Measurements of Heavy-Atom Isotope Effects Using 1H NMR Spectroscopy

Article abstract of DOI:10.1021/jo201226g

A novel method for measuring heavy-atom KIEs for magnetically active isotopes using ¹H NMR is presented. It takes advantage of the resonance split of the protons coupled with the heavy atom in the ¹H spectrum. The method is validated by the example of the ¹³C-KIE on the hydroamination of styrene with aniline, catalyzed by phosphine-ligated palladium triflates.

Full text of DOI:10.1021/jo201226g

NOTE
pubs.acs.org/joc
Measurements of Heavy-Atom Isotope Effects Using 1H NMR
Spectroscopy
Anna Pabis,† Rafaz Kamiꢀnski,† Grzegorz Ciepielowski,‡ Stefan Jankowski,*,‡ and Piotr Paneth*,†
†Institute of Applied Radiation Chemistry and ‡Institute of Organic Chemistry, Faculty of Chemistry, Technical University of Lodz,
Zeromskiego 116, 90-924 Lodz, Poland
S Supporting Information
b
ABSTRACT: A novel method for measuring heavy-atom KIEs for magnetically active isotopes
using 1H NMR is presented. It takes advantage of the resonance split of the protons coupled
with the heavy atom in the 1H spectrum. The method is validated by the example of the 13C-KIE
on the hydroamination of styrene with aniline, catalyzed by phosphine-ligated palladium
triflates.
inetic isotope effects (KIEs, ratios of rate constants of light-
to-heavy isotopologues) are very subtle tools for studying
Scheme 1
K
mechanisms of organic reactions that allow the deduction of the
structure and properties of transition states, entities not yet
amenable to direct experimental scrutiny.1 Their use is, however,
hampered by the high cost of isotope-ratio mass spectrometers
and the tedious procedure required frequently for conversion of
reactants into gaseous form suitable for analysis. Therefore, other
experimental techniques of measuring KIEs are being sought.
One of these emerging tools is nuclear magnetic resonance (NMR)
spectroscopy.2 Singleton has introduced 13C NMR using sam-
ples of natural isotopic abundance.3 Measuring deuterium iso-
tope effects by NMR using natural abundance was also proposed
in the literature.4 The main limitation of this approach is its low
sensitivity and the necessity of using large amounts of material. It
also suffers from the fact that only relative isotope effects can
be measured; thus, the isotope effect of a chosen reference
atom has to be assumed. Bennet improved this method to gain
higher sensitivity and to avoid dependence on the reference
KIE at the expense of isotopic synthesis by using 13C-labeled
material.5 Both of these methods suffer from the low sensitivity of
amination of styrene with aniline, catalyzed by phosphine-ligated
palladium triflates (Scheme 1), which has been shown to exhibit a
substantial 13C-KIE at the benzylic carbon.7
In the proposed methodology, we have used a combination of
two isotopologues, styrene of natural abundance and (α-13C)-
styrene, in order to obtain the 13C/12C ratio from H NMR
1
signals of benzylic hydrogen of the unreacted substrate. One of
these signals in the unlabeled isotopologue is a doublet of
doublets at 6.685 ppm, while it is additionally split with a
coupling constant of 153.3 Hz in the 13C-labeled reactant, as
illustrated in Figure 1.
The obtained value of 13C-KIE, 1.036, is in excellent agree-
ment with the value of 1.037 determined theoretically and is
slightly higher than 1.030 ( 0.003 (95% confidence level)
determined experimentally.7 The difference between the two
experimental KIE values may be due to the better sensitivity of
the method proposed herein or a small secondary isotope effect
of the reference carbon atom. This latter case would indicate that
the model in which aniline is truncated to ammonia does not
yield correct results. In order to find out if the theoretical model is
in fact the source of the discrepancy between theoretically pre-
dicted and experimentally observed KIE, we have performed
theoretical calculations extending the model to include the whole
aniline molecule. Three transition states, 1aꢀc, illustrated in
Figure 2 (detailed geometries, energies, and frequencies are given
1
13C NMR compared to H NMR. Recently, Bennet used the
splitting of 19F NMR signals for the purpose of measuring kinetic
isotope effects.6
Here we present a novel method for measuring KIEs for
magnetically active heavy-atom isotopes (e.g., 15N, 13C), which is
based on the isotopic change of the signal shape observed in the
1H NMR spectra. In this method we use a combination of a
compound at natural abundance and its heavy-atom isotopolo-
gue, labeled with 13C at a position for which the isotopic
fractionation is measured. The resonance split of the protons
coupled with the heavy atom in the 1H spectrum compared to the
unlabeled compound allows for direct measurement of the ratio
of concentrations of the two isotopologues (isotopic ratio, R). As
an example of this, we have chosen the 13C-KIE for the hydro-
Received: June 13, 2011
Published: August 22, 2011
r
2011 American Chemical Society
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The Journal of Organic Chemistry
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in the Supporting Information), have been found differing in
the C6ꢀC9ꢀN12ꢀC30 dihedral angle (atom numbering and
values of the torsional angle given in Figure 2). The most stable
structure 1a, given on the left, is more stable by 0.9 and 1.6 kcal/
mol than structures shown in the center (1b) and on the right
(1c) of Figure 2, respectively. The obtained 13C-KIEs for α and
para positions are 1.0372 and 1.0007, respectively, in agreement
with the results reported previously by Singleton. Thus, it seems
that the method presented herein yields results slightly more
accurate that those based on 13C NMR.
In conclusion, we present a new NMR approach to measure-
ments of heavy-atom isotope effects that takes advantage of the
high sensitivity of 1H NMR spectra. It should be noted, however,
that it can be applied only in the advantageous cases when the
proton signal of interest is not overlapping with any other signals,
a much less frequent case than in 13C NMR spectra, although
there is some flexibility here; only one satellite signal may be used
for kinetic studies if the other one overlaps with other peaks. In
such fortunate cases it can be applied to both chemical as well as
enzymatic reactions; we are currently working on the application
of this method to the studies of binding isotope effects on binding
inhibitors to lactic dehydrogenases that should allow us to
distinguish isoforms of the enzyme.8 On the other hand, mea-
surements of 15N KIEs using this method would be challenging
in systems that have exchangeable NꢀH protons in the reactant
and product.
’ EXPERIMENTAL SECTION
The reaction protocol followed that described in the literature7 with a
small modification that was made because we did not observe full
separation of the reactants until the reactant mixture was saturated with
gaseous HCl and amine reactants were converted into their respective
ammonium salts. Additionally, we used anhydrous deuterated benzene
(distilled from Na/benzophenone, 99.5% isotopic purity, The Radio-
isotope Production and Distribution Centre, Swierk, Poland) as the
reaction medium, which simplified the preparation of NMR samples,
and the scale was reduced to the 0.63 mmol level. The reaction was
carried out at 90 °C. Integrations of vinylic peaks of styrene (5.709 and
5.176 ppm) and the sum of quartet (4.323 ppm) and satellite quartets
(4.418 and 4.223 ppm) corresponding to benzylic protons of the
reaction product were used in the determination of the fraction of
reaction, f. The isotopic composition of the starting material at the α-13C
position was kept close to unity (see the Supporting Information).
Styrene samples with a natural abundance of isotopes (ReagentPlus
grade, g99%) and α-13C labeled (99% isotopic purity) were obtained
from Sigma-Aldrich and used without further purification.
All samples for NMR measurements were prepared using the same
amount of styrene (20 mg) in 0.7 mL of C6D6 in a 5 mm NMR tube. The
1H spectra were recorded at 700.21 MHz at 300 K, using 85.5 s delays
between 45° pulses, an 11.010 s acquisition time, and 131 072 points.
Because of the relatively high time constant T1 = 10.5 s of the proton
attached to the α-12C carbon atom, the 8T1 repetition time and a pulse of
π/4 instead of π/2 were applied to ensure the complete magnetization
recovery. A zeroth order baseline correction was applied, and integra-
tions of each doublet of doublets of 6.685 ppm signals were performed
using an integration region equal to the width of doublet of doublets
expanded by 10 half-widths of singlet lines. Spectra were recorded for 64
or more scans to achieve a signal-to-noise ratio of at least 1000. Isotopic
ratios were calculated from the ratio of sum of integrations of satellite
doublets of doublets and central doublet of doublets (Figure 1).
The 13C-KIE has been evaluated from the logarythmic dependence
correlating the isotopic ratios of the remaining reactant with the reaction
progress:
ꢀ
ꢁ
1
ln Rf ¼
ꢀ 1 lnð1 ꢀ fÞ þ ln R0
ð1Þ
KIE
where f is the extent of reaction, and Rf and R0 are the isotopic ratios of
styrene (ratios of concentrations of heavy to light species) determined
after fraction of reaction f and for the starting material, respectively. The
obtained dependence is illustrated in Figure 3. Triplicates of such
Figure 1. 1H NMR signals of benzylic hydrogen of a mixture of styrene
at natural abundance and (α-13C)styrene.
Figure 2. Transition state structures characterized by the C6ꢀC9ꢀN12ꢀC30 dihedral angle of ꢀ58.7 (1a), 40.4 (1b), and ꢀ162.9° (1c) (left to right).
Hydrogen atoms are omitted for clarity.
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1
Figure 3. Evaluation of the 13C-KIE from the H NMR determined
isotopic ratios.
measurements of the KIE (each based on six or seven points) have
yielded the 13C-KIE value of 1.0360 ( 0.0015 (95% confidence level).
Theoretical calculations were carried out at the DFT level using the
M05-2X functional9 expressed in the SDD with an effective core
potential10 (for Pd) and 6-31+G**11 (on remaining atoms) basis set
with the inclusion of the PCM solvent continuum model,12 as imple-
mented in Gaussian09.13 We have shown previously that this theory
level performs well in calculations of KIEs.14 Geometry optimizations
were carried out using default convergence criteria. Vibrational analysis
was performed to confirm that the obtained structures are stationary
points on the potential energy surface and correspond to either a local
minimum (3n ꢀ 6 real normal modes of vibrations) or a transition state
(exactly one imaginary frequency). KIEs were calculated using the com-
plete Bigeleisen equation15 as implemented in the ISOEFF program.16
€
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’ ASSOCIATED CONTENT
S
Supporting Information. Tables giving individual ex-
b
perimental points used in the determination of the 13C-KIE with
linear regression parameters, geometries of the transition state
structures, Gibbs free energies, and imaginary frequencies. This
material is available free of charge via the Internet at http://pubs.
acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: paneth@p.lodz.pl; stefan.jankowski@p.lodz.pl.
’ ACKNOWLEDGMENT
These studies were supported by the grant NN204/1579/33
from the Ministry of Science and Higher Education of Poland
and internal University grants DS/I18/10/2010 and DS/I19/7/
2011.
’ REFERENCES
(1) Wolfsberg, M.; Van Hook,W. A.; Paneth, P.; Rebelo, L. P. N.
Isotope Effects in the Chemical, Geological and Bio Sciences; Springer: New
York, 2010.
(2) Jankowski, S. Annu. Rep. NMR Spectrosc. 2009, 68, 149–191.
(3) Singleton, D. A.; Thomas, A. A. J. Am. Chem. Soc. 1995,
117, 9357–9358.
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