Optimized Interactions in a DNA Minor Groove Complex
A R T I C L E S
the d(GCGAATTCGC)2 oligomer DNA from IDT was used for Tm
and CD studies. Calorimetric experiments were performed with
d(GCCGCAATTCGC/iSp18)2 (iSp18 stands for internal spacer 18
which is the hexaethylene glycol linkage) hairpin duplex DNA (Figure
1). Solutions were prepared in degassed cacodylic acid buffer containing
0.01 M cacodylic acid, 0.001M EDTA, 0.1 M NaCl adjusted to pH
6.25. The concentration of the DNA solutions was determined spec-
trophotometrically at 260 nm using extinction coefficients per nucleotide
of AATT duplex DNA. The extinction coefficients were calculated on
a per strand basis by the nearest-neighbor method and divided by the
number of nucleotides per strand.17
were performed essentially as previously described.18 Briefly, reactions
were conducted in a total volume of 10 µL. Samples (3 µL) of the
labeled DNA fragments were incubated with 5 µL of compound solution
for 30 min of incubation. Digestion was initiated by the addition of 2
µL of a DNase I solution whose concentration was adjusted to yield a
final enzyme concentration of ∼0.01 U/mL in the reaction mixture.
After 3 min, the reaction was stopped by freeze-drying. Samples were
lyophilized and resuspended in 5 mL of an 80% formamide solution
containing tracking dyes. The DNA samples were then heated at 90
°C for 4 min and chilled in ice for 4 min prior to electrophoresis.
Thermal Melting (Tm). Tm experiments were conducted with a Cary
300 UV-visible spectrophotometer in 1-cm quartz cuvettes. The
absorbance of the DNA-compound complex was monitored at 260
nm as a function of temperature and DNA without compound was used
as a control. Cuvettes were mounted in a thermal block, and the solution
temperatures were monitored by a thermistor in a reference cuvette
with a computer-controlled heating rate of 0.5 °C/min. Experiments
were generally conducted at a concentration of 2 × 10-5 M base pair
for polydA‚polydT and 3 × 10-6 M duplex for d(GCGAATTCGC)2.
For experiments with complexes a ratio of 0.3 compound per base pair
for polydA‚polydT and ratio of one compound per oligomer duplex
for d(GCGAATTCGC)2 was generally used.
Preparation of Reversed Amidines: General Procedure. The new
compounds in this report were prepared following the general method
previously developed by us.16 The general procedure used follows along
with characterization data for DB1215 (structure in Table 1). The
reaction scheme employed and the data for all the new compounds
may be found in the Supporting Information.
Free Base Preparation. A chilled solution of the diamine (1.28
mmol) in dry MeCN (10 mL) and dry EtOH (15 mL) was treated with
S-(2-naphthylmethyl)thiobenzimidate hydrobromide (2.69 mmol). After
stirring at rt for 24 h, the solvent was evaporated to dryness, leaving
an oily residue. Treatment with ether gave the reversed amidine as the
hydrobromide salt, which was dissolved in EtOH, basified with 1 N
NaOH, extracted with EtOAc, and dried over Na2SO4; finally the solvent
was evaporated to dryness, giving the free base of the reversed amidine
in an analytically pure form.
Circular Dichroism (CD). CD spectra were obtained on a computer-
controlled Jasco J-710 spectrometer in 1-cm quartz cell. The DNA was
added to cacodylate buffer in a 1-cm quartz cuvette and scanned over
a selected wavelength range. The desired ratios of compound to DNA
were obtained by adding compound to the cell containing a constant
amount of DNA at 25 °C. The compounds at increasing ratios were
then titrated into DNA, and the complexes were rescanned under same
conditions.
Hydrochloride Salt. An ice-bath-cold solution of the free base in
dry EtOH was treated with HCl gas for 5-10 min, and the reaction
mixture was kept stirring for 5 h. Later, the solvent was concentrated
to near dryness, and then the reaction mixture was diluted with ether.
SPR-Biosensor Binding Determinations. Surface plasmon reso-
nance (SPR) measurements were performed with a four-channel
BIAcore 2000 optical biosensor system (BIAcore Inc.). 5′-Biotin-labeled
DNA samples (Figure 1) were immobilized onto streptavidin-coated
sensor chips (BIAcore SA) as previously described.19 Three flow cells
were used to immobilize the DNA oligomer samples, while a fourth
cell was left blank as a control. The SPR experiments were performed
at 25 °C in filtered, degassed, 10 mM cacodylic acid buffer (pH 6.25)
containing 100 mM NaCl, 1 mM EDTA. Steady-state binding analysis
was performed with multiple injections of different compound con-
centrations over the immobilized DNA surface at a flow rate of 25
µL/min and 25 °C. Solutions of known ligand concentration were
injected through the flow cells until a constant steady-state response
was obtained. Compound solution flow was then replaced by buffer
flow, resulting in dissociation of the complex. The reference response
from the blank cell was subtracted from the response in each cell
containing DNA to give a signal (RU, response units) that is directly
proportional to the amount of bound compound. The predicted
maximum response per bound compound in the steady-state region
(RUmax) was determined from the DNA molecular weight, the amount
of DNA on the flow cell, the compound molecular weight, and the
refractive index gradient ratio of the compound and DNA, as previously
described.20 The number of binding sites and the equilibrium constant
were obtained from fitting plots of RU versus Cfree. Binding results
from the SPR experiments were fit with either a single-site model (K2
) 0) or with a two-site model:
DB1215: 2,5-Bis[4-(2-naphthylimino)amino)phenyl]pyrrole: yield
61%, mp 275-277 °C; H NMR (DMSO-d6) δ 6.52 (s, 2H), 6.72 (br
s, 4H), 6.96 (d, J ) 8.1 Hz, 4H), 7.57-7.61 (m, 4H), 7.75 (d, J ) 8.1
Hz, 4H), 6.96-6.98 (m, 6H), 8.12 (d, J ) 8.1 Hz, 2H), 8.54 (s, 2H),
11.08 (br s, 1H). 13C NMR δ 154.3, 147.3, 133.7, 132.8, 132.7, 132.2,
128.6, 127.5, 127.3, 127.0, 126.7, 126.4, 124.7, 121.9, 106.4.
1
1
Salt: mp 247-249 °C; H NMR (DMSO-d6) δ 6.79 (s, 2H), 7.53
(d, J ) 8.1 Hz, 4H), 7.69-7.78 (m, 4H), 7.95 (d, J ) 8.1 Hz, 2H),
8.06-8.16 (m, 8H), 8.20 (d, J ) 8.1 Hz, 2H), 8.64 (s, 2H), 9.18 (br s,
2H), 10.01 (br s, 2H), 11.65 (br s, 1H), 11.71 (br s, 2H). HRMS Calcd
for C38H30N5 m/s 556.2501; observed 556.2498. Anal. Calcd for
C38H29N5‚2HCl‚2.5H2O: C 67.75, H 5.38, N 10.39. Found: C 67.68,
H 5.24, N 10.39.
Purification and Radiolabeling of DNA Restriction Fragments
and DNase I Footprinting. The plasmid was isolated from Escherichia
coli by standard sodium dodecyl sulfate-sodium hydroxide lysis and
purified by banding in CsCl-ethidium bromide gradients. The 265 bp
DNA fragment was prepared by 3′-[32P]-end labeling of the EcoRI-
PVuII double digest of the pBS plasmid (Stratagene) using R-[32P]-
dATP and AMV reverse transcriptase. The products were separated
on a 6% polyacrylamide gel under nondenaturing conditions in TBE
buffer (89 mM Tris-borate pH 8.3, 1 mM EDTA). After autoradiog-
raphy, the requisite band of DNA was excised, crushed, and soaked in
water overnight at 37 °C. This suspension was filtered through a
Millipore 0.22 mm filter, and the DNA was precipitated with ethanol.
Following washing with 70% ethanol and vacuum drying of the
precipitate, the labeled DNA was resuspended in 10 mM Tris adjusted
to pH 7.0 containing 10 mM NaCl. DNase I footprinting experiments
r ) (K1‚Cfree + 2K1‚K2‚Cfree2)/(1 + K1‚Cfree + K1‚K2‚Cfree
)
(1)
2
where r represents the moles of bound compound per mole of DNA
hairpin duplex, K1 and K2 are macroscopic binding constants, and Cfree
is the free compound concentration in equilibrium with the complex.
(16) (a) Arafa, R. K.; Brun, R.; Wenzler, T.; Tanious, F. A.; Wilson, W. D.;
Stephens, C. E.; Boykin, D. W. J. Med. Chem. 2005, 48, 5480-5488. (b)
Arafa, R. K.; Brun, R; Werbovetz, K. A.; Wilson W. D.; and Boykin D.
W. Heterocycl. Comm. 2004, 10, 423-428. (c) Stephens, C. E.; Tanious,
F.; Kim, S.; Wilson, W. D.; Schell, W. A.; Perfect, J. R.; Franzblau S.G.;
Boykin, D. W. J. Med. Chem. 2001, 44, 1741-1748.
(18) Bailly, C.; Kluza, J.; Martin, C.; Ellis, T.; Waring, M. J. Methods Mol.
Biol. 288, 2004, 319-342.
(19) Nguyen, B.; Tanious, F. A.; Wilson, W. D. Methods 2006, in press.
(20) Davis, T. M.; Wilson, W. D. Methods Enzymol. 2001, 340, 22-51.
(17) Fasman, G. D. Handbook of Biochemistry and Molecular Biology, Nucleic
Acids, 3rd ed.; CRC Press: Cleveland, OH, 1975; Vol. 1, p 589.
9
J. AM. CHEM. SOC. VOL. 129, NO. 17, 2007 5691