The Journal of Organic Chemistry
Article
Autopol V polarimeter. Mass spectra for reaction intermediates were
obtained using Synapt G2 high definition mass spectrometry.
Solid Phase Synthesis of Bimodal Cα-PNA Oligomers. The
synthesis of PNA oligomers was carried out using the solid phase
synthesis protocol using the Boc strategy on MBHA (4-methyl-
benzhydryl amine) resin with 0.20 mmol/g loading in a glass-
sintered flask. The deprotection of the N-t-Boc group from the resin-
bound lysine with 50% TFA in dichloromethane (DCM) (3 × 15
min) was followed by washing with DCM and dimethylformamide
experiment was repeated at least twice. The normalized absorbance at
260 nm was plotted as a function of the temperature. The Tm was
determined from the first derivative of normalized absorbance with
respect to temperature and is accurate to ±1.0 °C. The data were
processed using OriginPro 8.5, data were fitted by the sigmoidal
curve, and the functions used were Boltzmann for one-face binding
and biphasic dose response for two-face binding. The concentrations
of all oligonucleotides were calculated on the basis of absorbance from
the molar extinction coefficients of the corresponding nucleobases
16
2
2
2
(
DMF) (3 × 10 mL) to give a TFA salt of amine, which was
that is T = 8.8 cm /μmol; C = 6.6 cm /μmol; G = 11.7 cm /μmol;
2
29
neutralized using 10% DIPEA in DCM (3 × 10 min) to liberate free
amine. After washing with DCM and DMF (3 × 10 mL), the free
amine was coupled with carboxylic acid of the incoming monomers
and A = 13.7 cm /μmol.
CD Spectroscopy. CD spectra were recorded on a JASCO J-815
spectropolarimeter connected with a Peltier. The calculated amounts
of PNA oligomers and the complementary DNA were mixed together
in a stoichiometric ratio (1:1 for duplex) in sodium cacodylate buffer
(10 mM) containing NaCl (10 mM); pH 7.2 to achieve a final strand
concentration of 10 μM for each strand. The samples were annealed
by heating at 90 °C for 10 min followed by slow cooling to room
temperature for at least 8−10 h. The cooled samples were transferred
to the refrigerator for at least 8−12 h. To record the CD spectra of
PNA/DNA duplexes and single stranded PNAs, the temperature was
maintained at 10 °C. The CD spectra were recorded as an
accumulation of three scans from 300 to 190 nm using a 1 mm
quartz cell, a resolution of 0.1 nm, bandwidth of 1 nm, sensitivity of 2
m deg, response of 1 s, and a scan speed of 50 nm/min.
(
1−6, 3 equiv) in DMF (500 μL) using HOBt (3 equiv), HBTU (3
equiv), and DIPEA (15 μL). After coupling reaction for 6 h, the
reagents were removed by filtration and the resin was washed with
DMF.
Solid Phase Click Reaction. After consecutive coupling of six units
of monomer 1 (Scheme 2), the resin bound oligomer (10 mg resin,
0
.20 mmol/g) having azido sidechain was subjected to the click
reaction with nucleobase C-alkyne 9 (4.2 mg, 6 equiv) in the presence
of CuI (12 mg, 18 equiv), ascorbic acid (3.0 mg, 5 equiv), and DIPEA
(
15 μL) in DMF:pyridine (1:1, v/v, 100 μL). The reaction was
maintained for 5 min in the microwave at 65 °C and 25 W and then
4 h at room temperature. Excess reagents were removed by filtration,
2
and the resin was washed with DMF, DCM, MeOH, and saturated
EDTA. The bm-Cα-PNA 2 was synthesized similarly by stepwise
coupling with desired monomers (1−4) followed by the click reaction
ITC Study of Complexes of bm-Cα-PNA 1 with Comple-
mentary DNA. Thermodynamic properties of bm-Cα-PNA 1
complexation to cDNA were determined using ITC. The hybrid-
ization and binding studies of bm-Cα-PNA 1 with its complementary
DNA oligonucleotides were carried out on Malvern MicroCal PEAQ
ITC instrument. All titration experiments were performed at 15 °C in
12a
at each step with appropriate nucleobase alkynes 9.
Cleavage of the bm-Cα-PNA Oligomers from Solid Support. The
MBHA resin (10 mg) after assembly of bm-Cα-PNA oligomers was
stirred with thioanisole (20 μL) and 1,2-ethanedithiol (8 μL) in an ice
bath for 10 min. TFA (200 μL) was added and cooled in an ice bath.
TFMSA (16 μL) was added slowly with stirring, and the reaction
mixture was stirred for 1.5−2 h at room temperature. The resin was
removed by filtration under reduced pressure and washed twice with
TFA, and the filtrate was evaporated on a rotary evaporator at
ambient temperature. The filtrate was transferred to the microfuge
tube, and the peptide was precipitated with cold diethyl ether. The
peptide was isolated by centrifugation, and the precipitate was
10 mM sodium cacodylate buffer (pH 7.2) containing NaCl (10
mM). The buffer was used to prepare all solutions used in the
experiment. The sample cell was loaded with bm-Cα-PNA 1 solution,
and the reference cell contained only the buffer. The syringe was
loaded with DNA solution (40 μL). The instrument was equilibrated
at 15 °C until the baseline was flat and stable. The stirring speed was
maintained at 700 rpm during the titrations.
Dilution experiments (DNA vs buffer) were performed at the same
condition, and the measured heat of dilution was subtracted from the
corresponding sample experiment. The binding isotherm was fitted to
a “one set of binding sites” model for duplexes and triplexes. However,
for double duplex of triplex, data best fitted in two sets of binding sites
dissolved in 40% MeNH solution to deprotect the isobutyl protecting
2
group of nucleobases at rt for 8 h and again concentrated on speed
vacuum, filtered, and purified by HPLC.
using MicroCal data analysis software to determine K , ΔG, ΔH, or
Purification of the PNA Oligomers by RP-HPLC. The purification
of PNAs was carried out on a Dionex ICS 3000 HPLC system with
semipreparative BEH130 C18 (10 × 250 mm) column using solvents
D
−
ΔS for all binding experiments. N corresponded to the ratio of
number of nucleobases involved in binding from DNA and the
number of nucleobases involved in binding from bm-Cα-PNA 1.
water and acetonitrile with composition A: 0.1% TFA in CH CN/
3
H O (5:95) and B = 0.1% TFA in CH CN/H O (1:1). The gradient
2
3
2
for elution was 100% A to 100% B in 20 min, with a flow rate of 2
mL/min. The HPLC elutions were monitored at 220 and 254 nm
wavelengths. The purity of oligomers was checked by reinjecting the
sample on the C18 analytical column.
Characterization of the PNA Oligomers. MALDI-TOF mass
spectrometry was used to confirm the integrity of the synthesized
PNA oligomers using sinapinic acid (3,5-dimethoxy-4-hydroxycin-
namic acid), 2,5-dihydroxybenzoic acid, or α-cyano-4-hydroxycin-
namic acid as the matrix.
Temperature-UV Absorbance Measurements. UV-melting
experiments were carried out on a Varian Cary 300 UV
spectrophotometer equipped with a Peltier. The samples for Tm
measurement were prepared by mixing the calculated amounts of
respective oligonucleotides in the stoichiometric ratio (1:1, duplex) in
sodium cacodylate buffer (10 mM) and NaCl (10 mM); pH 7.2 to
achieve a final strand concentration of 3 μM for each strand. The
samples were annealed by heating at 90 °C for 10 min followed by
slow cooling to room temperature for at least 8−10 h and then
refrigerated for at least 12−24 h. The samples (500 μL) were
transferred to the quartz cell and equilibrated at the starting
temperature for 5 min. The OD at 260 nm was recorded in steps
from 20−92 °C with temperature increment of 0.5 °C. Each melting
ASSOCIATED CONTENT
■
*
sı Supporting Information
Synthesis schemes and characterization data (NMR, MS
of all new compounds, HPLC, MALDI-TOF of PNA
oligomers, UV-melting curves, CD spectra, ITC data,
■
Corresponding Author
Krishna N. Ganesh − Department of Chemistry, Indian
Institute of Science Education and Research (IISER) Pune,
Pune 411008, India; Department of Chemistry, Indian
M
J. Org. Chem. XXXX, XXX, XXX−XXX
Gupta, Manoj Kumar
