S. Kumar et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
[
pyridine, DCM (dichloromethane)]. EtOH (Ethanol) was first dis-
and added to an oligomeric duplex solution with final concentra-
0
0
tilled with sodium metal and then redistilled over magnesium
turnings. Reactions were conducted under N
unless otherwise noted.
tion of 1
l
M/strand (for d[5 -A12-x-T12-3 ]) or 1
l
M/duplex (for
0
0
2
using a dry solvent,
d[5 -G
A
2 6
T
6
C
2
-3 ]). The fluorescence of the solution in each well
was measured and normalized to a 100% relative fluorescence.
An aliquot of the stock solution of ligand (5–200 M) was added
l
2
.2. Nucleic acids
The 12-mer duplex d[5 -A12-x-T12-3 ] (x = hexaethylene glycol)
into the pre-incubated solution of DNA+TO and the fluorescence
was measured after 5 min at 20 °C. The FID assay were conducted
in triplicate. For FID titrations, the addition of ligand was continued
until no more change in the fluorescence was detected. For FID
assay, the final concentrations were corrected for dilution (less
than 5% of the total volume). Fluorescence readings were reported
as a percentage of fluorescence relative to the control wells. The
reference fluorescence was defined as [TO+DNA] providing 100%
fluorescence and [TO] alone provides 0% fluorescence. The reported
error for FID assay was standard error which is calculated from
three independent experiments.
0
0
oligonucleotide was synthesized on an Applied Biosystem 8890
using standard phosphoramidite chemistry and purified by HPLC
on a Gen-Pak FAX (4.6 ꢂ 100 mm) ion exchange column, eluting
3
with buffer A (25 mM Tris HCl, 1 mM EDTA, 10% CH CN, pH 8.0)
from 98% to 50% and buffer B (25 mM Tris HCl, 1 mM EDTA, 1 M
NaClO , 10% CH CN, pH 8.0) from 2% to 50% in a 15 min process.
All other oligonucleotides were purchased from IDT DNA
Coraville, IA), the concentrations of which were determined by
4
3
(
UV using the following extinction coefficients [L/(moleꢃcm)]:
0
0
0
e
260 = 157,900 for d[5 -G
2
A
6
T C
6 2
-3 ],
e
260 = 59,844 for d[5 -A12-x-
4
4
. Results and discussion
0
0
0
T
12-3 ],
e
260 = 605,700 for d[5 -A30
T
30-3 ].
.1. Synthesis of neomycin dimers
2
.3. Instrumentation
1H NMR spectra were collected on a Bruker 500 MHz FT-NMR
The library of neomycin dimers was synthesized using
thiourea- and urea-based coupling chemistry. Hexa-N-Boc deoxy-
neomycin-5 -amine with short and long (Fig. 1) linkers were syn-
thesized from commercially available neomycin sulfate using pre-
viously published methods.
used in the synthesis of neomycin dimers were prepared by react-
ing commercially available diamines with TCDP (1,1 -thiocar-
bonyldi-2(1H)-pyridone) in almost quantitative yields (see
Supplementary information Table S1). The hexa-N-Boc deoxy-neo-
mycin-5 -amines were then reacted with diisothiocyanates and
diisocyanates in the presence of pyridine (dry) and DMAP (catalytic
amounts) for 12 h at room temperature to produce the Boc pro-
tected neomycin dimers with various linker lengths (DPA71–
DPA80, see Supporting information Scheme S1). The acid labile
Boc protected neomycin dimers were then reacted with 4 M
HCl/dioxane for 30 min to obtain the neomycin dimers as their
hydrochloride salts in good yields (60–71% yield for the two steps)
(Fig. 1).
spectrometer, and the MS (MALDI-TOF) spectra were collected
using Bruker Omniflex MALDI-TOF mass spectrometer.
00
a
1
3,18,19,21,22
Isothermal titration calorimetric measurements were performed
on a MicroCal VP-ITC (MicroCal, Inc. Northampton, MA). FID assays
were performed in 96-well plates (Black w/flat bottom, Greiner
Bio-one, Monroe, NC) on a Genios Multi-Detection Microplate
Reader, TECAN (McLean, VA) with Magellan software.
The diisothiocyanate linkers
0
0
0
3
3
. Methods
.1. Isothermal titration calorimetry (ITC)
In a typical experiment, an aliquot (9
125 M in 100 mM KCl, 10 mM SC, 0.5 mM EDTA, pH 6.8) was
injected at 25 °C into an isothermal sample chamber containing
an oligonucleotide duplex solution (1.42 mL, 4 M/duplex in
00 mM KCl, 10 mM SC, 0.5 mM EDTA, pH 6.8) via a rotary syringe
300 rpm, vol. 296 L). The interval between each injection was
00 s, the duration of each injection was 10 s, and the initial delay
lL) of neomycin dimer
(
l
l
1
(
l
4.2. FID assay reveals longer linker neomycin dimers are
stronger binder than short linker dimers
3
prior to the first injection was 60 s. Injection of neomycin dimer at
identical concentrations into a buffer solution at 25 °C was used as
a blank. Each injection generated a heat burst curve of microcalo-
ries per second. The area under each heat burst curve was deter-
mined by integration using the Origin (version 5.0, MicroCal, Inc.
Northampton, MA) software to obtain a measure of the heat asso-
ciated with that injection. The heat associated with each drug-buf-
fer injection was subtracted from the corresponding heat
associated with each drug-DNA injection to yield the heat of drug
binding for that injection. The final corrected injection heats were
plotted as a function of molar ratio ([drug]/[DNA]) and either fitted
with a single binding site model or with two independent binding
site models. In the case of two binding site model, the heat curves
were fit by allowing free floating of N1 and N2 (binding stoichio-
metric ratios, drug/DNA). The fits were optimized until no more
change in the chi-squared value was observed.
A single point FID assay23 was performed with the neomycin
dimers and DNA duplexes ([5 -G A T C -3 ] and [5 -A30T30-3 ]).
2 6 6 2
DNA duplexes were incubated with thiazole orange, an exogenous
intercalator, at a stoichiometric ratio of 1:0.5 (DNA base pair: thi-
azole orange) followed by the addition of neomycin dimers. The
0
0
0
0
binding site size for neomycin dimer is ꢁ12 base pairs/molecule,
0
therefore we used molar ratios of 1:1 and 1:5 for [5 -G
2
A
T
6 6
2
C -
0
0
0
3 ] and [5 -A30T30-3 ] respectively. The decrease in fluorescence
intensity can be directly correlated with the affinity of neomycin
dimers (Fig. 2). In all the studies carried out, including ITC and
FID, neomycin a weak binder of AT-rich DNA served as a control.
There are few important trends that emerged from the FID study:
(1) neomycin dimers, in general, exhibit stronger binding to AT-
rich DNA than neomycin (2) neomycin dimers with long linkers,
specifically L = 16–22, exhibit 30–50 fold higher affinity to AT rich
DNA sequences than neomycin. (3) Neomycin dimers (L = 17–22)
displayed higher affinity than short/rigid linker based neomycin
3
.2. Fluorescence intercalator displacement assay (FID)
0
0
2 6 6 2
dimers (L = 1–12). In the case of d[5 -G A T C -3 ], DPA312
FID assay for oligomeric DNA duplexes were conducted in a 96
(L = 19) attenuated the fluorescence intensity by a factor of ꢁ25
than DPA71 (L = 1). (4) The activity trend observed for neomycin
dimers is similar toward both the AT-rich DNA target sequences,
however there is a slight difference in % fluorescence change which
well plate. For FID single point assay, a thiazole orange (TO) solu-
tion (0.5 equiv/base pair, 200 L) in sodium cacodylate buffer
100 mM KCl, 10 mM SC, 0.5 mM EDTA, pH 6.8) was prepared
l
(