Raman studies on anticancer inorganic ring–dna interactions

of Molecular Structwre, 71 (1981) 39-49 Elsevier Scientific Publishing Company, Amsterdam -

Journal

RAMAN STUDIES INTERACTIONS

ON ANTICANCER

INORGANIC

Part 1. Hexaziridinocyclotriphosphazene

MICHEL MANFAIT

and ALAIN

Laboratoire de Recherches Cedex (France)

JEAN-LUC

Printed in The Netherlands

RING-DNA

N3P3( N&H&

J. P. ALIX

Optiques.

Facultk des Sciences,

B.P.

347, 51062

Reims

BUTOUR

Labomtoire de Pharmacologic de Narbonne, 31078 Toulouse

JEAN-FRANCOIS

LABARRE*

et de Toxicologic Cedex (France)

and FRANCOIS

Laboratoire Structure et Vie, Universite 31062 Toulouse Cedex (France)

Fondamentales

du CNRS,

205, Route

SOURNIES

Paul Sabatier,

118, Route

de Narbonne,

(Received 12 June 1980)

ABSTRACT A Raman investigation of hexaziridinocyclotriphosphazene-DNA interactions in vitro suggests that the alkylating sites on DNA for this powerful antitumour agent are the N(7) and NH, positions of adenine. INTRODUCTION

We recently reported on the antitumour activity of some cyclophosphazenes, namely N,P,Az6 (MYKO 63), N4P4Az8 (MYKO 83), N,P4Pyrro8 (MYKO 85) and NJP, (MeAz), (MYKOMET 63)**, on murine L1210 and P383 leukaemias and on B16 melanoma [l-3]. The most successful compound, in all the cases investigated, was MYKO 63 (Fig. 1) which in addition has the advantages of high solubility in water and saline solutions, activity by both the intraperitoneal and intravenous routes and, probably, the ability to reach all regions of the body, particularly the brain, since it exhibits no blood-brain barrier.

*Author for correspondence. **AZ = Aziridino, MeAz = methyl-2-aziridino, Pyrro = pyrrolidino. The name MYKO was chosen because of the striking similarity of the X-ray crystal structures of these compounds with Mykonos windmills [4-6 1. The first figure after this name refers to the number of atoms within the phosphazenic ring (6 = N,P,, 8 = N,P,) and the second figure is connected with the number of atoms in the cyclic amino ligand (3 = AZ, 5 = Pyrro). 0022-2860/81/0000-0000/$02.50

@ 1981 El sevier Scientific Publishing Company

40

AZ

AZ

1 /Yp

I

AZ ‘I

I‘Az I\:

AZ

LpHN /\

kz

CM2

= --N 3

AZ

Fig. 1. Formula

=‘-‘2

of hexaziridinocyclotriphosphazene

[2 J.

Up to now, MYKO 63 has been tested successfully on six animal tumours and its efficiency has been demonstrated on 26 colon carcinoma, ependymoblastoma, P815 mastocytoma, osteosarcoma and Lewis lung carcinoma [ 71. Furthermore, MYKO 63 was found to cure, within a monoinjection protocol, the Yoshida sarcoma [S] which may be considered as a model for scrotum tumour at the human level. Consequently, this drug is currently being tested on other tumours by several laboratories belonging to the pharmacology screening groups of E.O.R.T.C. and N.C.I. The object of the present work was to investigate the targets of MYKO 63 activity. Assuming DNA to be one such target, we investigated in a first step the nature and intensity of the interactions between MYKO 63 and DNA by the Scatchard technique [ 91. This method consists of studying the difficulties encountered by a fluorescent dye, ethidium bromide, with respect to its intercalation between the base pairs of DNA when the secondary structure of the macromolecule is modified by the grafting (or intercalation) of a drug. It was shown by this means that MYKO 63 interacts with DNA as a dialkylating agent [lo] but it was not possible to elucidate with what sites on DNA the MYKO 63 molecule is interacting_ It was considered that Raman spectroscopy would be a suitable technique to determine these sites, as it has been used previously to study the interactions of other biological systems with nucleosides or DNA [ 111. MATERIALS

Synthesis

AND

METHODS

and purity

of N$&z6

N3P3Azg was prepared by the procedure of R%tz et al. [ 121 in the presence of ammonia. ActuaIly, an improved procedure has been proposed [ 131 which leads to extremely pure samples, as checked by mass spectrometry [ 143 _ Pure N3P,Az, is highly soluble in water, about 100 g 1-l. 31P NMR spectroscopy gives a unique signal at -37.2 ppm with 85% H3P04 as a standard. The melting point of N3P3Az6 is 150°C. Nucleic

acid and salts

Stock solutions of calf thymus DNA (Sigma Chemical Co., St. Louis, Missouri) and salmon sperm DNA (Worthington Biochemical Co., Freehold,

41

New Jersey) were prepared by dropping a 10 mM solution of NaC104 (Fluka) on the DNA fibres to give an approximate concentration of 1.5 mg ml-’ for the salmon sperm (set I) and 4.5 mg ml -I for the calf thymus (set II) DNA. These solutions were gently agitated at 4°C for 48 h, centrifuged at 8000 rpm for 10 min and their concentrations determined spectrophotometrically. N3P3Az,-DNA

complexes

Hexaziridinocyclotriphosphazene-DNA complexes were made by mixing solutions of the drug and DNA in a molar ratio of approximately one base of DNA per drug molecule_ The reactions were run at room temperature in the dark for 1-6 days. The final DNA and MYKO 63 concentrations in the complex were 1 mg ml-’ and 3.2 X 10e3 M (set I), and 3 mg ml-’ and 9.6 X 10m3M (set II). In the experiments, it was ascertained that laser irradiation had no effect on the UV absorbance spectra of the samples. Raman

instrumentation

Raman spectra were obtained on a Coderg PHO Raman spectrometer equipped with a Coherent Radiation Model 52B Ar’ laser using 800-1200 mW of power from the 488.8-nm line; a small two-prism monochromator was used to remove background plasma lines. The Raman scattering was detected with a cooled EM1 9558 QB photomultiplier. Signal pulses from the photomultiplier were amplified and then counted digitally and stored in the computer memory. The computer (Alcyane, MBC, France) automatically coordinates the scanning of the spectrometer (steps of 2 cm-‘). The maximum counting time for each step was 2 s. In general, data accumulation (time averaging) was used to obtain the spectra and to improve the signal-to-noise ratio. This computer enables several operations to be carried out on the stored spectra, particularly the difference between two spectra (see ref. 15 for all the applications available). The sample was placed in a quartz cell in a thermostatted holder (25 f 0.5%). The laser line was focused by a lens and mirror system and the scattered light collected at right-angles to the incident radiation. The normal slits were 6 cm-‘. The Raman frequencies reported here are accurate to *l cm-’ for intense and/or sharp lines and to +2 cm-’ fdr weak and/or broad lines. The Raman band intensities were measured by their peak heights. ANALYSIS

METHODS

Analysis of binding experiments required a careful comparison of (i) the MYKO 63 bands, either in the presence or absence of DNA bands and (ii) the DNA Raman bands, either in the presence or absence of MYKO 63 bands.

42

This comparison was achieved by computer-subtracting variable amounts of one spectrum from another. Previously, the various spectra were normalized to the same relative Raman intensity, with the 934-cm-’ band (CIO, symmetric stretch) as an internal standard. The intensity of the ClO, scattering measures the combined effect of such experimental factors as counting time, optical aiignment and laser power. Comparison of the complete spectra of the solvent (10 mM NaC104 in water) and of the solutions (MYKO 63 or MYKO 63-DNA in the.solvent) showed that the region around 1850 cm-’ can be considered to have nearly “zero Raman intensity” at least for the lower wavenumbers. Thus, for each spectrum, a horizontal base-line is drawn from this point, then the solvent spectim is subtracted taking into account a coefficient determined from the compound concentration of the solution in order to keep the horizontal base-line unchanged for the new spectrum. Figure 2 compares the spectrum of free MYKO 63 with that of MYKO bound to calf thymus DNA. Both spectra have been normalized to the same Raman scattering intensity and drug concentration. The spectrum of free MYKO 63 was obtained by subtracting the solvent spectrum from the MYKO solution,spectrum. The spectrum of the bound drug was obtained by a two-step process: (i) the solvent spectrum was subtracted both from the spectrum of the MYKO-DNA complex and from the DNA solution spectrum; (ii) subtraction of these two new spectra was then performed until most of the DNA bands were removed. Step (ii) is applicable only’ if the spectral differences between unbound and partially bound DNA are negligible. This is strictly correct only for DNA bands unaffected by drug

250

500

750

1000

1250

1500

1750

cm-’

Fig. 2. Ramanspectraof unbound(A) and bound(3) MYKO calf thymusDNA and NaClO, backgrounds and normalization scattering

intensity

and drug concentration.

63 aftersubtractionof to the same Raman

43

binding. As can be seen from Fig. 2, the background and the base-line of the spectra around 1850 cm-’ are not affected by the subtraction process. For comparison of bound and unbound DNA (see Figs. 3 and 4) the procedure was performed in the same way. The spectrum of partially bound DNA was obtained by subtracting the free MYKO difference spectrum (Fig. 2A) from the spectrum of the MYKO-DNA complex (Fig. 3, set I and Fig. 4, set II).

1800

1400

1600

1200

1000

cm-’ Fig. 3. Raman spectra of set I: (A) salmon sperm DNA (1 mg ml-‘) in 10 mM NaCIO, solution; (B) partially bound DNA after subtraction of free MYKO 63 and solvent (10 m.M NaClO, in water); (C) complex MYKO 63-DNA (one base of DNA per drug molecule).

cm-’ Fig. 4. Raman spectra of set II: (A) calf thymus DNA (3 mg ml-‘) in 10 mM NaCIO, solution; (B) partially bound DNA after subtraction of free MYKO 63 and solvent;

(C)complex

MYKO

( one base of DNA per drug molecule).

63-DNA

1

250

500

750

I

1000

1250

1500

1750

cm-’ Fig. 5. Raman spectrum of 30 mM N,P,Az, in 10 mM NaCIO, solution. (-) with 10 mM NaClO, in H,O; (---*) 10 mM NaC10, in H,O.

N,P,Az,

45 RESULTS

AND DISCUSSION

Raman scattering data were obtained for the first time for NXPXAz6 f16] for N3P,Az,-DNA complexes (sets I and II). Raman spectrum

of hexaziridinocyciotriphosphazene

and

(N3P3Az6)

Figure 5 illustrates the spectrum of N3P3Az6 in 10 mM NaClO, solution (concentration 3.87 X lo-*M). In order to draw some conclusions about binding changes by Raman spectroscopy it was necessary to assign the observed Raman bands to the corresponding normal modes of vibration. Taking into account the previous frequency assignments appearing in the literature for N3P3Az6 [ 171 and N3P3F6 [ 181 as well as for aziridine itself [ 191, we have classified the group frequencies of N3P3Az6 as follows. (i) Framework frequencies: N3P3 ring (local symmetry &,); r(fr) = 4A f 4.E. (ii) Ligand frequencies: NC2H4 (local symmetry Cs); r(lig) (-N&Ha) = 344 + 34.E; r(lig) (-NC,) = 1OA + 1OE;r(lig) (CH,) = 24A + 24.E. (iii) Ligand framework couplings: r(cpl) = 8A + 8E. The overall structure is assumed to belong to C3 symmetry: r = 46A + 46E (R + IR). A complete normal coordinate analysis is in progress to assign the Raman spectrum of this compound (see ref. 20 for the theoretical aspects which comprise the determination of symmetry coordinates without redundancies and a treatment involving complex representations). The most important group frequencies which are of use in the present work are listed in Table 1. When assigned, each Raman band serves as an indicator of what is happening to specific parts of the N,P,Az, molecule when it is interacting with DNA. During a study of N,P,Az,-DNA complexes by the Scatchard technique [lo], it was shown that an incubation time of at least 5 days is necessary after mixing the MYKO 63 with DNA on day D before complete interaction is observed. These results are clearly confirmed by the present work as no fundamental discrepancy was observed between the spectra of the free and TABLE

1

Main-group frequencies of N,P,Az,

and their assignments

uj(cm-‘)

ZVia

aZb

Assignment

ui (cm-‘)

ZVi

aZ

Assignment

487 706

37 65

18 11.5

N,P,-AZ coupling N,P, ring mode

828 844

35 31

11.5 11.3 3 AZ

1100 1158 1276 1452 1475

21.5 21 71 12 9

7 7 21 -0 -0 3

AZ pl(CH,), pw(CHz) N,P, ring mode AZ ring breathing 6,(CH,) Az $(CH,)

pr(CHz) sym. ring. def.

aRelative intensity (a) of free MYKO 63, referred to ~$210; (936 cm-‘. bDecrease in intensity (%); (Zvi(free) - Z;i(bound))/Zq(free).

Z = 100).

46

bound MYKO 63 when the latter was deduced from the recorded spectrum of the MYKO 63-DNA complex on day (13 + I). Figure 2 shows the spectra of free and partially bound N3P3Az6 obtained by the process described above. The spectrum of the bound MYKO was obtained from the spectrum of the complex recorded on day (D + 6). The main results provided by the comparison between the free and bound MYKO spectra are as follows. (i) The intensity and position of some lines are practically unaltered (AI < 7%, see Table 1) on binding: this is the case for the 1452- and 1475-cml’ lines assigned to the 6 (CH,) deformation mode (ligand), and for the llOO- and 115&cm-’ lines a~~bu~d to the twisting and wagging of CH, (ligand) and to one of the degenerated stretch modes of the N3P3 ring skeleton, respectively. (ii) A relatively weak decrease in intensity is observed for the 706-cm-’ line (12%) (N,P, ring mode) as well as for the 828~-844cm-’ group (11.5%) (rocking CH2 and NC2 ring modes of AZ). (iii) The main alterations in intensity are seen for the 487-cm-’ line assigned to the framework-ligand coupling (N,P,-AZ) and for the 1276-em-’ band corresponding to the ring brea~~g of aziridine; these decrease by 18% and 21%, respectively. Thus, the largest decrease in intensity in the 1276-cm- E band is due to the mechanism of interaction of aziridino ligands in the complex as described previously [S] , following the scheme: H’ , -NH--CH,--CH;

’

CH,

Furthermore, in the case of the bound MYKO, a small broadening of that band is observed; subtraction of the bound and unbound spectra around the 1276-cm-’ wavelength yields a very weak band shifted to the highfrequency side. This can be seen in Fig. 4B, where the 1304-cm-* band of DNA shows a shoulder on the low-frequency side. This effect is interpreted as a type of aziridino ligand-DNA interaction involving neither a binding to DNA nor an opening of the NC,H, ring.

Figures 3A and 4A illustrate the spectra of salmon sperm DNA (1 mg ml-l) and calf thymus DNA (3 mg ml-‘) at pH 6.9, 25°C in the presence of 10 mM NaClO,. Contributions to the various bands by the individual bases are indicated (G = guanine, C I=cytosine, A = adenine, T = thymine). The assignment of Raman bands is based on similar previous studies by Lord and Thomas [Zl] , Small and Peticolas [ 22, 233, and Erfurth and Peticolas [24] _ Figures

47

3C and 4C give the spectra of the MYKO 63-DNA complexes (sets I and II, respectively) in 10 mM NaClO, solution. Figures 3B and 4B represent the parGaIly bound DNA spectra obtained by subtracting the spectrum of the free MYKO 63 from Figs. 3C and 4C, respectively (see Analysis Methods). A comparison of the unbound and bound DNA (set I, Fig. 3A, B; set II, Fig. 4A, B) leads to the following conclusions. (i) The very small increase in intensity of the 1240-cm-’ band, which is the most sensitive line to unwinding of the DNA double helix, indicates that DNA is probably slightly denatured in solution with MYKO 63 (a large increase in intensity is observed when DNA melts [ 241). The calf thymus DNA solution spectrum (Fig. 4A) exhibits a weak band at 835 cm-’ which is the line that appears to be indicative of the normal B conformation of DNA [25] (i.e. it disappears on melting). In the complex (see Fig. 4B) this line is slightly shifted to lower frequency. This effect, connected with what was observed for the 1240-cm-’ line, indicates a very weak denaturation of the DNA by disruption of the B conformation_ (ii) The band at 1492 cm-’ seems to be unaltered on binding. As this Raman band of guanine in DNA is a strong indication of binding to the N( 7) position of guanine (see refs. 26, 27), it may be concluded that there is no interaction between MYKO 63 and guanine. (iii) The major relative effects in intensity are seen for the 1340-cm-’ band, which decreases by 13% and 22%, and for the 1304-cm-’ band, the corresponding decreases being 31% and 30%, for set I and set II, respectively. These two bands are characteristic of adenine; more precisely, the C( 5)N( 7) stretching mode is predominant in the 1340-cm-* vibration whereas the 1304_cm-’ band strongly involves the C( 8)-N( 7) bond stretch [ 28, 291. As these two bands were shown to be predominantly related to the bond vibrations attached to the N( 7) position, it may be concluded that interactions between MYKO 63 and adenine occur in the N( 7) position_ It should be noted that the N(3) position of adenine is not a binding site for MYKO: the 1580-cm-’ band, which is very sensitive to binding to the adenine base in the N(3) position [30], shows no significant shift or decrease either in frequency or in intensity in any of the spectra. (iv) Finally, th e spectra of the complexes in solution (see Figs. 3B and 4B) exhibit a 20% decrease of the 1180-1182cm-’ band. This band is related both to the stretch modes of vibration of the C-NH, groups of the individual bases and to thymine itself. An overall survey of all the spectra presented here prompts us to postulate that the decrease of the 1180-1182-cm-’ band could be attributed to perturbation of the C-NH1 vibration mode of adenine. in other words, the nitrogen atom of the NH, group of adenine would be the second alkylating site of DNA for MYKO 63. Such a dialkylation on the N( 7) and NH, sites of an adenine residue has been reported for K*PtCI, by Theophanides et al. [31]. The fact that adenine appears to be the target for MYKO 63 through a dialkylating process on the N(7) and NH2 sites is consistent with the very

48

low kinetics of complexation of MYKO 63 to DNA mentioned above: Lawley and Brookes [32], Maxam and Gilbert [33] and Goodwin et al. [30] have clearly demonstrated that methylation on adenine happens very slowly, five times slower, for example, than on guanine. CONCLLJSION

The present Raman investigations show direct interactions between N3P3 AZ, and the N( 7) and NH2 sites of adenine. Further similar investigations are now in progress with several other anticancer inorganic ring compounds with the aim of elucidating the origin of their antitumour properties_ REFERENCES 1 J-F. Labarre, S. Cros, J-P. Faucher, G. Francois, G. Levy, C. Paoletti and F. Sournies, 2nd International Symposium on Inorganic Ring Systems (IRIS Meeting), Giittingen, August 1978, Proceedings, p. 44. 2 J-F. Labarre, J-P. Faucher, G. Levy, F. Sournies, S. Cros and G. Francois, Eur. J. Cancer, 15 (1979) 637. 3 J-F. Labarre, R. Lahana, F. Sournies, S. Cros and G. Francois, J. Chim. Phys., 77 (1980) 85. 4 J. 0. Bovin, J. GaIy, J-F. Labarre and F. Sournies, J. Mol. Struct., 49 (1978) 421. 5 J. 0. Bovin, J-F. Labarre and J. Galy, Acta CrystaUogr.. Sect. B, 35 (1979) 1182. 6 J. Galy, R. Enjalbert and J-F. Labarre, Acta Crystallogr., Sect. B, 36 (1980) 392. 7 E.O.R.T.C. results from Dr. F. Spreafico, Mario Negri Institute, Milano. 8 E.O.R.T.C. results from Dr. B. W. Fox, Paterson Laboratories, Manchester. 9 G. Scatchard, Ann. N-Y. Acad. Sci., 51 (1949) 660. 10 J-L. Butour, J-F. Labarre and F. Sournies, J. Mol. Struct., 65 (1980) 51. 11 T. Theophanides, N. Hadjiliadis, M. Berjot, M. Manfait and L. Bernard, J. Raman Spectrosc., 5 (1976) 315; L. Bernard, M. Manfait, M. Berjot, I’. K. Ganguli and T. Theophanides, Biochemistry, 60 (1978) 1139. 12 R. Ritz, E. Kober, C. Grundmann and G. Ottmann, Inorg. Chem., 3 (1964) 757. 13 F. Sournies, These de Doctorat d’Universit8 no. 350, Paul Sabatier University, Toulouse, (April 25, 1980). 14 B. Monsarrat, J-C. Prome, J-F. Labarre, F. Sournies and J. C. van de Grampel, Biomed. Mass Spectrom., in press. 15 M. Manfait, J-L. Beaudoin and L. Bernard, in J-P. Mathieu (Ed.), Advances in Raman Spectroscopy, Heyden, London, 1972, p. 76. 16 M. Manfait, A. J. P. Alix and J-F. Labarre, to be published. 17 D. M. Adams and W. S. Fernando, J_ Chem. Sot. Dalton Trans., 22 (1972) 2503. 18 J. Emsley, J. Chem. Sot. A, (1970) 109. 19 R. W. Mitchell, J. C. Burr, Jr. and J. A. Merritt, Spectrochim. Acta, Part A, 23 (1967)195. 20 A. J. P. Alix, M. Manfait and J-F. Labarre, to be published. 21 R. C. Lord and G. J. Thomas, Jr., Spectrochim. Acta, Part A, 23 (1967) 2551. 22 E. W. Small and W. L. Peticolas, Biopolymers, 10 (1971) 69. 23 E. W. Small and W. L. Peticolas, Biopolymers, 10 (1971) 1377. 24 S. C. Erfurth and W. L. Peticolas, Biopolymers, 14 (1975) 247. 25 S. C. Erfurth, E. J. Kiser and W. L. Peticolas, Proc. Nat. Acad. Sci. U.S.A., 69 (1972) 938. 26 S. Mansy, S. K. Engstrom and W. L. Peticolas, Biochem. Biophys. Res. Commun., 68 (1976) 1242. 27 S. Mansy and W. L. Peticolas, Biochemistry, 15 (1976) 2650.

49 28 M. Tsuboi,

S. Takahashi and I. Harada, in M. Duchesne (Ed.), Physico-chemical of Nucleic Acids, Academic Press, New York, 1973, p_ 91. 29 M. Tsuboi, A. Y. Hirakawa, Y. Nishimura and I. Harada, J. Raman Spectrosc., 2 (1974) 609. 30 D. A. Goodwin, J. Vergne, 18 (1979) 2057.

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31 T. Theophanides, M. Berjot and L. Bernard, J. Rarnan Spectrosc., 6 (1977) 109. 32 P. D. Lawley and P. Brookes, Biochem. J., 89 (1963) 127. 33 A. M. Maxam and W. Gilbert, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 560.

Properties