Chem. Res. Toxicol. 1992,5, 597-607
597
Synthesis and Sequence-Specific DNA Binding of a Topoisomerase Inhibitory Analog of Hoechst 33258 Designed for Altered Base and Sequence Recognition Malvinder P. Singh, Tomi Joseph, Surat Kumar, Yadagiri Bathini, and J. William Lown* Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2 Received March 11, 1992
The preparation and DNA binding characteristics of a structural analog of Hoechst 33258 bearing two pyridinic nitrogen atoms are described. The lH NMR signals of the complex formed between the new ligand 1 and decadeoxyribonucleotide d(CATGGCCATG)2 were assigned by employing one- and two-dimensional NMR techniques. Intermolecular nuclear Overhauser effects (NOE) between the ligand and the DNA receptor fragment confirm that the ligand binds in the minor groove of the DNA, interacting with the centrally located 5’-GGCCA segment. In contrast to the steric clash between the benzimidazole rings of the parent Hoechst 33258 molecule and the guanine 2-NH2 groups, which renders it G.C avoiding and thus A-T base pair preferring, the ligand 1 described here overcomes these unfavorable interactions and instead exhibits a marked preference for G C base pairs. This behavior appears to arise from additional stabilization due to H-bonding with the guanine 2-NH2 groups. Although a ligand-induced distortion a t the binding site is qualitatively assessable, the overall B-type conformation of the DNA fragment is retained upon complexation. The structural conclusions drawn from the NOE-NMR evidence were confirmed by molecular mechanics and molecular modeling studies.
Introduction Genetic targeting constitutes the basis of recent strategies of developing a novel approach to chemotherapy (1, 2). Initial efforts were based on the concept of antisense oligo- and polynucleotide probes to selectively bind and inhibit mRNA target regions;however, more recent studies have been directed at selectiveinhibition of gene expression via suppressions at the level of double-stranded DNA itself, constituting what is known as the antigene strategy. For example, the development and use of triplex-forming oligonucleotides is aimed at inhibiting DNA replication in the cell, thus affecting the origin of cellular expression most efficiently (3, 4). However, the use of oligo- and polynucleotides has potential obstacles in terms of their nuclease degradation, cellular impermeability, and the optimal requirement of -25 base pair length for efficient hybridization. A complementary approach which circumvents these problems is the use of non-nucleotidic DNA minor groove binding agents (5-8). We are concentrating on developing this methodology (7,9,10) based on extensive analysis of various components of drug-DNA binding interactions (11-14). The major impetus behind the design and development of sequence-specific DNA-binding drugs, based upon naturally occurring lead compounds, is the understanding of several factors responsible for the drug-DNA interactions at the molecular level (7, 14). Analysis of the structural elements for the molecular recognition of DNA by some naturally occurring antibiotics has been utilized in making appropriate modification of the parent compounds resulting in predictable changes in DNA recognition (6,7,10). For example, incorporation of hydrogenbond acceptor sites and alterations in the electrostatic components in oligopeptidic antibiotics netropsin and
* Author t o whom correspondence should be addressed.
distamycin A have provided “lexitropsins”or informationreading molecules with varying degrees of G-C base pair acceptance in contrast to the inherent strict affinity of the parent compounds for A.T-rich sites in DNA (15-18). One important consequence of specific binding to the DNA target sites is the ability to affect the activities of the enzymes that require DNA templates as their substrates. Type I and I1 topoisomerases form a class of essential nuclear enzymes acting to alter DNA topology, via catenation, overwinding, and underwinding, during DNA transcription and replication (19,20). Inhibition of such activities, therefore, offers an attractive target for developing chemotherapeutic agents (21,221. Indeed, a number of widely used clinical anticancer agents e.g., m-AMSA, doxorubicin (adriamycin), ellipticines, and epipodophyllotoxins, etc., are now known to induce topoisomerase-mediated DNA lesions (23). These agents generally exhibit mixed modes of binding to DNA helices, e.g., intercalation, minor-groove binding, etc, Recent reports suggest that purely minor groove binding agents, including certain lexitropsins related to distamycin and structural analogs of Hoechst 33258, are also potent inhibitors of topoisomerases both in vitro and in intact cells (24-27). In our first series of Hoechst 33258 analogs (281, the high DNA binding affinities correlate positively with their ability to inhibit topoisomerase 11and with in vitro cytotoxicity (29). In addition, an examination of the pBR322 cleavage recognition sequence reported for Drosophila topoisomerase I1 revealed the presence of two binding sites for the inhibitor described in this report. This sequence with t h e binding sites underlined is ACAATGJCGCTCATC (the arrow denotes the enzyme cleavage site). In order to begin exploring the structure of the threecomponent systems of protein-template-ligand, we report the details of the high-field lH NMR study of interactions 0 1992 American Chemical Society
598 C h e m . Res. Toxicol., Vol. 5, No. 5, 1992
S i n g h et al.
Scheme Ia
7 a
1
(a) PhN02, 150 O C .
Scheme IIa
nNo2 b
ij
3
2
OHC
a-ewHa -
1.
6
I
I OHC )3)"H3
l
*
CH2OCH,
I
4
pJ)mwH
8rCti*
' N
I
CHPCHl
6
5
(b) 10% PdlC, H2; (c) p-(CH30)PhCHO, PhN02, 150 "C; (d) CH30CH2C1, Et3N; (e) NBS; (f) NaHC03, DMSO; (g) dilute HCl.
between ligand 1 and a synthetic decadeoxynucleotide sequence. The selection of the DNA sequence d(CATGGCCATG)zwas based on our previous experience with its utility in probing the binding interactions of the modified ligands with high affinity for G C sites (16, 17, 30, 31). Synthesis. The reported methods (32,331for obtaining benzimidazole/pyridoimidazoles (PPAl-induced cyclocondensation of aromatic diamines and carboxylic acids) were not used owing to their relative inefficiency and the use of rather harsh conditions. Instead, compound 1 was prepared by a convergent approach outlined in Scheme I, via our recently reported method based on nitrobenzenemediated cyclodehydrogenationof the condensation products obtained from a diaminopyridine derivative and aromatic aldehydes (28,34). This method is analogous to an earlier report on the use of FeC13 in preparation of benzimidazole derivatives (35). The precursor aldehyde 7 required for the final reaction was obtained in five highyielding steps starting from 2-amino-6-methyl-3-nitropyridine (36) as shown in Scheme 11. The use of a methoxymethyl protecting group was critical in the success of the bromination reaction (step e).
Materials and Methods General Comments. In the procedures described below, all the reactions were carried out under Nz atmosphere and the evaporations were performed in vacuo using a rotary evaporator. All starting organic chemicals were obtained from Aldrich Chemical Co. (Milwaukee, WI) and used without further purification. Solvents used for chromatography were of HPLC grade. Anhydrous benzene and T H F were distilled prior to use from sodium benzophenone ketyl. Melting points were recorded on a Fisher-Johns capillary apparatus and are uncorrected. The IR spectra were obtained with a Nicolet 7199 FT spectrophotometer, and only the principal absorptions are reported. The lH NMR spectra for characterizing the reaction products were recorded 1 Abbreviations: COSY, correlated spectroscopy; DMSO, dimethyl sulfoxide;FID,free induction decay;HOD,deuteratedwater signal;HPLC, high-performanceliquid chromatography; HRMS, high-resolutionmass spectrometry; IR, infrared spectrophotometry; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect;NOESY, nuclear Overhauser and exchange spectroscopy; PPA, polyphosphoric acid; THF, tetrahydrofuran; TMS,tetramethylsilane.
on a Bruker WH-300 spectrometer. All lH chemical shifts are reported relative to tetramethylsilane (TMS) as internal standard when CDCq or DMSO-dewas used as the solvent. High-resolution mass spectra were determined on Associated Electrical Industries (AEI) MS-9 and MS-50 focusing mass spectrometers. Kieselgel 60 (230-400 mesh) obtained from E. Merck (Cherry Hill, NJ) was used for flash chromatography. Note: Methoxymethyl chloride and nitrobenzene are suspected toxins and should be carefully handled in a fumehood with proper ventilation. Benzoyl peroxide is potentially explosive on shock treatment and should be carefully handled. 2,3-Diamino-6-methylpyridine (2) was prepared by catalytic (36)(3.06 g, hydrogenation of 2-amino-6-methyl-3-nitropyridine 20 mmol) dissolved in 100 mL of EtOAc, using 10% Pd/C (320 mg). At the completion of the reaction, the catalyst was removed by filtration through Celite and the filtrate was evaporated to afford 2.34 g of the product 2 (95% yield) which was used in the subsequent step without further purification: mp 70-71 "C; IR (KBr) u, 3380,3320, 1650, 1600,1480 cm-l; lH NMR (CDCl3) d 2.30 (s, 3 H, CH3), 3.10 and 4.21 (2 br s, 2 H each, 2 X NH2, exchange), 6.40 (d, 1 H, Ar-H), 6.78 (d, 1 H, Ar-H). 2-( 4-Methoxyphenyl)-5-methyl-3H-imidazo[ 4,5-b]pyridine (3) was prepared by heating a mixture of 2,3-diamino6-methylpyridine (1.48 g, 12 mmol) and 4-methoxybenzaldehyde (1.63 g, 12 mmol) in 25 mL of nitrobenzene at 150 "C for 18 h. The reaction mixture was then cooled, and nitrobenzene was evaporated under reduced pressure. The residue obtained was purified by silica gel flash chromatography (1:l EtOAc/hexane eluent) to afford 2.04 g of 3 (71% yield): mp 231-233 "C; IR (KBr) u,, 3440,1610,1490,1390,1250 cm-l; lH NMR (CDC13) d 2.56 (5, 3 H, CH3), 3.84 ( ~ , H, 3 OCH3), 7.06 (d, 1H, Cs-H), 7.12 (d, 2 H, CrICy-H), 7.82 (d, 1 H, C7-H), 8.14 (d, 2 H, Cy/Ce,-H); HRMS calcd for CI4H13N30m l z 239.1058, found m/z 239.1058 (M+, loo), 224 (34), 196 (19). 3-(Met hoxymet h y l ) -2-(4-methoxyphenyl)-5-methyl3H-imidazo[4,5-b]pyridine(4). To a stirred suspension of the preceding imidazopyridine derivative 3 (1.196 g, 5 mmol) in 80 mL of anhydrous benzene containing 2 mL of Et3N was added dropwise a solution of methoxymethyl chloride (1.6 g, 20 mmol) in 5 mL of benzene at 0 "C. The reaction mixture was allowed to warm to 25 O C and then heated under reflux for 4 h. At the completion of the reaction, the reaction mixture was partitioned between water and EtOAc, and the organic layer was removed, washed with water, dried (Na2SO4),and evaporated to afford a solid residue which was purified by silicagel flashchromatography (EtOAcihexane eluent) to obtain 1.25 g of 4 (88% yield): mp
NMR Analysis of Hoechst Analog-DNA Complex 109-111 "(2; IR (KBr) vm= 2980, 2940, 2830, 1600, 1500, 1460, 1410 cm-1; 1H NMR (CDC13) 6 2.64 (s,3 H, CS-CH~), 3.54 (5, 3 H, OCH3),3.86 (s, 3 H, C4,-OCH3),5.60 (s, 2 H, CHz), 7.02 (d, 2 H, Cy/CU-H),7.10 (d, 1 H, Cs-H), 7.91 (d, 1 H, C7-H), 8.02 (d, 2 H, CZ,/C6,-H); HRMS calcd for Cl6HI7N302m/z 283.1320, found m/z 283.1320 (M+, loo), 252 (97), 238 (21). B-(Bromomet hyl)-3-(methoxymethy1)-2- (4-methoxyphenyl)-3H-imidazo[4,5-b]pyridine(5). A mixture of the preceding compound 4 (1.132 g, 4 mmol), N-bromosuccinimide (780 mg, 4 mmol), and benzoyl peroxide (50 mg) in 60 mL of CC14was heated under reflux for 4 h, followed by cooling and treatment with 3% aqueous NaHC03 solution (30 mL). The combined organic extracts were washed successively with brine (50 mL) and water, dried (Na2SO4),and evaporated to afford a residue which was purified by silica gel flash chromatography (EtOAc eluent) to obtain 1.15 g of 5 (80% yield): mp 106-108 "C; IR (KBr) vmar 1610,1480,1420,1280,1250 cm-1; 'H NMR (CDC13) 63.6O(s,3H,OCH~),3.91(~,3H,C4-OCH3),4.70 (s,2H,CHzBr), 5.65 (9, 2 H, CHz), 7.06 (d, 2 H, C~/CV-H), 7.40 (d, 1 H, c~j-H), 8.05 (2 d, 3 H, CZ,/C~,-H and C7-H); HRMS calcd for C16H16BrN302m/z 361.0425, found m/z 361.0439 (M+,12), 282 (M - Br, loo), 252 (28). 3 4 Methoxymet hyl)-2-(4-methoxyphenyl)-3H-imidazo[4,5-b]pyridine-5-carboxaldehyde (6) was obtained by stirring a mixture of the preceding compound 5 (361 mg, 1 mmol) and NaHC03 (92 mg, 1.1 mmol) in 10 mL of DMSO a t 130 "C for 2 h. The solvent was then removed under reduced pressure and the residue was extracted with a 1:l mixture of T H F and EtOAc (3 x 30 mL). The organic extracts were dried (Na2SO4)and evaporated to obtain a residue which was purified by silica gel flash chromatography (EtOAc eluent) to give 180 mg of 6 (64% yield): mp 184-186 "C; IR (KBr) vmax 1695, 1610, 1580, 1470 cm-1; lH NMR (CDC13) 6 3.64 (9, 3 H, OCH3), 3.90 (s, 3 H, C4,OCH3),5.72 (s, 2 H,CHz), 7.08 (d, 2 H, C3&-H), 8.0-8.2 (m,4 HI Ar-H), 10.12 (s, 1 H, CHO); HRMS calcd for C16H15N303m/z 297.1113, found m/z 297.1115 (M+, 91), 283 (lo), 267 (39), 266 (loo), 254 (12). 2- (4-Met hoxyp henyl) -3H-imidazolo[ 4,5-b]pyridine-5-carboxaldehyde (7). The preceding imidazopyridine aldehyde derivative 6 (170 mg, 0.57 mmol) dissolved in 5 mL of THF was treated with 18% aqueous HCl solution, and the mixture was stirred at 30 "C for 18 h. The solution was neutralized to pH 7 with KzCO3, and the mixture was extracted with EtOAc. The EtOAc extracts were dried (NazS04) and evaporated to give a solid residue which was purified by silica gel flash chromatography (EtOAc eluent) to afford 136 mg of 7 (94% yield): mp 232-234 "C; IR (KBr) vmar 3420,1710,1620,1500cm-l; lH NMR (DMSOds) 6 3.88 (8, 3 HI OCHa), 7.18 (d, 2 H, CT/Cy-H), 7.86 (d, 1 H, Cs-H), 8.14 (d, 1 H, C.I-H),8.24 (d, 2 H, Cp/C.yH), 10.04 ( 8 , 1H, CHO), 13.7 (s,1H, NH, exchange); HRMS calcd for C14HllN302 m/z 253.0851, found m/z 253.0856, (M+, loo), 225 (421,210 (23). 2-(4-Methoxyphenyl)-5-[ (4-methylpiperazin- 1-y1)-3H-imidazo[4,5-b]pyridin-2-yl]-3H-imidazo[4,5-b]pyridine ( 1). This was prepared by heating a mixture of 2,3-diamino-6-(4-methylpiperazin-1-y1)pyridine (28) (104 mg, 0.5 mmol) and the preceding aldehyde derivative 7 (127 mg, 0.5 mmol) in 15 mL of nitrobenzene at 150 "C for 18 h. The reaction mixture was cooled and nitrobenzene was removed under reduced pressure to afford a residue which was purified by flash chromatography on a column of fluorisil (EtOAc/MeOH eluent) to obtain 160 mg of 1 (73% yield): mp 280 "C dec; IR (KBr) vma. 3400, 2930, 2840, 2800, 1620,1590,1490,1440,1400cm-'; lH NMR (DMSO-&) 6 2.22 (s, 3 H, N-CHS), 2.42 (t, 4 H, CHz), 3.50 (t, 4 H, CHz), 3.80 (8, 3 H, OCH3), 6.74 (d, 1 H, Ar-H), 7.00 (d, 2 H, Ar-H), 7.6-7.8 (2 d, 1 H each, Ar-H), 7.88 (d, 1 H, Ar-H), 8.24 (d, 2 H, Ar-H), 12.60 (br s, 2 H, NH, exchange);HRMS calcd for Cz4Hz4N*O m/z 440.2073, found m/z 440.2069 (M+, 21, 370 (10). DNA Synthesis. The self-complementary decamer d(CATGGCCATG):!was synthesized and purified as previously described (16).The purity of the DNA fragment was confirmed by reversephase HPLC and 'H NMR for any residual organic impurities.
Chem. Res. Toxicol., Vol. 5, No. 5, 1992 599 High-Field NMR Measurements. The proton one-dimensional and two-dimensional spectra were obtained on a Varian Unity 500-MHz spectrometer. The one-dimensional 'H NMR spectrum of the decamer and that of the decamer-ligand complex was acquired during a titration experiment involving stepwise additions of a freshly prepared stock solution of the ligand to the NMR sample of the oligonucleotide. The spectra were obtained at 294 K with a sweep width of 5000 Hz and 32K data points. A 90" pulse width of 8 ps and a total recycle delay of 3 s were used. The data were processed with a Gaussian apodization function [interactively adjusted to ensure decay of free induction decay (FID) to zero] to provide resolution enhancement. Spectra showing the exchangeable imino protons of the oligomer and the oligomer-ligand complex were obtained by using the Sklenar-Bax water suppression scheme (37) on the sample dissolved in 90% H20-Dz0. These spectra were collected with a sweep width of 10 000 Hz, a 90" pulse of 8.5 ps, and 32K data points. The delay time between the 90" pulses was optimized to 7.4 ms with a spin-lock pulse of duration 2 ms, in order to maximize the HOD suppression and selective observation of the imino signals. The data were Fourier-transformed with 3-Hz line broadening. The 1D-NOE measurements used a 0.3-9 preirradiation of selected resonance frequencies at a power level suitable for 90% saturation of the corresponding signals. Two-dimensional NOESY experiments on the oligonucleotide and the oligo-ligand complex were performed on nonspinning samples in the phase-sensitive mode according to the hypercomplex method (38, 39) by use of standard NOESY pulse sequence provided in the Varian VNMR software. For each sample a series of such experiments were performed for mixing intervals of 80, 150, 250, and 40 ms. The cross peak volume integrals for assessment of relative intensities were determined by evaluating the average volumes for a series of equally sized rectangles circumscribing each peak. The longest mixing time NOESY spectra were obtained for the purposes of complete assignment of the nonexchangeable proton signals. Data were acquired with the carrier frequency at the center of the spectrum and quadrature detection for a sweep width of 4500 Hz (adjusted to cover the entire range of the signals due to the nonexchangeable protons) in both the tl and t2 dimensions. Data sets consisted of 512 FIDs ( t l )and 2048 data points in t2. A relaxation delay interval of 2.0 s was set for each pulse sequence. The spectra were zero-filled to 2048 points in tl prior to Fourier transformation, thus relating to equal digital resolution of 4.1 Hz in both dimensions. In order to avoid truncation of the FIDs, a skewed sine bell apodization function was set interactively to ensure that the interferograms decayed to zero in both t l and t2 dimensions. The base line corrections were performed according to the procedure of Otting (40). The contour plots for 2D experiments were constructed for publication after symmetrization. Molecular Mechanics a n d Molecular Modeling. Molecular mechanics calculations were carried out on an IRIS 4D/ 70GT workstation (SiliconGraphics) using the BIOSYM Software package Insight I1 (Version 2.0.0, Biosym Technologies Inc., San Diego, CA). The standard BIOSYM force field was used in all energy calculations. The structure of ligand 1 was constructed using the Builder module of Insight I1 and energy minimized with the Discover module until all the derivatives converged to 0.1 kcal/(mol.A) or less. The DNA decamer d(CATGGCCATG)z was built with the aid of the Biopolymer module and energy minimized until all the derivatives converged to 0.5 kcal/(mol.A) or less. In all the energy calculations a distance-dependent dielectric constant of 2r was used to account for the solvent screening effects. The minimized structure of the ligand was docked into the minor groove of the energy-minimized DNA duplex. The docking was carried out with the aid of extensive 3D visualization. In order to dock the ligand into the minor groove of the B-DNA duplex, the location of the binding site was selected on the basis of the NMR data. The NMR data were used in obtaining a starting structure for the molecular mechanics calculations. Since no NOE constraints were imposed during
600 Chem. Res. Toxicol., Vol. 5, No. 5, 1992
Singh et al.
H
Hoechst 33258 7
4
11
17
23
Ligand 1 5'-C
I 3'-G
A
T
G
G
C
C
A
T
I I m 1 v v v
IVIIIII
T
G
A
C
C
G
T
A
G-3'
I
T3Me 1
i
TgMe~
C-5'
Figure 1. Chemical structures of Hoechst 33258 and 2-(4-
methoxyphenyl)-5-[(4-methylpiperazin-l-yl)-3H-imidazo[4,5-b] ppidin-2-yl]-3H-imidazo[4,5-b]pyridine, depicted as ligand 1. Also shown is the duplex DNA sequence used in this study, with the roman numerals reflecting the imino protons of the base pairs in the symmetrical self-complementary d(CATGGCCATG)*. the minimization procedure, this permitted us to use molecular mechanics as an independent method to check the validity of the structural conclusions derived from the NMR results.
Results The structures and numbering schemes for the decadeoxynucleotide and the ligand 1 are shown in Figure 1. The roman numerals identified for the oligonucleotide sequence designate the imino protons at each of the basepair steps and reflect the 2-fold symmetry of the selfcomplementary duplex sequence. The numbering scheme for the ligand is based on our previous NMR studies on Hoechst 33258 and ita analogs ( 3 0 , 3 1 ) . Assignment of DNA 'H Resonances in the Free Form. In order to characterize the interaction of ligand 1to d(CATGGCCATG)zby NOESY-based analyses, the corresponding spectrum of the free oligonucleotide shown in Figure 2c had first to be assigned completely, which was accomplished by using a combination of 2D NMR experimenta. A typical contour plot of a NOESY spectrum is shown in Figure 3. The subsection of a typical NOESY spectrum showing the NOE cross peaks is illustrated in Figure 4A along with the magnetization-transfer pathways involving the purine/pyrimidine base protons and the deoxyribose H1' protons. A continuous connectivity pathway involving intra- and interresidue H8/H6-H1' proton pairs extends along the full length of the oligonucleotide (from C1to GlO),which constitutes the strategy of making unambiguous assignments based upon the welldocumented sequential NOE connectivity approach to right-handed B-type duplex DNA conformations (41-43). This assignment procedure was further extended to identify additional connectivities from base protons to glycosidic H2'/H2" (Figure 4B)and H3' protons within the same residues and to the adjacent 5'-neighbor residues. The characteristic intra- and internucleotide NOES involving the relevant protons are summarized in the form
7
8
6
5
4
3
2
1
6 (PPW Figure 2. 500-MHz lH NMR spectra of the nonexchangeable protons of ligand 1 (a), d(CATGGCCATG)z (c), and their 1:l complex (b) in DzO a t 294 K. Selected resonance assignments for the DNA fragment are indicated (see Materials and Methods for details on data acquisition and processing). H6/H8/H2
H1 /H5
2-
3-
H3iH4iH5
. ,'.": 4 . 1 ,
b
,'
1i
P
' I
1 *a*,
.
j
6'
"3
II
a
'
t
H2 IH2'IMe
&g
H2 H2
w*
&
1
/
a.
0
7
6
5
4
3
2
I
1
F1 (PPm)
Figure 3. Contour plot of aphaae-sensitive 1H NOESY spectrum a t 500 MHz of a Dz0 solution of duplex d(CATGGCCATG)zat 294K and pH 7.0. The mixing time, T, was 0.25 s (see Experimental Section for spectral parameters and other acquisition methods). The boxed regions A and B correspond to expanded plots given in Figure 4, and individual spectral regions grouped according to the types of protons are identified a t the axes.
of an NOE-grid diagram (Figure 5), and the individual assignments of the proton resonances are given in Table
Chem. Res. Toxicol., Vol. 5, No. 5, 1992 601
NMR Analysis of Hoechst Analog-DNA Complex A
B
7.1
C6lC7
a
7.5
C6lC7
D
F2
(PPW 7.9
I
8.3
l
6.4
6.0
5.6
5.2
,
,
3.0
F1 (PPm)
,
,
2.6
1 .a
2.2
14
F1 (PPm)
Figure 4. Expanded subsections of the NOESY contour plot of Figure 3 showing the cross peaks between the purine/pyrimidine base protons and glycosidic H1’ protons (A), and between the same base protons and H2’/H2”/T-Me protons (B). Continouous lines illustrate the sequential NOE connectivities from C1 through G10. Additional cross peaks marked by arrows in panel A correspond to those involving cytosine H5 protons.
Figure 5. Summary of intra- and internucleotide NOES observed among the nonexchangeable protons in d(CATGGCCATG)2.The NOE contacts involving deoxyribose protons H3’/H4’/H5’ are not shown.
Table I. Chemical Shift Values of Protons in d(CATGGCCATG)zat 294 K,and Its 1:l Complex (in Parentheses) with Ligand 1
C A T G G C C A T G
H6/H8 7.59 (7.56) 8.33 (8.35) 7.08 (7.19,7.07) 7.75 (7.76) 7.67 (7.66) 7.27 (7.26) 7.44 (7.43) 8.29 (8.28,8.11) 7.07 (7.06) 7.80 (7.77)
H5/H2/Me 5.80 (5.77)
1.35 (1.35) 5.19 (5.18) 5.54 (5.54) 1.46 (1.45, 1.42)
H1’ 5.56 (5.55) 6.24 (6.22) 5.64 (5.62) 5.59 (5.57) 5.81 (5.79) 5.85 (5.85) 5.38 (5.40) 6.22 (6.21) 5.73 (5.71) 6.04 (6.03)
I. These assignments are in agreement with those obtained earlier using 1D NOE difference measurements (16). Characteristic Ligand-Induced Effects on DNA Resonances. Initial indication of binding of ligand 1to the oligonucleotide was provided by an NMR titration experiment performed by adding small aliquots of a solution of 1 to a sample of oligonucleotide in DzO. Comparison of the ‘HNMR spectrum (500 MHz) of the mixture containing equivalent amounts of the two components with those of individual samples in the free form (Figure 2) shows some characteristic changes that are attributed to a direct interaction between the ligand and
H2” 2.35 (2.34) 2.88 (2.89) 2.27 (2.32, 2.22) 2.68 (2.65) 2.63 (2.55) 2.39 (2.35) 2.32 (2.34) 2.87 (2.86) 2.24 (2.26) 2.51 (2.57)
H2’ 1.93 (1.91) 2.68 (2.72) 1.85 (2.00, 1.80) 2.60 (2.51) 2.49 (2.37) 1.99 (2.03) 2.02 (2.06) 2.65 (2.64) 1.81 (1.88) 2.28 (2.28)
imino 13.51 (13.48) 12.96 (12.87) 12.90 (12.73)
13.61 (13.58) (13.06)
the DNA fragment. For instance, there is increased line broadening of the resonances over much of the spectrum as a result of an increased correlation time (16, 17). Notably, a “doubling” of resonances is also observed for DNA protons, e.g., for A8H8 and T3H6 determined by the 2D experiments described below, and for the thymidine methyl group a t 1.47 ppm discernible even in the onedimensional spectrum of the complex. Such doubling apparently results from the magnetic nonequivalence of the two strands (originally identical in the free form of oligonucleotide) of the DNA duplex in the presence of the asymmetric ligand which eliminates the 2-fold pseudo-
602 Chem. Res. Toxicol., Vol. 5, No.5, 1992 6 7.65
Singh et al. CH3
6 7.22 6 11.97
6 7.49
6 6.88
6 3.25
62.85
6 11.16
{c) *\
CHI
Figure 6. Assignments of the ligand protons in its complex form with the oligonucleotide d(CATGGCCATG)*.
dyad symmetry of the self-complementary duplex sequence (44-47). Ligand ‘HResonance Assignments in the Complex. The nonexchangeable proton resonances of ligand 1 in the complexed form were assigned by a combination of COSY and NOESY experiments. The three pairs of directly coupled aromatic spin systems were first identified via their respective cross peaks in a phase-sensitive COSY spectrum. These same correlations were subsequently identified in a NOESY contour plot and individually assigned on the basis of through-space interactions with the aliphatic protons in the upfield regions. Accordingly, proton H3 could be identified via an NOE to the methylene protons (H-26) of the N-methylpiperazine ring, and the methoxyphenyl H-21/23 protons through an NOE from the OCH3 signal. The N-methyl (H-28) and the second set of methylene protons (H-27) were also confirmed via their NOESY cross peak. The assignment of H-4 and H-20/24 then follows directly since they are spin-coupled with H-3 and H-21/23 respectively. Protons H-11 and H-12 could not be assigned individually since they do not correlate (via NOE) with any other reference point in the molecule. The proton resonances of ligand 1 in the complexed form are summarized in Figure 6. DNA 1H Resonance Assignments in the Complex and Overall Conformation. In addition to the aforementioned spin-coupled proton pairs of the ligand in the COSY experiment, cross peaks involving the spin systems of the DNA fragment were also identified. Thus, the aromatic H6 and H5 protons for all three sets of cytosine (Cl, C6, and C7) residues and the deoxyribose Hl’-H2’ and Hl’-H2’’ correlations for each nucleotide were distinguishable through their cross peaks. Besides the identification of the cytosine aromatic protons in the complexed form of the oligonucleotide, an interesting feature pertains to a significant reduction in the cross peak intensities (compared with those for the ligand-free oligomer) of C6 and C7 residues. The reason for this is not entirely clear, but such characteristic changes upon ligand binding have been consistently observed in a number of previous investigations (16,17, 30, 31). Borah et al. (48)have postulated that the intensity of cytidine H5-H6 cross peaks is quite sensitive to the local environment and mobility of cytosine residues in drug-DNA complexes and is reduced upon binding of ligands in close proximity to these residues. Thus, these correlation peaks in a COSY experiment provide an approximate marker for the location of ligands on DNA sequences. In the present example a
significant difference in the relative intensities observed for two adjacent cytosine residues C6/C7 suggests a direct interaction between the ligand and these cytosine residues. The connectivities mentioned above for the spin-coupled proton pairs were next identified in the NOESY spectra and further used for elucidating the sequence-specific magnetization-transfer pathways. One such connectivity pattern involving the DNA base protons (H6/H8) and anomeric sugar H1’ protons along the full length of the duplex oligonucleotide is outlined in the NOESY contour plot (Figure 7). The previously identified resonances for the aromatic protons H5 and H6 of the cytosine (Cl) residue provided a starting point for analyses of the cross peaks in the 2D-NOESY spectra. Thus, C1H6 gives a cross peak to ClH1’ witha subsequent connection toA2H8, which in turn shows a cross peak to A2H1’, and the mapping continues through T3H6, etc. The connectivity pattern is similar to that outlined for the free oligonucleotide, which indicates that the right-handed B-type DNA conformation is retained upon complexation with the ligand. This is further confirmed by an inspection of the relative intensities of the NOESY cross peaks. Accordingly, it was observed, among various NOESY spectra obtained by using progressively shorter mixing times, that the cross peaks corresponding to intranucleotide H6/H8H2’ and internucleotide H2”-H6/H8 proton pairs were present even at short mixing times, while those corresponding to intranucleotide H6/H8-H2” and internucleotide H2’-H6/H8 start to build up only at mixing times of 100 ma or greater. Such a pattern of NOE cross peak intensities is consistent with a right-handed B-DNA conformation (41-43)and is further supported by an overall order of relative cross peaks intensities of H2’ >> H1’ > H3’ at short mixing times (100-150 ms) where the intensities are not severely overestimated due to spin diffusion. The characteristic intra- and interresidue NOES involving the nonexchangeable protons are summarized in Figure 8, and the individual assignments are provided in Table I. In the case of the complex, several cross peaks in the NOESY spectra acquired at short mixing time intervals show extremely low intensities, which makes it difficult to make complete assignments of the spectrum from a single low-mixing-time NOESY experiment. It was therefore necessary either to approach the noise levels or to acquire NOESY data at progressively longer mixing times to ensure the detection of these cross peaks. Although the loss of NOE cross peak intensities could arise from line broadening, it can also be attributed to the presence of the ligand at its preferred site, which causes structural distortions along the length of the duplex, e.g., stretching out along the ligand binding region (i.e., unwinding). This is consistent with the observation that the intensity loss described above relates to the proton pairs at the ligand binding site, e.g., G5Hl’-C6H6, C6Hl’-C7H6, C7H1’A8H8, etc. The elongation along the helix axis is evidently to the extent that the corresponding proton-proton distances are forced out to >5 A, the range of NOE detection limits. More accurate estimation of the ligandinduced distortion can only result from determination of individual proton-proton distances generally obtainable by correlating the NOES measured for these vectors to those for cytosine H6-H5 vectors, whose lengths are essentially fixed. However, in the present case, we observe a significant suppression of the cytosine H6-H5 cross peak
Chem. Res. Toxicol., Vol. 5, No. 5, 1992 603
NMR Analysis of Hoechst Analog-DNA Complex
B
A 7.
i
7.5
@C6iC7
C6lC7@
I
T3,M e G4iG5
F2 (PPm) 7.9
8.3
6.4
6.0
5.6
5.2
3.0
2.6
2.2
i.a
1.4
F1 (PPm) Figure 7. Expanded regions of the phase-sensitive NOESY spectrum of the 1:lcomplexformed between ligand 1and d(CATGGCCATG)2. The spectrum was recorded with mixing time, T , = 0.25 s. The regions correspond to the spectral windows containing the base protons, and sugar H1’ protons (A) and H2’/H2’’/Me protons (B). Sequential NOE connectivities along the full length of DNA fragment are illustrated in panel A. The dashed lines forming part of the pattern join the cross peaks which experience significant loss in intensities as a result of ligand binding. The doubling of T3H6 and ASH8 signals is clearly evident by the cross peaks corresponding to thymidine methyl protons.
F1 (PPm)
Figure 8. Summary of intra- and internucleotide NOES observed in the NOESY spectrum shown in Figure 7. For the purposes of simplicity, the NOES involving glycosidicH3’/H4’/H5’ protons are omitted. Open circles denote the NOESY cross peaks that experience significant reduction in their intensities under the influence of ligand 1.
intensities, which presents a potential problem in their use as reference distances. For these reasons, only a relative comparison of cross peak intensities is valid for qualitatively assessing the structural distortions in the DNA fragment. Intermolecular NOE Contacts and Minor Groove Binding. As demonstrated in a number of previous NMR investigations on solution structures of noncovalently bound ligand-DNA complexes (16,17,30,31,44-47), the observation of intermolecular NOE contacts forms the basis of defining the location and relative orientation of the ligand with respect to the oligonucleotide. In the present study numerous close intermolecular contacts between the ligand and the DNA fragment via observation of pertinent NOES are evident over the entire length of the ligand molecule. For instance, the protons from the methoxyphenyl terminus showed NOES to the glycosidic H1’ protons of residues G4 and C7 while the N-methylpiperazine protons at the other end showed NOES to the adenine (A8)-H2 proton. Additional through-space proximities were determined through 1D-NOE measurements involving the exchangeable protons, e.g., NOE
Table 11. Intermolecular Proton-Proton NOE Contacts in the 1:l Complex between Ligand 1 and d(CATGGCCATG)z Observed in 2D-NOESY and 1D-NOE Difference Measurements ligand oligonucleotide ligand oligonucleotide proton proton proton proton C21123-H C7-HI’ C27-H A8-H2 C20124-H C6-H1’ N28-CH3 A&H2 imino V N18-H N9-H imino IV
interactions between the N9H at 11.16 ppm, and N18H at 11.97 ppm with DNA imino protons marked IV and V, respectively (see Figure 1for numbering). All the intermolecular NOES observed are listed in Table 11. Such an array of close contacts involving the ligand protons along its concave surface and DNA protons associated with the minor groove is most consistent with a model orientation illustration (Figure 9) which highlights the ligand spanning the central 5’-GGCCA segment on the basis of aforementioned arguments. No NOE effects are observed between the aromatic protons of the two pyridoimidazole units and any of the DNA protons, which is also consistent with the
604
Chem. Res. Toxicol., Vol. 5, No. 5, 1992
Singh et al.
G-4
G-5
C6 c-7 AD
'
5'
Figure 9. Schematic representations of the inferred binding region of ligand 1 to 5'-GGCCA within the minor groove of d(CATGGCCATG)2.The model orientation of 1 relative to the DNA fragment is deduced from experimentallyobserved intermolecular NOE contacts indicated by double-headed arrows. Dashed lines and arrows represent the possible hydrogen bonding between the ligand and DNA. model interaction of Figure 9, indicating that both the heterocyclic rings present the same pyridinic nitrogencontaining edges toward the floor of the minor groove. This is in contrast to the case of a benzoxazole analog of Hoechst 33258 where the existence of two rotational isomers was proposed on the basis of a conformationally labile C-C bond linking the two heterocyclic rings (30). Exchange Processes for the Ligand-DNA Complex. As described above, a "doubling" of selected resonances is due to the loss of dyad symmetry in the self-complementary dupiex sequence in the presence of ligand 1. In addition, the preferred binding site for ligand 1 is the centrally located 5'-GGCCA segment, and the duplex DNA sequence contains two such equivalent sites (one on each strand). The exchange of the ligand between two equivalent sites was examined using temperature variable 'H NMR experiments. Upon increasing the temperature the aforementioned "doubled" resonances were observed to coalesce, suggesting a faster rate of exchange of 1between the two equivalent sites. The exchange rate and the free energy of activation for this exchange phenomenon could then be estimated by employing reported equations for kinetics of such dynamic processes. Sutherland (49) has proposed the relationship for evaluating the rate constant for the exchange at coalescence temperature (Tcoal,) between two equivalent sites as
kcoal,= ( a / h ) A u = 2.2Au where Au denotes the separation in Hz of the coalescing signals in the limits of minimal rate of exchange. Gunther (50) has further proposed the relationship for estimating the free energy of activation for the exchange:
(x)]
AG* = 19.14TCod,[9.97+ log TCOd.
The two signals corresponding to the DNA proton A8H8 in the complex are separated by 40 Hz, and their coalescence sets in at 301 K, which corresponds to the values of -88 s-l and 15 kcal-mol-1for the rate constant and activation energy, respectively. The actual mechanism for exchange processes cannot be unambiguously deduced
I
Figure 10. Skeletal depiction of the energy-minimized 1:l complex of ligand 1-DNA complex constructed with the aid of NMR data. The ligand binds in the minor groove along the 5'-GCCA sequence. from these data but is likely to involve transient dissociation of the complexes, followed by reorientation of the ligands before binding again to the equivalent target sites on the opposite strand. Molecular Mechanics and Molecular Modeling. The energy-minimizedstructure, shown in Figure 10,shows that the ligand binds in the minor groove of the duplex along the 5'-GCCA sequence. The ligand inserts into the groove with the concave side facing the floor of the groove, and the torsion about the central carbons (C8410)is about loo, facilitating the positioning of the ligand parallel to the walls of the groove. The snug fit of the ligand in the groove is clearly seen in the CPK model, shown in Figure 11. A detailed analysis of the molecular mechanics results shows that the dominant factors in the binding of ligand 1 to the DNA duplex are the two strong hydrogen bonds involving the NH hydrogens of the ligand and 0 2 of the two adjacent CC base pairs. The 5.3-Aseparation between the two donor nitrogens in the ligand makes it an ideal hydrogen bond donor for the cytosine oxygens in the minor groove. The two nitrogens introduced by replacing the methine carbons of the original Hoechst compound act as additional hydrogen-bond acceptors for the G-2NHz groups of the complementary strand. These hydrogen bonds are a major factor in specifying the location and orientation of the ligand in the groove. Neither of the ends of the ligand appears to play a major role in this overall binding process. The OCH3 group projects out of the groove with no close contacts. The puckered piperazine ring lies parallel to the groove with several hydrophobic contacts corresponding to the NOESobserved in the NMR experiments. This is clearly indicated in the reduction in cross peak intensities of C6 and C, residues observed in the NMR.
NMR Analysis of Hoechst Analog-DNA Complex I-
Figure ll. CPK model of the energy-minimized 1:l complex of ligand 1-DNA complex showing location and snug fit of the drug in the 5’-GCCA sequence in the minor groove.
Discussion Recent advances in automated DNA synthesis and molecular biology (1,2),computational modeling (51),and high-field NMR (52,531 have provided opportunities for investigating both the structural and biochemical consequences of ligand binding to DNA fragments (7,10,14). The information gained concerning the individual components of drug-DNA binding interactions (7,11-14) has further resulted in a rational approach to designing newer and improved DNA binding agents with the aim of increasing their therapeutic potential (6-9, 15-18). Encouraged by our results in the development of lexitropsins based on the naturally occurring pyrrole-amidine class of DNA minor groove binders netropsin and distamycin (7, IO), we recently reported analogous structural modifications in another type of DNA minor groove recognizing molecule, namely, Hoechst 33258 (28). The DNA-binding interactions of the bis-benzimidazole, Hoechst 33258,initially developed as a chromosomal stain (54),have themselves been widely explored by footprinting, by biophysical methods (551, and structurally by X-ray (56,57) and NMR techniques (58-60), which all provide evidence of its markedly preferential binding to the minor groove at AST-rich sequences of DNA. Footprinting (28) and physicochemical studies (61) on the first generation of Hoechst 33258 analogs developed in our laboratories have provided promising results in terms of their specific interactions through the minor groove of B-DNA and alterations in sequence selectivity toward G-C bases. As a bonus, the new compounds exhibit significant topoisomerase activities and are now being screened for their anticancer and antiviral potency (29). On the basis of these investigations and on the general principle that incorporation of hydrogen-accepting heterocycles permits the recognition of G-C sites, it was envisaged that the replacement of both the benzimidazole rings in the parent molecule with pyridoimidazoles should show increasing amounts of G C base pair acceptance due
Chem. Res. Toxicol., Vol. 5, No. 5, 1992 605 to incorporation of two suitably located pyridinic nitrogen centers. The preceding sections describe the preparation of this ligand, 1, and results from the NMR investigations on its binding interactions with a synthetic decadeoxyribonucleotide sequence. Structural information on the complex formed between ligand 1and d(CATGGCCATG)? has been obtained from selected NMR experiments. The presence of the ligand results in selective chemical shift perturbations and removal of the degeneracy in the palindromic decanucleotide sequence. Judging by the doubling of the resonances due to the loss of the 2-fold symmetry of the self-complementary DNA duplex, it is concluded that the residence time of the ligand on the DNA is long relative to the NMR time scale. Supporting evidence is provided by a downfield shift of the DNA imino protons that is consistent with a minor groove binding mode (62). Precise information about the nature of binding interactions is obtained by observing intermolecular proton-proton NOE contacts, thus providing the basis for elucidating the 5’-GGCCA segment as the ligand binding site. The relative orientation of the ligand is deduced from intermolecular NOES observed at both the N-methylpiperazine and methoxyphenyl ends of the ligand. On the basis of these results we infer that, in the initial stages of DNA recognition, the cationic N-methylpiperazine group in its protonated form anchors at the highly negative electrostatic potential at the A.T sites in the minor groove of DNA (63),followed by alignment of the rest of the molecule onto the floor of the minor groove. The overall binding is facilitated by the natural crescent shape of the ligand and is further stabilized by favorable H-bonding interactions between the heteroatom-containing edge of the ligand molecule and potential H-bond donors of the DNA. The results from the present study are in general agreement with those from related structures recently reported (30,311 where the 1:l complexes of two different topoisomerase I1 inhibitory analogs of Hoechst 33258were examined with the same decadeoxynucleotide sequence as employed in the present study. These analogs were designed to alter the parent sequence preference of A.T base pair recognition to G.C by incorporating H-bond acceptor moieties in appropriate positions in the structure of Hoechst 33258. The two compounds have a common feature in the form of a pyridinic nitrogen center (pyridoimidazole in the place of one benzimidazole),and despite the difference in the second substitution (benzoxazole vs benzimidazole), they both exhibit preferential binding on the 5’-CCAT segment. Such partial selectivity for G C base pairs observed by NMR is in agreement with conclusions drawn from footprinting studies (28). The analog 1 used in this study was designed with the incorporation of two pyridinic nitrogens by substituting both the benzimidazole rings by pyridoimidazole with the expectation of an increased recognition and/or selectivity for G-C base pairs. As anticipated, the binding region on the same DNA fragment has shifted to 5’-GGCCA, indicating a marked preference of ligand 1 for GC-rich segments. In addition to exhibiting such affinity for G-Cbase pairs, ligand 1 is also among the most potent topoisomerase inhibitors in the series of Hoechst 33258 analogs examined to date. Further biophysical investigations are now in progress to delineate what structural features correlate with potent inhibition of topoisomerases. Interestingly,
606 Chem. Res. Toxicol., Vol. 5, No. 5, 1992
the 5'-GGCCA DNA binding domain of ligand 1also forms part of the recognition sites for two restriction endonucleases MscI and StuI (64). We are currently examining the extent of protection provided by this ligand against cleavage of pBR322 and yIP5 DNA by these restriction enzymes. Thus, in addition to their potential use as chemotherapeutics, the novel ligands based on Hoechst 33258 may find utility as useful selective probes in molecular biology. Acknowledgment. This research was supported by a grant (to J.W.L.) from the Medical Research Council of Canada.
References (1) Cohen, J.S. (Ed.) (1989) Oligodeoxynucleotides: Antisenseinhibitors of gene expression. Topics in Molecular and Structural Biology, Vol. 12, MacMillan Press, London. (2) Barkel, C. L. (Ed.) (1989) Discoveries in antisense nucleic acids. Advances in Applied Biotechnology Series, Vol. 2, Gulf Publishing Co., Houston, TX. (3) Moser, H. E., and Dervan, P. B. (1987) Sequence-specific cleavage of double helical DNA by triple helix formation. Science 238,645650. (4) Cooney, M., Czemuszewicz, G., Postel, E. H., Flint, S.J., and Hogan, M. E. (1988) Site-specific oligonucleotide binding represses transcription of the human c-myc gene in vitro. Science 241,456-459. (5) Dervan, P. B. (1986) Design of sequence specific DNA binding molecules. Science 232, 464-471. (6) Dervan, P. B. (1987) Design of synthetic sequence specific DNA binding molecules. I n Molecular Mechanisms of Carcinogenic and Antitumor Actiuity. International Symposium of the Pontifical Academy of Sciences (Chagas, C., and Pullman, B., Eds.) pp 365384, Vatican Press and Adenine Press, New York. (7) Lown, J. W. (1988) Lexitropsins: Rational design of DNA sequence reading agents as novel anticancer agents and potential cellular probes. Anti-Cancer Drug Des. 3, 25-40. (8) Nielsen, P. E. (1990)Sequence selective DNArecognition by synthetic ligands. Bioconjugate Chem. 2, 1-12. (9) Lown, J. W. (1989) Synthetic chemistry of naturally occurring oligopeptide antibiotics and related lexitropsins. Org. Prep. Proced. Int. 21, 1-46. (10) Lown, J. W. (1990) Molecular mechanism of DNA sequence recognition by groove binding ligands: biochemical and biological consequences. In Molecular Basis of Specificity in Nucleic AcidDrug Interactions (Pullman, B., and Jortner, J., Eds.) pp 103-122, Kluwer Academic Publishers, Dordrecht, The Netherlands. (11) Kopka, M. L., Yoon, C., Goodsell, D., Pjura, P., and Dickerson, R. E. (1985) The molecular origin of DNA-drug specificity in netropsin and distamycin. Proc. Natl. Acad. Sci. U.S.A. 82, 1376-1380. (12) Goodsell, D., and Dickerson, R. E. (1986) Isohelical analysis of DNA groove binding drugs. J . Med. Chem. 29, 727-733. (13) Zakrezwska, K., and Pullman, B. (1988) Theoretical study of the sequence selectivity of isolexins, isohelical DNA groove binding ligands. Proposal for the G:C minor groove specific compounds. J . Biomol. Struct. Dyn. 5, 1043-1058. (14) Pullman, B. (1989) Molecular mechanisms of specificity in DNAantitumor drug interactions. Adv. Drug Res. 18, 1-113. (15) Kissinger, K. L., Krowicki, K., Dabrowiak, J. C., and Lown, J. W. (1987)Molecular recognition between oligopeptidesand nucleic acids. Monocationic imidazole lexitropsins that display enhanced G:C sequence dependent DNA binding. Biochemistry 26, 5590-5595. (16) Lee, M., Hartley, J. A., Pon, R. T., Krowicki, K., and Lown, J. W. (1988) Sequence specific molecular recognition by a monocationic lexitropsin of the decadeoxyribonucleotide d- [CATGGCCATG]*: structural and dynamic aspects deduced from high field 1H-NMR studies. Nucleic Acids Res. 16, 665-684. (17) Lee, M., Krowicki, K., Shea, R. G., Lown, J. W., and Pon, R. T. (1989)Molecular recognition between oligopeptidesand nucleic acids. Specificityof binding of a monocationic bis-furan lexitropsin to DNA deduced from footprinting and lH NMR studies. J . Mol. Recognit. 2, 84-93. (18) Rao, K. E., Krowicki, K., Burckhardt, G., Zimmer, C., and Lown, J. W. (1991) Molecular recognition between oligopeptides and nucleic acids: DNA binding selectivity of a series of 1,2,4-triazole-containing lexitropsins. Chem. Res. Toxicol. 4, 241-252. (19) Wang, J. C. (1985) DNA toposiomerases. Annu. Reu. Biochem. 54, 665-697. (20) Osheroff, N. (1989) Biochemical basis for the interactions of type I and type I1topoisomerases with DNA. Pharmacol. Ther. 41,223241.
Singh et al. (21) Lock, R. B., and Ross, W. E. (1987) DNA topoisomerases in cancer therapy. Anti-Cancer Drug Des. 2, 151-164. (22) Hsiang, Y.-H., Wu, H.-Y., and Liu, L. F. (1988) Topoisomerases: Novel therapeutic targets in cancer chemotherapy. Biochem. Pharmacol. 37, 1801-1802. (23) D'Arpa, P., and Liu, L. F. (1989) Topoisomerase-tmgetingantitumor drugs. Biochim. Biophys. Acta 989, 163-177. (24) Woynarowski, J. M., McHugh, M., Sigmund, R. D., and Beerman, T. A. (1989) Modulation of topoisomerase I1 catalytic activity by DNA minor groove binding agents distamycin, Hoechst 33258, and 4',6-diamidine-2-phenylindole.Mol. Pharmacol. 35, 177-182. (25) Woynarowski, J. M., Sigmund, R. D., and Beerman, T. A. (1989) DNA minor groove binding agents interfere with topoisomerase IImediated lesions induced by epipodophyllotoxin derivative VM-26 and acridine derivative m-AMSA in nuclei from L1210 cells. Biochemistry 28, 3850-3855. (26) McHugh, M. M., Sigmund, R. D., and Beerman, T. A. (1990) The effect of minor groove binding drugs on Camptothecin induced DNA lesions in L1210 nuclei. Biochem. Pharmacol. 39, 707-714. (27) Fesen, M., and Pommier, Y. (1989) Mammalian topoisomerase I1 activity is modulated by the DNA minor groove binder distamycin in simian virus 40 DNA. J. Biol. Chem. 264, 11354-11359. (28) Bathini, Y., Rao, K. E., Shea, R. G.,andLown, J. W. (1990) Molecular recognition between ligands and nucleic acids: Novel pyridine- and benzoxazole-containingagentsrelated to Hoechst 33258 that exhibit altered DNA sequence specificity deduced from footprinting and spectroscopic studies. Chem. Res. Toxicol. 3, 268-280. (29) Beerman, T. A., Lown, J. W., Rao, K. E., Bathini, Y., Sigmund, R. D., and McHugh, M. M. (1992) Effects of analogs of the DNA minor groove binder Hoechst 33258 on topoisomerase I1 and I. Biochim. Biophys. Acta (in press). (30) Kumar, S.,Bathini, Y., Zimmermann, J., Pon, R. T., and Lown, J. W. (1990) Sequence specific molecular recognition and binding by a G:C recognizing Hoechst 33258 analogue to the decadeoxyribonucleotide d-ICATGGCCATGl2: structural and dynamic aspects deduced from high field 'H-NMR studies. J . Biomol. Struct. Dyn. 8, 331-357. (31) Kumar, S., Joseph, T., Singh, M. P., Bathini, Y., and Lown, J. W. (1992) Structure and dynamics of ligand-template interactions of topoisomerase inhibitory analogs of Hoechst 33258: high-field 'HNMR and restrained molecular mechanics studies. J . Biomol. Struct. Dyn. 9, 853-880. (32) Middleton, R. W., and Wibberley, D. G. (1980) Synthesisof imidazo[4,5-b]-and [4,5-c]pyridines. J . Heterocycl. Chem. 17, 1757-1760. (33) Hisano, T., Ichikawa, M., Tsumoto, K., and Tasaki, M. (1982) Synthesis of benzoxazoles, benzothiazoles, and benzimidazoles and evaluation of their antifungal, insecticidal and herbicidal activities. Chem. Pharm. Bull. 30, 2996-3004. (34) Bathini, Y., and Lown, J. W. (1990) Convenient routes tosubstituted benzimidazoles and imidazo[4,5-b]pyridines using nitrobenzene as oxidant. Synth. Commun. 20,955-963. (35) Coville, N. J., Neuse, E. W. (1997) Oxidative cyclodehydrogenation of aromatic bis(o-aminoanils). J . Org. Chem. 42, 3485-3491. (36) Parker, E. D., and Shive, W. (1947) Substituted 2-picolines derived from 6-amino-2-picoline. J . Am. Chem. SOC. 69, 63-66. (37) Sklenar, V., and Bax, A. (1987) A new water suppression technique for generating pure-phase spectra with equal excitation over a wide bandwidth. J . Magn. Reson. 75, 378-383. (38) States, D. J., Haberkorn, R. A., and Reuben, D. J. (1982) A twodimensional nuclear Overhauser experiment with pure absorption phase in four quadrants. J. Magn. Reson. 48, 286-292. (39) Keeler, J., and Neuhaus, D. (1985) Comparison and evaluation of methods for two-dimensional NMR spectra with absorption-mode lineshapes. J . Magn. Reson. 63, 454-472. (40) Otting, G., Widmer, H., Wagner, G., and Wuthrich, K. (1986) Origin of tl and t 2 ridges in 2D NMR spectra and procedures for suppression. J . Magn. Reson. 66, 187-193. (41) Reid, B. R. (1987) Sequence-specific assignments and their use in NMR studies of DNA structure. Q.Reu. Biophys. 20, 1-34. (42) Patel, D. J., Shapiro, L., and Hare, D. (1987) Nuclear magnetic resonance and distance geometry studies of DNA structures in solution. Annu. Reu. Biophys. Biophys. Chem. 16, 423-454. (43) Gronenborn, A. M., and Clore, G. M. (1985) Investigation of the solution structures of short nucleic acid fragments by means of nuclear Overhauser enhancement measurements. Prog. Nucl. Magn. Reson. Spectrosc. 17, 1-32. (44) Leupin, W., Chazin, W. J., Hyherts, S., Denny, W. A.,and Wuthrich, K. (1986) NMR studies of the complex between the decadeoxynucleotide d-(GCATTAATGQ2 and a minor-groove-binding drug. Biochemistry 25, 5902-5910. (45) Patel, D. J., and Shapiro, L. (1986)Sequence-dependent recognition of DNA duplexes. Netropsin complexation to the AATT site of the d(GGAATTCC) duplex in aqueous solution. J . Bioi. Chem. 261, 123&1240.
NMR Analysis of Hoechst Analog-DNA Complex (46) Lee, M., Chang, D.-K., Hartley, J. A., Pon, R. T., Krowicki, K., and Lown, J. W. (1988) Structural and dynamic aspects of binding of a prototype lexitropsin to the decadeoxynucleotide d(CGCAATTGCG), deduced from high-resolution 1H NMR studies. Biochemistry 17,445-455. (47) . . Pelton. J.G..andWemmer.D.E. (1990)Bindinr!modesofdistamvcin A with d(CGCAAATTTGCG)Z.determinedby two-dimensional NMR. J. Am. Chem. SOC. 112, 1393-1399. (48) Borah, B.,Roy, S., Zon, G., andCohen, J. S.(1985) Unique pyrimidine 2D-COSY aromatic cross-peaks as monitors of pyrimidine environmenta and mobility in oligc- and polynucleotides. Biochem. Biophys. Res. Commun. 133, 380-388. (49) Sutherland, I. 0. (1981) The investigation of the kinetics of conformational changes by nuclear magnetic resonance spectroscopy. Ann. Rep. NMR Spectrosc. 4, 71-225. (50) Gunther, H., Ed. (1984) NMR Spectroscopy, pp 234-280, Wiley, New York. (51) Barlow, D. J., and Perkins, T. D. J. (1990)Applicationsof interactive computer graphics in analyses of bimolecular structures. Nat. Prod. Rep. 7, 311-326. (52) Patel, D. J. (1979) Nuclear magnetic resonance studies of drugnucleic acid interactions a t the synthetic DNA level in solution. Acc. Chem. Res. 12, 118-125. (53) Harris, T. M., Stone, M. P., and Harris, C. M. (1988) Applications of NMR spectroscopy to studies of reactive intermediates and their interactions with nucleic acids. Chem. Res. Toricol. 1, 79-96. (54) Comings, D. E. (1975) Mechanisms of chromosome binding. VII. Hoechst 33258-DNA interaction. Chromosoma 52, 229-243. (55) Harshman, K. D., and Dervan, P. B. (1985) Molecular recognition of B-DNA by Hoechst 33258. Nucleic Acids Res. 13, 4825-4835. (56) Pjura, P. E., Grzeskowiak, K., and Dickerson, R. E. (1987) Binding of Hoechst 33258 to the minor groov of B-DNA. J. Mol. Biol. 197, 257-271. (57) Teng, M.-K., Usman, N., Frederick, C. A., and Wang, A. H. J. (1988) The molecular structure of the complex of Hoechst 33258 and the
Chem. Res. Toxicol., Vol. 5, No. 5, 1992 607 DNA dodecamer d(CGCGATATCGCG). Nucleic Acids Res. 16, 2671-2690. (58) Parkinson, J. A., Barber, J., Douglas, K. T., Rosamund, J., and Sharples, D. (1990)Minor-groove binding of the self-complementary duplex d(CGCGAATTCGCG)*by Hoechst 33258 a high-field NMR study. Biochemistry 29, 10181-10190. (59) Searle, M. S., and Embrey, K. J. (1990)Sequence-specificinteraction of Hoechst 33258 with the minor groove of an adenine-tract DNA duplex studied in solution by 1H NMR spectroscopy. Nucleic Acids Res. 18, 3753-3762. (60) Fede, A,, Labhardt, A., Bannwarth, W., and Leupin, W. (1991) Dynamics and binding mode of Hoechst 33258 to d(GTGAATTCCAC)z in a 1:l solution complex as determined by two-dimensional 'H-NMR. Biochemistry 30, 11377-11388. (61) Rao, K. E., and Lown, J. W. (1991) Molecular recognition between ligands and nucleic acids: DNA binding characteristics of analogues of Hoechst 33258 designed to exhibit altered base and sequence recognition. Chem. Res. Toxicol. 4, 661-669. (62) Feigon, J., Denny, W. A,, Leupin, W., and Kearns, D. R. (1984) Interactions of antitumor drugs with natural D N A 'H NMR study of binding mode and kinetics. J. Med. Chem. 27, 450-465. (63) Pullman, A., and Pullman, B. (1981)Molecular electrostatic potential of the nucleic acids. Q. Rev. Biophys. 14, 289-297. (64) Roberts, R. J. (1990) Restriction enzymes and their isoschizomers. Nucleic Acids Res. 18, 2331-2365.
Registry No. 1, 142764-75-8;2, 33259-72-2; 3, 129011-99-0; 4, 142764-76-9; 5, 142764-77-0; 6, 142764-78-1; 7, 142764-79-2; 4-MeOCsH4CH0, 123-11-5; MeOCH2Cl, 107-30-2; PhC000COPh, 94-36-0; 2-aminoS-methy1-3-nitropyridine, 21901-29-1; N-bromosuccinimide, 128-08-5; 2,3-diamino-6-(4-methylpiperazin-1-yl)pyridine, 126824-12-2.
