well as a geometrical model for the Yb(II1)-Adm complex based on our analysis. (8) Fritzsche, H.; Triebel, H.; Chaires, J. B.; Dattagupta, N.; Crothers,
D.M. Biochemistry 1982, 21, 3940-3946. (9) Lenkinski, R. E., manuscript in preparation. (10) Reuben, J. "Handbook on the Physics and Chemistry of the Rare Earths", Gschneidner, K. A,, Jr., and Eyring, L., Eds.);North Holland: New York, 1979; 515-552. (1 1) Reuben, J. Naturwissenschaften 1975, 62, 172-178. (12) Lenkinski, R. E.; Sierke, S.; Vist, M. R. J . Less.-Common Met. 1983, 94, 359-365. (13) Barthelemy-Clavey, V.; Maurigot, J.; Dimicoli, J.; Sicard, P. FEBS Lett. 1974, 46, 5-9. (14) Martin, S. R. Biopolymers 1980, 19, 713-721. (15) Chaires, J. B.; Dattagupta, N.; Crothers, D. M. Biochemistry 1982, 21, 3927-3932. (16) Lee, L.; Sykes, B. D. Biochemistry 1981, 20, 1156-1162. (17) Lee, L.; Sykes, B. D. In 'Biomolecular Structure Determination by NMR" Bothner-By, A. A., Glickson, G. D., Sykes, B. D., Eds.; Marcel Dekker: New York, 1982; pp 169-188. (18) Lee, L.; Sykes, B. D. Biochemistry 1983, 22, 4366-4373.
0 1984 American Chemical Society
6906 J . Am. Chem. SOC.,Vol. 106, No. 23, 1984
McLennan and Lenkinski
I
'
7
9
. -
PPM
3
1
Figure 1. The 400-MHz 'H spectrum of 4 mM adriamycin in methanol, 128 scans.
J 7 I I 10
0
'
-20
0
PPM
Table I. IH Chemical Shift Data for Adriamycin in Methanol position H1 H2 H3 HI' H7 HI4 H5' 4-OCHp
6 (PPm from DSS) 7.97 7.85 1.59 5.45 5.1 1 4.71 4.28 4.04
position H4' H3'
HI0 H8 H8 H2' H 2' 5'-CH3
8 (PPm
from DSS) 3.66 3.56 3.05 2.36 2.18 2.04 1.88 1.29
+
Figure 2. The 400-MHz 'Hspectrum of 5 mM Adriamycin 2.5 m M YbCI3 in methanol, 500 scans, sweep width 40000 Hz. The two peaks marked with an X do not arise from the Yb(II1)-Adm complex. In comparison to the other peaks at -32 and -39 ppm the aforementioned peaks arise from more than one proton.
Materials and Methods Adriamycin hydrochloride was kindly provided by Dr. F. Arcamone of Farmitalia Carlo Erba (Milan, Italy). The concentration of Adm was determined spectrophotometrically at a wavelength of 477 nm (t = 11 500 M-' cm-I).l9 The solutions of Adm and Adm-Yb(II1) were made up within 24 h prior to use in deuterated methanol (Merck Sharpe and Dohme). The pH of the solutions was adjusted with either 0.01 M DCI or 0.01 M NaOD (Merck). For convenience we will use the term p H to refer to the pH meter reading obtained in methanol although we realized that this does not give the hydrogen ion concentration directly. No corrections were made for deuterium isotope effects. YbCI3 (99.99%) was obtained from Ventron (Danvers, MA). The concentrations of stock solutions of Yb(II1) were determined by titration with EDTA with arsenazo as an indicator. The UV-vis and N M R experiments were performed at 22 i 1 OC. The UV-vis absorption spectroscopy was performed on a Perkin Elmer Lamda 3 spectrophotometer. The ' H N M R experiments were performed on a Bruker WH-400 spectrometer operating at 400.13 M H z in the Fourier transform mode with quadrature detection. All ' H N M R spectra were referenced to internal DSS (sodium (trimethylsi1yl)propanesulfonate). For the transfer of magnetization experiments the decoupler power was adjusted so that one of the three aromatic protons of Adm was completely saturated but no visible perturbation in the intensities of the other peaks in the N M R spectrum was observed (on the spectrometer being used in these studies, the power level was ca 17 dB attenuation below 0.2 W). The shifted resonances of the Yb(II1)-Adm complex were selectively saturated by using gated homonuclear decoupling with a decoupling time of 1 s. The IH spin-lattice relaxation times were determined by the inversion recovery method (180°-r900)20 in which there were at least 10 T values and a recycle time of 5 X T I . The data were subsequently fit to a three-parameter exponential relationship based on a plot of the peak intensity vs. T.
Results and Discussion Figure 1 shows a 'H N M R spectrum of 5 mM Adm in deuterated methanol at 400 MHz. Since Adm self-associatesin water, (19) Gabbay, E. J.; Grier, D.; Fingerle, R. E.; Reiner, R.; Levy, R.; Pearce,
S.W.; Wilson, W. D. Biochemistry 1976, 15, 2062-2070.
(20) Vold, R. L.;Waugh, J. S.; Klein, M. P.; Phelps, D. E. J . Chem. Phys.
1968, 48, 3831-3832.
Figure 3. UV-vis absorption spectrum of adriamycin in methanol 5.3 X lo-' M Adm, and with the addition of 1 X lo-' M YbCI,.
the present study was performed in methanol where we have found no evidence for self-association.* The peak assignments are presented in Table I. Upon the addition of 2.5 mM YbC13 a new set of peaks is seen at lower frequency (Figure 2). It can be seen by a comparison of Figure 2 with Figure 1 that the resonance positions of the peaks in the original diamagnetic spectrum of Adm are not altered. Thus we can conclude that the paramagnetic Yb(II1)-Adm complex is in slow exchange on the 'H N M R time scale with free Adm under these conditions. It is likely that the Yb(II1)-Adm complex in methanol still may have chlorines in some of the coordination sites. However, this will not affect any of the geometrical conclusions reached regarding the site of complexation of Yb(II1) on Adm. With Yb(II1) in particular, the paramagnetic interactions can give rise to large shifts which are dipolar in origin with minimal broadening of the shifted resonances. This is seen in Figure 2, and therefore the set of peaks at lower frequency are resonances arising from the paramagnetic complex (Yb( 111)-Adm). Analogous experiments were performed with La(II1) in order to determine the magnitude of the diamagnetic contribution to the overall shift. Upon the addition of La(II1) there was found to be little (
