Biochemical Pharmacology, Vol. 38, No. 21, pp. 3683-3687, 1989. 0(I)6--2952/89 $3.00 + 0.00 Printed in Great Britain. I~) 1989. Pergamon Press plc ARISTOLOLACTAM-fl-D-GLUCOSIDE A NEW DNA BINDING MONOFUNCTIONAL INTERCALATING ALKALOID SASWATI CHAKRABORTY, RUMA NANDI, MOTILAL MAITI,* BASUDEB ACHARI, CHITI'A R. SAHA and SATYESH C. PAKRASHI Indian Institute of Chemical Biology, Calcutta 700 032, India (Received 11 January 1989; accepted 16 May 1989) Abstract--The binding of aristololactam-fl-D-glucoside to DNA is characterized by hypochromism and bathochromism in the absorption band, quenching of the fluorescence intensity, increase in the positive and negative ellipticity of DNA, enhancement of thermal transition temperature, sign and magnitude of thermodynamic parameters, increase of the contour length of sonicated rod-like DNA and induction of the unwinding-rewinding process of covalently closed superhelical DNA. Binding parameters deter- mined from absorbance and fluorescence titration by Scatchard analysis, according to an excluded-site model, indicate a very high affinity towards DNA. The binding of the alkaloid is an exothermic process with Gibbs free energy of -7.4 kcal/mol, van't Hoff enthalpy of -13.8kcal/mol and entropy of -21.5 cal/degree/moi at 25°. On the basis of these observations it is concluded that aristololactam-fl- D-glucoside binds to DNA by a mechanism of intercalation. Biological activities of many naturally occurring and synthetic compounds are presumed to be due to their interaction with DNA, interfering with its expression in the cells [1, 2]. The binding of these compounds to DNA is a topic of wide interest and investigation in order to establish a structure-function relationship. One important class of these compounds is com- prised of those that bind to DNA by intercalation. Such compounds are important tools in molecular biology, and some are used for the treatment of cancer in man [1-3]. Aristolochia group of alkaloids and their glucoside derivatives has attracted recent attention for their antimicrobial, antitumor and various other biological properties [4]. This group of compounds has been successively tested for the therapy of tuberculosis, chronic bronchitis, pneumococcal disease and in the treatment of cancer [4]. Although there are reports for their clinical and pharmacological uses, the physi- cal and molecular basis of their interaction with DNA are still unexplored. Among this group of alkaloids, aristololactam-~D-glucoside (ADG)t or 6-fl-D- glucopyranosyl-8-methoxy benzo (f)-l,3-benzo- dioxolo(6,5,4-cd) indol-5(6H)-one (Fig. 1), a phen- anthrenic lactam derivative is of particular interest to us for its attached glucoside ring in the structure. As a part of our investigation on the interaction of alkaloids with nucleic acids [5-11], we report here, for the first time, the binding properties of ADG to DNA from the measurements of spectrophotometry, * Author to whom correspondence should be addressed. t Abbreviations: ADG, aristololactam-fl-D-glucoside; CT, calf thymus; CD, circular dichroism; CCS Col El, covalently closed superhelical Col E1 plasmid; DMSO, dimethyl sulphoxide; EB, ethidium bromide; GC, guanine- cytosine content; Tin, thermal melting temperature. o H H 3 4 6 9 Fig. 1. Chemical structure of aristololactam-fl-D-glucoside. spectrofluorimetry, spectropolarimetry, thermal melting profiles, thermodynamic parameters and viscometric studies. MATERIALS AND METHODS ADG was extracted from Aristolochia indica and crystallized from ethanol. Its purity was checked by thin layer chromatography, melting point, and it was characterized by mass and NMR spectra respectively [12, 13]. It is slightly yellowish in colour, freely sol- uble in DMSO but sparingly soluble in water. EB was obtained from Sigma Chemical Co. (St Louis, MO) and was used without further purification. The ligand concentrations were determined spectro- photometrically using a molar extinction coefficient (e) of 10930/M/cm at 398 nm for ADG in DMSO and of 5680/M/cm at 480 nm for EB in water. CT DNA (type I, 42 mole % GC), CCS Col El DNA (53 mole % GC) and DMSO were obtained from Sigma Chemical Co. and were used as such. DNA concentrations in terms of nucleotide phos- 3683