V.V. Krishnan et al. / Chemical Physics Letters 689 (2017) 148–151
149
2
. Materials and methods
For each value of
a calculated, the dihedral angle b was changed
ꢀ180° to +180° in steps of 5 °C, leading a matrix of 33 ꢁ 33.
2.1. Synthesis
3
. Results
All chemicals were purchased from Sigma Aldrich or MERCK
and were used as received without further purification. Ortho-
DBET: N, N-dibenzyl-o-methyl benzamide: To a solution of o-toluic
acid (2.08 g, 15.3 mmol) in methylene chloride (28 mL), a catalytic
amount of DMF (2 drops) was added with stirring. Freshly distilled
thionyl chloride (2.3 mL, 31.0 mmol) was added to the flask, and
the resulting solution was gently refluxed for two hours. The reac-
tion mixture was concentrated under reduced pressure to remove
excess thionyl chloride using a warm water bath (55 °C). The crude
acid chloride was redissolved in methylene chloride (28 mL), and
dibenzyl amine (5.88 mL, 30.6 mmol) was carefully added to the
flask, dropwise, over the course of 2–3 min (Note: exothermic, pro-
duces gaseous HCl). The solution was allowed to stir at room tem-
perature overnight. The organic layer was washed with water (30
mL) followed by 5% HCl (30 mL), 10% NaOH (30 mL), finally with
Fig. 1 shows the variable temperature NMR experiments of the
ortho (o), meta (m) and para (p) – DBET molecules over the temper-
ature range recorded by the variable temperature, between 1 °C
and 51 °C. At low temperature (1 °C) the chemical shifts of the
chemically exchanging methylene protons at the two sites are of
m-DBET are at 5.45 ppm and 5.15 ppm, which reaches an interme-
diate exchange region at the high temperature (51 °C) (Fig. 1, panel
m). The p-DBET also shows a similar chemical exchange behavior
with the two distinct sites at 5.65 ppm and 5.38 ppm at low tem-
perature (1 °C) and a coalesced single peak at higher temperatures
(
51 °C) (Fig. 1, panel p). At high temperature (51 °C), o-DBET mole-
cule shows a broad resonance (5.32 ppm) representing an interme-
diate exchange and a sharp resonance (4.77 ppm) similar to a third
high-energy conformation observed previously in the case o-DEET
2 4
brine (30 mL), and dried with anhydrous Na SO . The solution
[
11]. Upon lowering the temperature, a complex peak pattern
was filtered and concentrated under reduced pressure to yield a
granular, slightly yellow solid before being dissolved in a minimum
amount of hot hexanes. The solution was allowed to cool, and crys-
tals were collected and dried to yield 1.98 g (41% overall) of the
pure N, N-dibenzyl-o-toluamide.
Each DEET analog was synthesized by following the above
method and purified by crystallization (hot hexanes) or column
chromatography (10% ethyl acetate in hexanes and 230–400 mesh
silica gel) as required. Approximately 10 mg of each sample was
emerges (Fig. 1, panel o). When the probe temperature reaches
approximately below 10 °C, in addition to the splitting of down-
field resonance (5.78 ppm), the upfield peak shows a complex line
shape (centered at 4.81 ppm).
The two distinct set of exchange peaks from o-DBET are seen in
the two-dimensional exchange spectrum (Fig. 2) recorded at 1 °C
with a mixing time of 300 ms. Two sets of spin systems identified
2
are as follows: an (AX) spins system (dAX = 400.2 Hz and JAX = 12
Hz) and an (AB) spin system (dAB = 56.2 Hz and JAB = 16 Hz). These
2
3
dissolved in CDCl (total volume of 600 lL) for the NMR experi-
NMR parameters are determined by simulating the 1D NMR spec-
trum in the absence of chemical exchange (Fig. S1). Contribution
from zero-quantum coherences in the EXSY experiment is
expected to be significantly reduced due to long mixing time
ment. Each NMR tube was glass-sealed by first applying the
freeze-thaw technique using liquid nitrogen to expel any dissolved
air/oxygen, followed by sealing them using a small butane torch.
(
300 ms) and the effect of solvent viscosity over the temperature
range (Chloroform viscosity 0.699 mPa s at 0 °C–0.389 mPa s at
2.2. NMR spectroscopy
6
0 °C) is also expected to be insignificant [11].
Increasing the temperature alters the kinetics of the sub-spin
1
All the NMR experiments were performed in a 400 MHz ( H res-
systems separately (Fig. 1). Fig. 3 shows variable temperature
NMR experiments of o-DBET (red lines) along with the correspond-
ing line shape fitted using a three-site exchange model (black
lines). At temperatures below 11 °C, a three-site model was unable
to reproduce the line shape (Fig. 3). In contrast, the temperature-
dependence of the m-DBET and p-DBET spectra follows the charac-
teristic features corresponding to a two-site chemical exchange
process. Fig. 4 shows the Eyring analysis plot (ln (kex/T) vs. 1000/
onance frequency) VNMRS spectrometer (Varian-Agilent) and
using a one-NMR probe. The probe temperature was calibrated
using MeOH [13]. The probe temperature was varied from 1 °C to
5
5 °C (in steps of 3 °C). One-dimensional, variable-temperature
experiments were performed with 16 transients over 16 K complex
points after calibrating the 90° pulse at each temperature. Samples
were equilibrated for 20 min at each temperature, and a relaxation
delay of 30 s was used between the transients. WinDNMR [14,15]
ꢀ1
was used to estimate the exchange rates (kex
s ). A three-site
exchange model was used to fit the variable temperature line
shape data of o-DBET, and a two-site exchange model was used
to fit for m-DBET and p-DBET molecules. The constant line width
of 6 Hz was used for all the fits. In the three-site model with a slow
exchange between the third spin to first two spins was assumed. It
is necessary to adopt a three-site model for o-DBET, due to chem-
ical shift overlap between the spins in the up-field part of the
exchange spectrum. The activation energy was estimated following
the Eyring analysis (exchange rates vs. inverse of temperature in
K).
o
m
p
5
1°C
Two-dimensional exchange spectroscopy (EXSY) [16] was per-
formed at three different temperatures (1 °C, 25 °C, and 55 °C)
using a standard Nuclear Overhauser Effect Spectroscopy (NOESY)
pulse sequence [17] using the procedure described previously [11].
Molecular mechanics calculations were performed using Avo-
1
°C
ppm
Fig. 1. Variable temperature DNMR spectra. (o) Temperature dependence of the
NMR line shapes of the methylene protons in N, N-dibenzyl-o-toluamide (o-DBET,
black), (m) N, N-dibenzyl-m-toluamide (m-DBET) and N, N-diethyl-m-toluamide
gadro as a function of two dihedral angles:
Aromatic-CO) and b (OACANAC) [18]. The dihedral angle
a
(CACACAO/
was
(
m-DEET, red) and (p) N, N-dibenzyl-p-toluamide (p-DBET, blue). The stacked plot
a
for each molecule was recorded from 1 °C to 51 °C and in steps of 2 °C. (For
interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)
changed from ꢀ180° to +180° in steps of 5 °C. The calculations
can only be considered qualitative or at most semi-quantitative.