Deconvolution of Complex NMR Spectra in Small Molecules by Multi
Frequency Homonuclear Decoupling (MDEC)
Ana Paula D. M. Espindola,†Ronald Crouch,‡John R. DeBergh,†Joseph M. Ready,†and
John B. MacMillan*,†
Department of Biochemistry, UniVersity of Texas Southwestern Medical Center at Dallas,
Dallas, Texas 75390-9038, and Applications Laboratory, Varian, Inc., Palo Alto, California 94303
Received August 22, 2009; E-mail: email@example.com
Advances in NMR instrumentation have pushed the limits of
small molecule NMR, with higher fields allowing better signal
dispersion and modern probe design permitting analysis1and
quantification2on the nanomolar level. One of the remaining
limitations of small molecule NMR is the ability to obtain relative
configuration of small molecules. Since the 1959 report by Karplus3
the use of coupling constants to determine the relative configuration
in conformationally rigid small molecules has become routine. More
recently, J-based configuration analysis4has been applied to
determine the relative stereochemistry of complex acyclic and
macrocylic small molecules and natural products.5The utility of
J-based methods relies on the ability to measure discrete coupling
constants between protons (JHH), which can be difficult in molecules
with complex multiplets and significant signal overlap. The existing
methods, such as E.COSY6and 2D J-resolved, suffer from lack of
sensitivity and complex data analysis. Alternatively, classic homo-
nuclear decoupling can be used to simplify a complex multiplet
but is limited to irradiating a single proton.7Due to the challenges
of measuring coupling constants, many reports of natural products
and synthetic small molecules do not report vital coupling informa-
tion; rather they only assign signals as multiplets. In this paper we
report an experiment with selective homonuclear decoupling of
multiple protons simultaneously that allows a fast and reliable
determination of specific coupling values from complex spectra.
The development of this experiment was inspired by our studies
to determine the relative stereochemistry of unsaturated fatty acids
in which we were faced with complications due to multiple3JHH
and allylic couplings, making assignment of individual coupling
constants difficult. Advances in NMR technology allowed us to
utilize a q3 shaped pulse to obtain a multi frequency homodecou-
pling (MDEC) during the acquisition time. In combination with
the standard1H experiment, it results in a complete simplification
of a complex multiplet. The usefulness of our MDEC experiment
is demonstrated with three compounds of increasing structural and
spectral complexity: menthol (1), cholesteryl acetate (2), and a
synthetic C16fatty acid (3) (Figure 1).
As proof of principle, menthol (1) offers a1H NMR spectrum
that is well resolved but has complex multiplets. In particular, the
proton H-2 (δ 0.99 ppm) appears as a dddd, due to coupling to
four distinct protons (Figure 2a).8Application of homonuclear
decoupling with a hard pulse at H-1 (δ 3.25 ppm) causes H-2 to
collapse to an apparent doublet of triplets (Figure 2b). Application
of a selective q3 shaped pulse to irradiate protons H-1 and H-7
(δ 2.11 ppm) results in the H-2 multiplet collapsing to a distinct
doublet of doublets with coupling to H-3b (3JHH) 12.2 Hz) and
H-3a (3JHH) 3.3 Hz) (Figure 2c).9Additional decoupling of H-3a
(δ 1.49 ppm) collapses H-2 to a doublet with a 12.2 Hz coupling
(Figure 2d). An advantage of this experiment is the sensitivity,
requiring only eight scans (60 s) for a 20 mM solution of menthol.
In a matter of minutes, it is possible to reliably ascertain all3JHH
from a complex multiplet. This compares to an E.COSY (Supporting
Information Figure 6), which required multiple hours to obtain
sufficient S/N and resolution to reliably measure coupling constants.
It should be noted that E.COSY offers the advantage of analyzing
all coupling information for a molecule in a single experiment,
although due to optimization parameters this may require multiple
experiments for complex small molecules.10
Cholesteryl acetate (2) offers a more complex spectral pattern,
due to the overlapping methylenes that comprise the tetracylic ring
system. The1H NMR region from δ 1.40-1.60 ppm is comprised
of four overlapping multiplets that prohibit reliable measurements
of coupling constants (Figure 3a). Application of the MDEC
methodology alone would not provide sufficient deconvolution of
the signals; however, MDEC can be incorporated in other selective
excitation 1D experiments such as a 1D-TOCSY to isolate
individual spin systems prior to decoupling.11A selective 1D-
TOCSY of 2 by irradiation of H-3 (δ 4.57 ppm) results in the
isolation of the spin system from H-1 through H-4 (Figure 3b).
†University of Texas Southwestern Medical Center at Dallas.
Figure 1. Structure of compounds used for the MDEC experiments.
Figure 2. Menthol (1)1H NMR data in CDCl3. (a)1H NMR of 1. (b)
Homonuclear decoupling of 1 with irradiation at H-1. (c) MDEC irradiation
of 1 at H-1 and H-8. (d) MDEC irradiation of 1 at H-1, H-3a, and H-8.
Published on Web 10/21/2009
10.1021/ja907110e CCC: $40.75 2009 American Chemical Society
15994 9 J. AM. CHEM. SOC. 2009, 131, 15994–15995
The signal associated with H-2a (δ 1.56 ppm), obscured in the Download full-text
standard1H experiment, can be resolved to a complex multiplet in
the 1D-TOCSY experiment. The use of a selective 1D-TOCSY-
MDEC experiment with initial selection of H-3 for the TOCSY
followed by decoupling of H-3, H-2b, and H-1b results in the
subsequent collapse of the H-2a mutliplet to a doublet with a
coupling constant of 13.7 Hz, consistent with the trans-diaxial
relationship between H-1a and H-2a (Figure 3c).
Recently, the potent cytotoxic natural product nigricanoside A
was reported with only a planar structure. The small quantity of
material and the overlapping1H NMR precluded assignment of the
relative configuration of the C16and C20fatty components.12Our
efforts toward assigning the relative stereochemistry via NMR
studies of synthetic models were hindered by the inability to assign
the proton coupling constants of interest. We applied a combination
of MDEC and the 1D-TOCSY-MDEC to obtain the necessary
coupling information from the synthetic C16fatty acid 3 to assign
the C9/C10 anti stereochemistry. The key coupling values were
obtained from deconvolution of H-9 (δ 3.77 ppm). Systematic
decoupling of two protons simultaneously resulted in the assignment
of3JH9-H10) 3.5 Hz,3JH10-H11a) 4.7 Hz, and3JH10-H11b) 7.4 Hz
(Figure 4b-d). Further stereochemical and synthetic studies are
underway to assign the relative configuration of 3 and its diaster-
Our experiments started with the selection of a shaped pulse.
The q3 Gaussian was selected to invert the signal of interest, as it
efficiently irradiates the peak of interest but with little net effect
outside the chosen bandwidth, resulting in highly selective irradia-
tion.14In all of our experiments, we gave preference to a small
duty cycle (between 0.02 to 0.05) and higher decoupling power
and applied a correction for the Bloch-Siegert effect in our multiple
band excitation. This correction is necessary to account for small
changes in the chemical shift due to the closeness of the irradiation
frequency and the resonance frequency.15,16
The MDEC experiment fills a gap in the current methods to
measure proton homonuclear coupling constants. In a matter of a
few minutes complex multiplets can be simplified to easily
interpretable doublets. In comparison with the E.COSY experiment,
MDEC is faster, more sensitive, and easier to interpret. This is
especially the case when only a few coupling values are necessary.
Additionally, the single pulse MDEC experiment can be incorpo-
rated in other 1D experiments, increasing its power and allowing
a better tailoring of the experiments to solve specific problems. If
desired the sites where MDEC was applied could be effectively
removed from the spectra by presaturation with the same waveform
combined with a two-step phase cycle.
Robert A. Welch Foundation (I-1612 and I-1689) and the Chilton
Foundation (J.B.M.) and the NSF-CAREER (J.M.R.). J.R.D. is
supported by a fellowship from the Sara and Frank McKnight
Financial support was provided by the
Supporting Information Available: Experimental procedures,
representative NMR spectra, and experiment macros. This material is
available free of charge via the Internet at http://pubs.acs.org.
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(16) A stepwise procedure for acquiring the MDEC experiment and the 1D-
TOCSY MDEC experiment is included in the Supporting Information.
Figure 3. ( a)1H NMR of 2 in CDCl3. (b) 1D-TOCSY of 2 with selection
of H-3. (c) 1D-TOCSY with selection of H-3 followed by simultaneous
irradiation at H-1b, H-2b, and H-3. (d) A-ring of cholesteryl acetate (2).
Figure 4. (a)1H NMR of 3 in 25:2 C6D6/DMSO-d6. (b) Decoupling at
H-9, H-11b. (c) Decoupling at H-9, H-11a. (d) Decoupling at H-11a, H11b.
J. AM. CHEM. SOC. 9 VOL. 131, NO. 44, 2009