1344
J . Phys. Chem. 1994,98, 1344-1350
Comparative Studies of Antitumor DNA Intercalating Agents, Aclacinomycin and Saintopin, by Means of Surface-Enhanced Raman Scattering Spectroscopy Igor Nabiev,+ Igor Chourpa, and Michel Manfait’ Laboratoire de Spectroscopie Biomol&ulaire, UFR de Pharmacie, Universit6 de Reims, 51 rue Cognacq-Jay, F-51096 Reims Cedex, France Received: September 9, 1993@
The surface-enhanced Raman scattering (SERS) spectra of aclacinomycin, saintopin, and their complexes with D N A recorded a t the very low (ca. lo-’ M ) concentration allowed us to obtain, by the analysis of deuteration and p H effects, a vibrational assignment of the SERS active modes for both chromophores. W e interpret the SERS spectra of aclacinomycinf D N A complex as indicating that the chromophore is stabilized inside doublestranded helix by the hydrogen bond between OH group of the chelate-like system of aclacinomycin (Figure 1) and functional group (probably, free C==O group of thymine) of DNA. The structural model of the complex is proposed and found to be consistent with N M R studies published before. This model is discussed in terms of data for daunomycin and adriamycin complexes with D N A fragments already obtained from X-ray measurements. In contrast to aclacinomycin, the binding mode of partial intercalation has been found to be preferential for saintopin chromophore. The free carbonyl group and a part of the chelate system (Figure 1) are both buried in the interior of DNA. However, the periphery hydroxyl group is shown to be still accessible to the silver surface upon the chromophore intercalation. N o evidence for redistribution of intramolecular hydrogen bonds of the chromophore, or formation of new hydrogen bonds between saintopin and functional groups of D N A has been found. Differences between aclacinomycin and saintopin D N A intercalation modes revealed by the S E R S spectroscopic data are considered in connection with their biological effects, especially within a process in which recognition of D N A by enzymes such as D N A topoisomerases I and I1 occurs.
Introduction Many DNA intercalators have been shown to have antitumor activity.’ Among them, anthraquinone derivatives have been studied most extensively.2 Despite extensive effort in analogue synthesis, antitumor activity of these intercalators was not understood. No single known parameter (e.g., DNA-binding strength, drug hydrophobicity, ability to inhibit DNA synthesis, etc.) correlated with drug cytotoxicity or antitumor activity.2 However, structural specificity of the drugs was clearly noted,3 N\ CH, Qo and the biological activity has been attributed to the formation of the intercalation complexes between the chromophore framework and base pairs of DNA.4,5 The changes in the overall structures of the drugftarget complexeS amplify small chemical differences between antibiotics and provide a possible explanation for the differences in the clinical activity of the drugs.6 Much research has been carried out in order to elucidate the structural basis of intercalation mechanism.G8 In particular, ADNAMYCIN ACLACINOMYCIN resonance Raman spectroscopy has been widely used because of Figure 1. Structures of aclacinomycin, saintopin, and adriamycin. its selectivity which permits the observation of only bands corresponding to the vibrations of the chromophoric framework DNA complexes due to a short-range character of Raman and of its sensitivity to the structure of drug/ DNA c o m p l e x e ~ . ~ - ~ ~ enhancement effect.” Sequaris et al.15 were the first to analyze Unfortunately, it is quite difficult to record well-resolvedresonance the SERS spectra of complexes of some Pt-coordinatedcompounds Raman spectra and to propose accurate assignments of the bands with DNA and to correlate the antitumor activity of these species due to the strong fluorescence of anthraquinone derivatives in with their ability to intercalate inside DNA double helix. SERS water solutions. spectra of some anthracyclines systematically used in the clinical Surface-enhanced Raman scattering (SERS) has been used treatment of cancer have been reported by Smulevich et al.,I2J3 as a powerful method to obtain information for fluorescent Nonaka et a1.,I0 and Nabiev et al.11J4 The well-detailed SERS chromophores.1g14 It allows total quenching of the chromophore spectra of adriamycin,lg12 THP-adriamycin,” aclacinomycin,lOJl fluorescence and at the same time magnifies Raman scattering (Figure 1 ) and some derivativesI2J3allowed to obtain a nearly by several orders of magnitude. SERS technique has become complete vibrational assignment of the resonance Raman active very attactive also for investigation of the topology of drugmodes. Models of the intercalation between some drugs and DNA have been proposed and were found to be consistent with * Author to whom correspondence should be addressed. X-ray crystallography data.IgI3 + Permanent address: Optical SpectroscopyDivision, Shemyakin Institute In the present paper, we report a comparative SERS study of of Bioorganic Chemistry, Russian Academy of Sciences, 117871, Moscow, two anticancer drugs, structurally related to the anthracycline Russia. antibiotics, aclacinomycin and saintopin (Figure 1). Abstract published in Advance ACS Abstracts, January 1, 1994. @
0022-3654/94/2098- 1344$04.50/0
0 1994 American Chemical Society
SERS of Antitumor DNA Intercalating Agents Aclacinomycin is recognized as a strong DNA intercalator.' This drug at low concentration stimulates topoisomerase IImediated DNA cleavage and inhibits topoisomerase 11-mediated DNA cleavage at higher concentrations.IJ The interaction of aclacinomycin with DNA has been studied by resonance Raman,Io electronic absorption, fluorescence, and NMRI6J7spectroscopy. The results suggested that its chromophore is deeply intercalated preferentially into the T-A sequences of DNA.16J7 No SERS studies of aclacinomycin in the complex with DNA have so far been published. Saintopin chromophore is structurally similar to that of aclacinomycin (Figure 1). However, their biological effects are very different. Saintopin represents a new class of antitumor agents that can induce both DNA topoisomerase I- and DNA topoisomerase 11-mediated DNA cleavage a t relatively low concentration.I8 In contrast to aclacinomycin, DNA cleavage induced by saintopin is not suppressed at a high drug concentration. It is probable that the unusual mode of binding to DNAcontributes to its unique functions.'* As far as we know, no physicochemical studies of this drug have so far been published. In this paper we present the SERS spectra of aclacinomycin, saintopin, and their complexes with DNA. Vibrational assignment of the SERS active modes has been done on the basis of attributions proposed for adriamycin11-13 and aclacinomycinI0J1before and from our SERS measurements in water and deuterium oxide solutions. Models of interaction of aclacinomycin and saintopin with DNA double helix have been proposed and compared with those already obtained for adriamycin and daunomycin from X-ray measurements.c8 Biological effects of the drugs are concerned in connection with the differences between their DNA intercalation modes being demostrated by the SERS spectroscopic data. Experimental Section Aqueous silver hydrosol was prepared by reducing of silver nitrate with trisodium citrate as described p r e v i o ~ s l y ~and ~J~ preaggregated by the addition of sodium perchlorate up to the final concentration of 0.06 M. DzO colloids were made in exactly same fashion, substituting D20 (loo%, Sigma) for water. No sodium chloride was added. Aclacinomycin was kindly supplied by Roger Bellon Laboratories (Paris) and used without further purification. Saintopin was kindly supplied by RhBne-Poulenc Rorer (Vitry sur Seine) and used as received. The stock solutions of the drugs were prepared in twice distilled deionized water or in deuterium oxide M and diluted to the (loo%, Sigma) at the concentration ca. desired concentration before the experiment. Calf thymus DNA was purchased from Sigma. DNA was dissolved in potassiumbuffered saline. The concentration of DNA (phosphate) was estimated on the basis of a molar absorption coefficient of 6600 M-' cm-I at 260 nm. The complexes were prepared by mixing the drug solutions and DNA (the ratios are indicated in the figure legends). The absorption spectra were measured by means of a Philips P U 8720 UV/vis scanning spectrophotometer. The SERS and resonance Raman spectra were measured on a computer-controlled DILOR Omars-89 Raman spectrometer. Samples were excited with a Spectra-Physics Model 2020-03 argon laser. The experimental conditions are described in the figure legends. The resonance Raman and SERS band intensities were corrected by the monochromator-detector response. The highfrequency bands at ca. 3400 cm-' of water or at ca. 2500 cm-' of deuterium oxide were used as references for the measures in intensities. The bands of ring breathing vibrations of the chromophores were used for normalization of the SERS spectra of free drug and drug/DNA complex in order to obtain the difference spectra presented in Figures 5 and 6. These bands are not changed upon interaction of the drugs with DNA as was
The Journal of Physical Chemistry, Vol. 98, No. 4, 1994 1345 demonstrated before in the S E R S studies of related anthracy~lines.~-l~ Results General Characteristics of the Drugs and Silver Hydrosol. Aclacinomycin and saintopin chromophores are both derivatives of a quinolone differing in ring substituents (Figure 1). In contrast to saintopin, aclacinomycin has a trisaccharide residue at C7 position. In the both chromophores the OH bonds are located so that it is possible for them to form a part of chelated sixmembered rings by hydrogen bonding with the nearly quinonoid carbonyl groups5 We will call this part of the chromophores "chelate system". The absorption band of the T-T* transitions of the quinonoid structures of both drugs has a red shift at ca. pH 9. This shift is expected on successive proton dissociation (deprotonation) of the intramolecularly hydrogen bonded phenolic hydroxyl groups, and the quinonoid system may be more delocalized including the carbonyl and phenolic oxygen^.^,^ The atomic charges distribution in the aclacinomycin and saintopin chromophores is similar. As follows from our CNDO/2 calculations, the most negatively charged atom in both chromophores is a hydrogen-bonded oxygen of the carbonyl group. In contrast to aclacinomycin, negative charges are also located on the oxygens of the periphery hydroxyl groups of the saintopin chromophore (Figure 1). Silver hydrosol, used in our SERS experiments, is a colloid prepared by reducing of silver nitrate with trisodium citrate at the concentration corresponding to a silver particle density of 3.2 X 1014 L-I at the formation of spherical (80%) and ellipsoidal (20%) particles with the mean diameter of the spheres ca. 30 nm, and the average length of the rods ca. 60 nm.I9 The surface of colloidal silver is slightly positively charged. The effective potential at the colloid surface has been estimated to be about -110 mV, compared with -800 mV at the potential of zero charge.19 So, it could be reasonable to predict a preferential adsorption of compounds by hydrosol via the most negatively charged groups. As was noted by Smulevich et al.,lZJ3and Nonaka et al.,1° from comparison of the SERS spectra of related drugs with those previously reported in solution, an interaction with colloids does not disturb the complex of drugs with DNA. VibrationalAssignmentof SEW Active Modes. Aclacinomycin. In general, vibrational assignment of anthracyclines has been described before in terms of pseudosymmetry of the substituted anthraquinones chromophore^.^^^^^ The vibrational assignment of the resonance Raman active modes in the SERS spectrum of aclacinomycin was discussed also by Nonaka et a1.I0 Characteristic SERS spectral features of aclacinomycin (Figure 2, Table 1) are the following: (i) Aclacinomycin shows a free C=O stretching band at 1669 cm-I. This band becomes weaker and shifts to ca. 1631 cm-I when carbonyl becomes hydrogen-bonded with one adjacent hydroxyl group1I-l3 (like in adriamycin, Figure 1). This band is very weak (nondetectable) when carbonyl is hydrogen-bonded with two adjacent hydroxyl groups (like the intramolecular-bonded carbonyl in aclacinomycin (Figure l ) , or in 1l-deoxycarminomycin, which has the same chromophore as aclacinomycin12). (ii) The band of carbonyl in-plane bending motions is located a t ca. 487 cm-l. This band is mainly due to vibrations of the carbonyl group which are involved in intramolecular hydrogen bonding.l0JI It shifts to much lower frequency (ca. 461 cm-I) when carbonyl is hydrogen-bonded with one adjacent hydroxyl group (as in adriamycin, Figure 1). (iii) coupled C-0 stretching and OH in-plane bending vibrations show great contribution in the bands at 1242, 1300, and 1347 cm-I. (iv) The band at 1563 cm-' can be assigned to ring stretching mode coupled with v(C-0) vibration (Table 1).
1346 The Journal of Physical Chemistry, Vol. 98, No. 4, 199'4
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Nabiev et al.
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Laser power 15 mW.
TABLE 1: Vibrational Frequencies of Major Bands in the SEW Spectra of Aclacinomycin in H20 (ACM/H20) and D20 (ACM/D20) Solutions and in the Complex with the DNA (ACM/DNA).
1347sh 1370s 1409 sh I443 m
1370 s 1409 m 1443 m 1535 s
I367 s 1405 sh
1592 s I669 m
1669 m
1631 m 1670 m
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Figure 3. SERS spectra of saintopin in aqueous solution at pH 7.2 (1) and 10.0 (2) and indeuteriumoxidesolution (3). Experimental conditions were as for spectrum 2 in Figure 4.
TABLE 2: Vibrational Frequencies of Major Bands in the S E W Spectra of Saintopin in H20 (ST/H20) and D20 (ST/D20) Solutions and in the Complex with the DNA (ST/DNAP ST/HzO
assignments skel def b(C=o) skel def skel def
p H 7.2
+ b(C=O)
b(C-H) ring breathing
+
b ( 0 - H ) u(C-0) u(C-0) + b ( 0 - H ) u(C-0) + b ( 0 - H ) ring stretch ring stretch ring stretch u(C=C) v(C-0) u(C=c) + u(C-0) u(C=C) + v(C-0) u ( C = O ) , hydrogen bonded v(C=O), free
+
1563 us
1563 us
1000
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Figure 2. SERS spectra of aclacinomycin in aqueous solution (1) and in deuterium oxide solution (2) both ca. 5 X l t 7 M , excited at 457.9 nm.
A C M / H 2 0 ACM/DzO A C M / D N A 331 vw 331 vw 465 sh;uw 465 vw 461 w 486 w 487 m 478 m 915 w 937 m 952 w 1023 m 1018 w 1194w 1242 m I242 m 1302 s I300 s
1200
1400
Frequencies in cm-I. The italicized frequencies correspond to the bands that change in intensity upon complextion. vw, very weak; w, weak; m, medium; s, strong; vs, very strong; sh, shoulder. a
We were able to record for the first time the SERS spectra of aclacinomycin in DzO solution (Figure 2). Analysis of the effect of deuteration can help to evaluate an extent of coupling between ring stretching vibrations of chromophore and C=O, (2-0, and OH motions. The band of C=O stretching vibrations at 1669 cm-I is not affected by going from HzO to D20. This confirms previous conclusion about the main contribution of the free carbonyl vibration in this mode. From the other hand, in the 400-500 region, deuteration causes significant (9 cm-I) downward shift of the band at 487 cm-I. It is compatible with the assignment that this band is mainly due to the in-plane bending vibrations of the carbonyl group involved in intramolecular hydrogen bonding. The band a t 1242 cm-I completely disappears on deuteration and involves the hydroxyl bending. The strong band at 1300 cm-l and shoulder at 1347 cm-I both disappear on deuteration demonstrating a strong coupling with C-0 stretching
462 w 618 w 1023 m 1153 m 1278 sh 1316us
p H 11.O
ST/DzO
462 vw 620 w 934 m 1023 m
462 m 1033m
1023m
1278 sh 1322 vs
1275 m/s
1278 sh
ST/DNA 462 w 618 w
1316~s
1356 vs 1403 s 1423 sh 1508 m 1561s/sh
1423 m 1561 vs
I583 s I664 s
1423 s
1653 vs
1583 vs 1664 s
1561 vs 1580sh 1664 s
assignments skel def skel def
+ b(C4)
+b ( C 4 )
ring breathing b(0-H) u(C-0) ring stretch u(C-0) b ( 0 - H ) u(C-0) + b(0-D) u(C-0) b(O-H) v(C-0) b(O-H) u(C=c) u(C-0) v ( C 4 ) u(C-0) ring stretch v(C=O)
+
+ + + + +
a Frequencies in cm-l. The italicized frequencies correspond to the bands that change in intensity upon complexation. vw, very weak; w, weak; m, medium; s, strong; vs, very strong; sh, shoulder.
and OH bending motions (Table 1). The bands at 1370, 1409, and 1443cm-I can be assigned to the pure ring stretching vibrations of the chromophore being not affected by deuteration. The band a t 1563 cm-I on deuteration seems to split into two, 1535 and 1592 cm-I, demostrating a strong coupling of corresponding ring stretchings with C-O motions. Vibrational Assignment of SERS Active Modes. Saiitopin versus Aclacinomycin. Some of the SERS spectral features found in saintopin (Figure 3, Table 2) are similar to those in aclacinomycin (Figure 2, Table 1). The frequency of C=O stretching vibration in saintopin (1664 cm-I) corresponds to the vibration of the free carbonyl and is not affected by deuteration. But in contrast to aclacinomycin, C=O in-plane bending vibration in saintopin is very weak and does not shift in frequency on going from H 2 0 to DzO (Figure 3). It seems that in saintopin the C=O motions are much less coupled with OH vibrations than that in aclacinomycin. We speculate that the oxygens of the periphery hydroxy groups in the rings A and D of saintopin (Figure 1) shift an electron density from the chelate system to the center of the chromophore. Due to this fact the protons of the chelate
SERS of Antitumor DNA Intercalating Agents system of saintopin must be more strongly coupled with the C-0 groups than that in aclacinomycin. This increasing of coupling can be seen from the more high frequency of v(C-0) coupled with 6(OH) vibrations in saintopin (1316 and 1403 cm-I) than that in aclacinomycin (1300 and 1347 cm-l). The differences in the modes of coupling of C-0 and O H motions in aclacinomycin and saintopin are shown also by the experiments in D2O (Figures 2 and 3). The bands at 1300 and 1347 cm-I disappear in aclacinomycin, whereas the band a t 1403 cm-I disappears and the band at 1316 cm-I shifts to 1356 cm-I in saintopin on going from H2O to D2O. The other bands in the SERS spectra of saintopin can be assigned in the same manner as for aclacinomycin. As was noted before, the band at 1403 cm-l in assigned to coupled v(C-0) and b(0H) vibrations. The increasing of p H leads to the red shift of ~ .the ~ quinonoid structure. The the *--A* electronic t r a n s i t i ~ nof Raman intensities of the bands which are related to the T-T* transition decrease more rapidly than those which are assumed to be coupled to the n-T* t r a n ~ i t i o n .The ~ band at 1403 cm-I disappears upon deuteration and is coupled with the *--A* electronic transition of the chromophore, being strongly affected by its pH-induced shift in the red region (Figure 3). On the other hand, the shoulder at ca. 1423 cm-I has the same assignment but is not strongly affected by the r-r* transition being probably localized on the periphery C-0-H groups. The band at 1561 cm-I does not change its frequency and relative intensity on going from normal to alkali pH but disappears upon deuteration of the molecule. We attribute this band to a group vibration with the main contribution of C=C stretching coupled with periphery C-0 bending of saintopin chromophore. This band probably relates to the n-** electronic transition. In contrast to the 1561-cm-l mode, the 1583-cm-I band exists in the resonance Raman as well as in the SERS spectra of saintopin (see below), being not affected by deuteration of the chromophore, but disappears completely at the higher pH. We assign this band to the ring stretching fundamental strongly coupled with the T-T* transition of the chromophore. The bands a t 1508 and 1153 cm-I are related to the 7-T* electronic transition. They disappear at higher pH as well as upon deuteration of the molecule. So, we assign them to the ring stretching modes strongly coupled with the C-0 (former) and 0-H in-plane (later) bending vibrations. The main contribution in thesevibrations is, probably, the groups forming chelate system of intramolecular hydrogen bonds. Effect of Interaction of Drugs with the Silver Surface. The SERS spectrum of aclacinomycin recorded by us at pH 7.2 (Figure 2) is very similar to its resonance Raman spectrum reported previously.1° A slight shift (3-5 cm-I) toward lower frequencies is observed for the 6(OH), b(C=O), and v(C-0) groupvibrations on going from water solution to silver surface. Thus, a weakening must take place in one of aclacinomycin intramolecular hydrogen bonds on adsorption. These spectral changes suggest a direct interaction of aclacinomycin chromophore via one OH-O=C group with the hydrosol surface. The same conclusions have been made by Nonaka et a1.I0 and Smulevich et a1.I2 for their studies of related anthracyclines adsorbed on the borohydride hydrosol. We attempted to observe SERS of aclacinomycin at alkali pH. When aclacinomycin was introduced in the silver hydrosol with pH 10 at the concentration adjusted to have the same extinction a t the wavelength of excitation as compared with the sample a t pH 7.2, a SERS was certainly observed. Its intensity, however, was much weaker than those found at pH 7.2, and all the Raman frequencies and even relative intensities were found to be identical to the SERS spectra recorded a t normal pH. In addition, on going to pH > 10, the SERS intensities became even weaker, and when the pH reached 1 1.O, the SERS disappeared completely. Therefore, the observed SERS must be due to a small amount of nonionized aclacinomycin which is left on the silver surface, but not due to ionized aclacinomycin. We conclude that a n
The Journal of Physical Chemistry, Vol. 98, No. 4, 1994 1347
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Figure 4. Resonance Raman (1) and SERS (2) spectra of saintopin.
Excitation 514.5 nm. Resonance Raman: laser power 200 mW; concentration 10-4M; 100accumulations(1 s each). SERS: laser power 15 mW; concentration lo-' M; 25 accumulations (1 s each). Asterism on the resonance Raman spectrum indicate position of the bands of dimethyl sulfoxide.
aclacinomycin chromophore is adsorbed by the silver surface only via its chelate system of intramolecular hydrogen bonds being completely desorbed after ionization. For saintopin, we compared the resonance Raman spectrum recorded with a le3M water solution with its SERS spectrum M concentration of the drug in the hydrosol obtained at theca. le7 (Figure 4). The bands characteristic of this compound could still be detected by SERS when the concentration was as low as 10-8 M. The general features of the spectra, namely positions of the Raman bands, remain unchanged on going from aqueous solutions to silver surface. For the free carbonyl stretch and C=O bending vibrations no influence of silver surface is appreciable. In detail, however, some significant differences are found. As are seen here (Figure 4), upon interaction with the hydrosol, the bandsatca. 1316cm-Iandca. 1403 cm-',correlatedtothemcdes of ring stretching vibrations strongly coupled with v ( C - 0 ) and OH bendings (Table 2), appear to increase. By considering the very low concentration necessary for recording the SERS spectra, this could be due to the removal of the hypochromic effect in resonance Raman spectra and absorption, deriving from the dimer formation in concentrated aqueous solutions of anthraquinone derivatives.12J3 The SERS of saintopin differ from its resonance Raman spectrum specially by appearance of the new components at ca. 1561, 1508, and 1153 cm-I (Figure 4). An interaction with the silver surface leads to the reduction of the symmetry of the chromophore.12 So, some bands which were not seen in the resonance Raman spectrum can appear in SERS. As was noted in the previous subsection, the bands at 1508 and 1153 cm-1 could be attributed to the ring stretching vibrations coupled with v(C-0) (former), and 6(OH) (later) motions related to thechelate system of the molecule. This is not the case for the 1561-cm-I vibration. This band can also be characterized as a group vibration. It includes C-0-H bending, disappearing upon deuteration but not influenced by the shift of the T-T* electronic transition of the chromophore at higher pH. So, we attribute this band to the periphery C-0-H group motions in the A or D rings of saintopin chromophore. Appearance of this band in the SERS spectrum of saintopin is indicative of the interaction of the molecule via peripheral hydroxyl groups with the silver surface.
Nabiev et al.
1348 The Journal of Physical Chemistry, Vol. 98, No. 4, 1994 (D
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Figure 5. SERS spectra of aclacinomycin ( l ) , its complex with calf thymus DNA (2) and their differencespectrum (3 = 1- 2). Experimental conditions for curve 1 were as in Figure 2. Drug/DNA complex: ratio - 1 molecule of drug for 40 base pairs of DNA. Absolute concentration of the drug/DNA complex in the hydrosol (ca. 5 X 10-6 M in the drug concentration) was adjusted to have spectrum of the comparablequality
with the spectrum 1. These spectral changes suggest a direct interaction of saintopin chromophore both via the peripheral OH groups as well as through the chelate system with the hydrosol surface. Effects of Intercalation into DNA. SERS Spectral Intensity of Drug/DNA Complexes. The effect of decreasing the SERS intensity a t the intercalation of the drug into the DNA has been well documented and explained in terms of short-range character of Raman enhancement in colloid systems.” This effect was first observed by Koglin et a1.I5 and used by us for studies of topology of membrane proteins and drug/target complexes.lI Nonaka et a1.I0attempted to observe SERS of aclacinomycin/ DNA complexes. When the drug/DNA solution of molar ratio 1:20 was added to a silver hydrosol, all the Raman frequencies were found to be identical to the SERS spectrum of a free drug. Their intensities were much weaker than those found without DNA. In addition, on increasing the DNA concentration, the SERS intensities became even weaker, and when the drug/DNA ratio reached 1:100, no SERS was observed. We suggest that complete disappearance of the SERS signal of intercalated aclacinomycin in the described measurements was not only due to the short-range character of Raman enhancement. SERS media employed were not very convenient for analysis of intercalated complexes. Calf thymus DNA and drug have been prepared in the sodium chloride solutions.I0 Addition of a very small amount of sodium chloride strongly increases the silver hydrosol specificity to the chromophoric systems of the drugs.11J9 Consequently, the probability of silver micelle being adsorbed by DNA is decreased. So, the signal of the free drug gives the main contribution in the SERS spectra and the signal of the bound drug becomes undetectable. W e were able to record t h e S E R S spectra of bound aclacinomycin in the aclacinomycin/DNA complexes at the molar ratio (DNA in base pairs) 1:40 (Figure 5) by using silver hydrosol prepared as described above11J9 without addition of sodium chloride. A ‘nonactivated” hydrosol (without addition of sodium chloride) has less specificity with respect to the chemical nature of the adsorbate as compared with the “activated” (with sodium chloride) one. Use of the nonactivated hydrosol enhances the
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Figure 6. SERS spectra of saintopin (l), its complex with calf thymus DNA (2), and their difference spectrum (3 = 1 - 2). All experimental conditions for spectra 1 and 2 were identical to each other and the same as for curve 2 in Figure 4. Drug/DNA complex: ratio 1 molecule of drug for 200 base pairs of DNA.
adsorption capability of DNA. Consequently, it is possible to detect signals from the buried chromophores. In this case, the overall degree of decrement of the SERS intensity may be used as a diagnosis of the degree of the chromophore intercalation into DNA, when the concentration of the complex is adjusted to have the same extinction at the wavelength of excitation as compared with the free drug. In this sense aclacinomycin belongs to the deeply intercalating chromophores. The SERS intensity of its complex with DNA (at the drug/DNA (base pairs) ratio 1:40) is decreased a t ca. 20 times as compared with the SERS intensity of the free drug (data not shown). In order to obtain the SERS spectra of comparable quality between aclacinomycin and its complex with DNA, much larger amounts of the complex must be introduced into the hydrosol as compared with the free drug. These spectra have been used for the detailed analysis of the spectral differences between the complex and free drug (see next subsection). It was not the case for saintopin. We were able to record SERS spectra of its complex with DNA at the large excess (X200 in moles of the base pairs) of DNA duplex (Figure 6). At this ratio all the molecules of the drug are considered to be intercalated.I8 The SERS spectra of the complex show some decrement in intensity upon saintopin interaction but not more than 30% from the SERS intensity of the free drug (Figure 6). In contrast to aclacinomycin, saintopin chromophore seems not to be deeply buried into the double-stranded helix. Detailed analysis of the spectral differences between the free drugs and their complexes with DNA is presented below. Effects of Intercalationinto DNA. Aclacinomycin. On adding a large excess (X40 in moles of the base pair) of DNA duplex, the SERS spectrum of aclacinomycin shows the following prominent changes (Figure 5 , Table 1): (i) appearance of the new band at 1631 cm-l (this band does not exist in the SERS spectra of free aclacinomycin and free DNA alone) and decreasing of the intensity of the band at ca. 1669 cm-*; (ii) appearance of the band a t ca. 461 cm-’ and decreasing of the band at ca. 487 cm-I; (iii) disappearance of the band a t 1443 cm-1 and shoulder at 1347 cm-*. We speculate that changes (i) and (ii) are not independent. The band at ca. 1631 cm-1 may be used as a diagnosis of
SERS of Antitumor DNA Intercalating Agents intramolecular hydrogen bonding of carbonyl with the single adjacent hydroxyl group in diverse dihydroanthraquinone derivatives.lsi3 This band is prominent in the SERS and resonance Raman spectra of daunomycin and adriamycin being accompanied by6(C=O)vibrationatca.461 cm-l. Thesespectral changes show that one of the C-O-H groups of the chelate system of aclacinomycin chromophore form the hydrogen bond with functional group of DNA upon intercalation. The system of intramolecular hydrogen bonds of bounded aclacinomycin includes one hydrogen bond between carbonyl group and adjacent hydroxyl group. Decrease of the relative intensity of the band at 1669 cm-1 (free carbonyl stretching), and the bands at 1242, 1300, and 1370 cm-1 (ring stretchings coupled with C - O and O H motions), shows that the free carbonyl group and the chelate system of aclacinomycin are preferentially buried in the double-stranded helix. The bands at 1370 and 1443 cm-l are related to the pure ring stretching vibrations being not affected by deuteration (Figure 2, Table 1). These bands are correlated with the modes of A1 species under the C2"symmetry of aclacinomycin chromophore.I* The interaction between the drug and the DNA base pairs essentially affects the excited state via T-T interaction. So, the bands mentioned above could be indicative for this interaction. The potential curve shifts between the ground and excited states of the A1 modes are expected to be larger compared to those of B1 modes. The equilibrium position changes of the potential curveat theexcitedstateaddupuponinteractionforthesymmetric modes and nearly cancel for the antisymmetric ones.12 Effects of Intercalationinto DNA. Saintopin. The comparison between the SERS spectra of saintopin and saintopin/DNA complex (Figure 6) shows the enormous relative intensity reduction of the bands at 1153,13 16,1403,1508,1583, and 1664 cm-l upon complexation. What is noticeable is the fact that the intercalation leads to reduction of the 1583-cm-1 band which is strongly coupled with the ~ - rtransition * of the chromophore. Thecomponent ofthis bandat ca. 1561 cm-I in theSERSspectrum appeared to be related to the motions of the periphery COH group of A or D ring of the chromophore (Figure 1). It is not affected by intercalation. On the other hand, the bands at 1153 and 1508 cm-I, being strongly dependent on the T+* electronic transition, disappear upon intercalation. Intercalation also affects the intensities of the free C = O and C-0 stretching vibrations of the chromophore. The decrease of the intensity of v ( C l 0 ) and ring stretching coupled with v(C-0) motions could be explained by the partial penetration of saintopin inside the double-stranded helix. After the interaction with DNA the periphery O H group of saintopin is well accessible to the silver surface. Models of Binding with DNA. Aclacinomycin versus Saintopin. As mentioned in the previous sections, upon complexation, the following features can be summarized for aclacinomycin: (i) A new hydrogen bond between one O H group of the chromophore and the functional group of DNA appears. The system of intramolecular hydrogen bonds of bounded aclacinomycin includes only one hydrogen bond (between carbonyl and adjacent OH group), in contrast to that for the free drug, having hydrogen bonds between carbonyl and two adjacent OH groups (Figure 1). (ii) T-T interaction between the chromophore and DNA base pairs induces additional stabilization of an intercalation complex. (iii) The free carbonyl group is buried in the interior of DNA. These data are fully consistent with the results of 3IP and IH NMR studies of the interaction of aclacinomycin with a few sequence-controlled oligo-DNAs in aqueous ~ o l u t i o n s . ~In ~ Ja~ gradual addition of aclacinomycin to d(CCTAGG), the line widths of the exchangeable-proton resonances were examined as a function of the temperature of the solution. It was found that the signals from two protons assignable to the two OH'S of the
The Journal of Physical Chemistry, Vol. 98, No. 4, 1994 1349 L
=kw
BELIX AXIS
7
A
MWOR
(iRoovI:
m a r bp.
Figure 7. Proposed models of the intercalation between aclacinomycin and DNA (A) and saintopin and DNA (B). For orientation of chromophores between DNA base pairs (right part of the figure), X-ray crystal data of related anthracyclinesu have been used. RI= COOCH,; R2 = CH2CHo; R3 = trisaccharide residue. chromophoreremain sharp in the complex even at 50 OC, whereas all of these rapidly exchange with water at 35 OC in its free
state.I6J7 We propose the following model (Figure 7A) of orientation and conformation of aclacinomycin chromophore in the interior of DNA based on our SERS and literature data. Aclacinomycin chromophore is inserted into the T-A rich site of DNA, oriented with its long axis parallel to the dyad axis (y = 0), and fixed there by electrostatic and van der Waals forces. The relative position of the DNA base pairs and aclacinomycin is shown here on the assumption that the chromophore is intercalated in a similar manner to that found for daunomycin and adriamycin on the basis of X-ray data.- The free carbonyl group and chelate system of the chromophore are both buried deep inside double-stranded helix. At this configuration, the COH group of the C ring of aclacinomycin is hydrogen bonded with the C = O group of thymine which is not involved in the interbase hydrogen bonding in the T-A base pair. The corresponding C=O group of cytosine is involved in the interbase hydrogen bonding in the C-Gbase pair, so that it would not be available for attracting any OH group of the drug. The sugar residues have a very low Raman cross section. Their characteristic bands cannot be observed in SERS spectra because of the background of intense bands of the chromophore. Nevertheless, polysaccharide side chains of anthracycline-related drugs interfere both in the positioning of the chromophore within its intercalative site and in the drug-DNA binding affinity through additional interactions in the DNA minor groove, which is also documented in the case of daunomycin and adriamycin molecules.C8
1350 The Journal of Physical Chemistry, Vol. 98, No. 4, 1994
In saintopin, only a portion of the overall chromophore is involved in the intercalation (Figure 7b). This portion is formed by the rings A and B (Figure 1) and includes OH-.O intramolecular bond and free carbonyl group. The peripheral OH group of the ring D is not buried inside the DNA double helix, being well accessible to the silver surface. It should be pointed out that no evidence of redistribution of intramolecular hydrogen bonds of saintopin or formation of new hydrogen bonds between chromophore and DNA upon intercalation could be found. It could be probably thecaseof preferential intercalation of saintopin chromophore into the G-C sequences ofDNA (Figure 7b). In this arrangement, thenegatively charged functional groups of DNA are far away from the chromophore chelate system or are participated in the interbase hydrogen bonding. The 7r-x interaction between saintopin chromophore and DNA base pairs seems to give main contribution in the fixation of saintopin/DNA intercalation complex. Conclusion. Models and Biological Effects All together the evidence accumulated in the present study indicates that the modes of binding with DNA for aclacinomycin and saintopin are very different (Figure 7). The following differences in the biological effects of the drugs could be explained in terms of the differences between their intercalation modes: (i) aclacinomycin shows induction of DNA-topoisomerase I1 cleavable complex at the much lower concentration of the drug as compared with saintopin; (ii) saintopin induces cleavable complex with both DNA-topoisomerases I and I1 whereas aclacinomycin does not; (iii) aclacinomycin inhibits DNAtopoisomerase I1 cleavable complex at the high concentration of the drug whereas saintopin does not.lJ8 For aclacinomycin, the chromophore is selectively intercalating into the TpA site and is fixed here by hydrogen bond between OH group of the C ring and free carbonyl group of thymine, and also by electrostatic and van der Waals forces (Figure 7a). From the point of view of formation of ternary complex, it should be pointed out that aclacinomycin is deeply buried within base pairs. So, the enzyme access to DNA at the low concentration of drug is not hampered. Close contacts between topoisomerase and DNA backbone can be easily facilitated by the local and medium-range distortions induced by the deeply intercalated drug. Thestabilization of theDNA-topoisomerase11 cleavablecompelx can then result from the stiffening of DNA in the surroundings of the intercalation site and from the specific interaction of aclacinomycin via, for instance, its free carbonyl group (Figure 7a), with the functional group of DNA-topoisomerase 11. This functional group may be the hydroxyl of tyrosine residue which is involved in the formation of the ternary complex.’ According to the nearest-neighbor exclusion principle which seems to be applied to most good intercalators, only about half of all potential sites are filled by drug molecules.20 Hence, high concentration of aclacinomycin can inhibit topoisomerase IImediated DNA cleavage due to the template blockage by this intercalator. With regard to saintopin (Figure 7b), the spectral evidence indicates that its binding to DNA,fits in with an “outside” mode of the “three mode binding model” similar to the one previously suggested for porphyrins by Fiel et al.zl and applied to ellipticine derivatives by Monnot et a1.22 According to this mode of binding, partly intercalated chromophores are fixed inside DNA by only weak electrostatic and van der Waals forces. This arrangement presumably occurs in the minor groove (Figure 7b).8.9 As regards the stabilization of the DNA-topoisomerase cleavable complex, this arrangement
Nabiev et al. does not disturb DNA recognition by topoisomerases which, as illustrated by studies with other DNA-binding proteins, mostly concerns the major groove.23 Partly intercalated saintopin chromophores should interact with DNA a t higher concentration, as compared with aclacinomycin, to induce comparable local distortions of DNA structure being responsible for induction of DNA-topoisomerase cleavable complexes. On the other hand, partly intercalated chromophore does not penetrate through the DNA duplex and is not able to interact directly and specifically with the functional groups of DNAtopoisomerase (Figure 7b). Hence, in contrast to aclacinomycin, this type of interaction facilitates nonspecific DNA recognition by both DNA-topoisomerase I and 11. The nearest-neighbor exclusion principleZocannot be applied to the “outside mode” of binding between saintopin and DNA. So, the intervals between the drug molecules could be similar to that found between the base pairs in DNA with a native B-type conformation.22 Hence, many more molecules of the drug could be interacted with DNA without blockage of the template, as compared with aclacinomycin. That is why this type of binding leads to consecutive occupation of external sites, which are responsible for the induction of the topoisomerase-DNA complex but not suppress it at high concentrations of the drug. Acknowledgment. We are grateful to Jean-Franpis Riou for the saintopin sample and to Hamid Morjani for his technical assistance. Registry No. Saintopin, 131 190-63-1; aclacinomycin, 8138207-2; adriamycin, 23214-92-8. References and Notes (1) Liu, L. F. Annu. Reu. Biochem. 1989, 58, 351-375. (2) Remers, W. A. The Chemistry ofAntitumor Antibiotics; Wiley: New York, 1979; Vol. 1. (3) Wilson, W. R.; Baguley, B. C.; Wabelin, L. P. G.; Waring, M. J. J . Mol. Pharmacol. 1981, 20, 404-414. (4) Pigram, W. J.; Fuller, W.; Hamilton, L. D. Nature 1972,235,17-19. (5) Aubel-Sadron, G.; Londos-Gagliardi, D. Biochimie 1984.66, 333352, and references therein. (6) Wang, A. H.-J.; Ughetto, G.; Quigley, G. J.; Rich, A Biochemistry 1987.26. 1152-1163. (7) Moore, M. H.; Hunter, W. N.; Longlois d’Estainot, B.; Kennard, 0. J . Mol. Biol. 1989, 206, 693-705. (8) Frederick, C. A.; Dean Williams, L.; Ughetto, G.; van der Marel, G. A.;van Boom, J. H.; Rich, A.; Wang,A. H.-J. Biochemistry 1990,29,25382549, and references therein. (9) Manfait, M.; Bernard, L.; Theophanides, T. J . Raman Spectrosc. 1981, 11, 68-74. (10) Nonaka, Y.; Tsuboi, M.; Nakamoto, K.J. Raman Spectrosc. 1990, 21, 133-141. (1 1) Nabiev, I. R.;Sokolov,K. V.;Manfait, M.BiomolecuIarSpectroscopy; Wiley: London, 1993; Chapter 7, pp 267-338. (12) Smulevich, G.; Feis, A. J . Phys. Chem. 1986,90, 6388-6392. (13) Smulevich, G.; Feis, A.; Mantini, A. R.; Marzocchi, M. Indian J . Pure Appl. Phys. 1988, 26, 207-21 1. (14) Morjani, H.; Riou, J.-F.; Nabiev, I.; Lavelle, F.; Manfait, M. Cancer Res. 1993, 53, 47844790. (15) Sequaris, J.-M.; Koglin, E.; Malfoy, B. FEES Lett. 1984, 173, 9598. (16) Takahashi, S.;Nagashima, N.; Nishimura, Y.; Tsuboi, M.Chem. Pharm. Bull. 1986, 34,4494-4504. (17) Tsuboi, M.; Ikeda, T.; Shindo, H. Chem. Pharm. Bull. 1987, 35, 4405441 1. (18) Yamashita, Y.; Kawada, S.-2.; Fujii, N.; Nakano, H. Biochemistry 1991, 30, 5838-5845, and references therein. (19) Hildebrandt, P.; Stockburger, M. J. Phys. Chem. 1984, 88, 59355944. (20) Saenger, W. Principles of Nucleic Acid Structure; Springer-Verlag: New York, 1984. (21) Fiel, R. J. J . Biomol. Sfrucf.Dyn. 1989, 6, 1259-1274. (22) Monnot, M.; Mauffret,O.;Simon, V.;Lescot, E.;Psaume, B.;Saucier, J.-M.; Charra, M.; Belehradek, J., Jr.; Fermandjian, S.J . Biol. Chem. 1991, 266, 1820-1829. (23) Schleif, R. Science 1988, 241, 1182-1187.
