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Two-dimensional concurrent HMQC-COSY as an approach for small molecule chemical shift assignment and compound identification
Kaifeng Hu
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William M. Westler
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John L. Markley
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K. Hu W. M. Westler J. L. Markley (&) National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison
,
Madison, WI 53706, USA
Chemical shift assignment is the first step toward the structure elucidation of natural products and other chemical compounds. We propose here the use of 2D concurrent HMQC-COSY as an experiment for rapid chemical shift assignment of small molecules. This experiment provides well-dispersed 1H-13C peak patterns that are distinctive for different functional groups plus 1H-1H COSY connectivities that serve to identify adjacent groups. The COSY diagonal peaks, which are phased to be absorptive, resemble 1H-13C HMQC cross peaks. We demonstrate the applicability of this experiment for rapidly and unambiguously establishing correlations between different functional groups through the analysis of the spectrum of a metabolite (jasmonic acid) dissolved in CDCl3. In addition, we show that the experiment can be used to assign spectra of compounds in a mixture of metabolites in D2O.
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2D COSY spectra (Aue et al. 1976) provide important
information on the connectivity between different
functional groups; however, 2D 1H1H COSY spectra suffer
from phasing issues, arising from intrinsic differences in
the relative phase of diagonal and cross peaks, and peak
overlap issues, resulting from the low proton chemical shift
dispersion. Heteronuclear 2D HSQC or HMQC
experiments yield better-resolved spectra of metabolites and
metabolite mixtures because of the higher chemical shift
dispersion of carbon; however, these experiments do not
offer information on the connectivity between different
functional groups. Heteronuclear multiple-bond correlation
spectra, HMBC type experiments (Bax and Summers 1986)
can show heteronuclear long-range indirect
connectivities, but these are not always unambiguous. The
INADEQUATE experiment (Bax et al. 1980), which makes use of
direct 13C13C coupling, can provide very important
information on 13C13C connectivities within the
molecular skeleton, but its application is limited by the low
sensitivity of the experiment at natural abundance 13C.
To overcome the peak overlap issue in 2D COSY, the
3D 13C-edited HMQC-COSY experiment was proposed
and demonstrated on kanamycin A with natural abundance
13C (Fesik et al. 1989). A 13C-resolved COSY experiment
involving selective excitation of a particular carbon signal
by the SELINCOR technique was proposed as a means for
overcoming spectral overlaps (Facke and Berger 1995).
The 3D HMQC-COSY experiment was later modified with
gradient-enhanced coherence order selection and further
demonstrated on sucrose and menthol to facilitate
assignments (Hurd and John 1991).
In 2D HMQC-COSY, the initial 1H magnetization is
transferred concurrently to 13C through 1JCH (HMQC) and
to 1H through 3JHH (or nJHH, COSY). The 1H13C
multiplequantum period is exploited for both 13C chemical shift
coding and for achieving a maximal COSY effect. Similar
information on the connectivity can be obtained from
HSQC-COSY to HSQC-TOCSY (Bax and Davis 1985)
experiments. For HSQC-COSY and HSQC-TOCSY, the
initial 1H magnetization is transferred sequentially to
bonded 13Cs (by HSQC) and to homonuclear J coupled
1Hs (by COSY or TOCSY). Considering the relatively
small 3-bond 1H1H homonuclear J-couplings, usually the
coupling Hamiltonian has to be allowed to evolve for a
considerably long time to achieve reasonable COSY or
TOCSY effects. In addition, to achieve reasonable
resolution along the indirect 13Cdimension, magnetization on
13C (single-quantum coherence) has to evolve for a long t1
time.
Here we present 2D HMQC-COSY experiments that can
be run as constant-time (Fig. 1a) or non-constant-time
(Fig. 1b) versions and used for rapid spectral assignment of
small molecules. In the constant-time version of 2D
HMQCCOSY to achieve maximum COSY effect, given the
relatively small 3-bond 1H1H homonuclear J-couplings, the
coupling Hamiltonian evolves during both the 1H13C
heteronuclear multiple quantum generation period (ab) and the
1H13C multiple quantum constant-time period. In the
nonconstant-time version of 2D HMQC-COSY, the 1H1H
homonuclear J-coupling runs in accordion mode (Mandel
and Palmer 1994). The constant-time version is superior for
the analysis of small molecule spectra because of its higher
COSY transfer efficiency with small J-couplings and its
higher resolution along the indirect 13C-dimension
compared to the non-constant-time version.
We illustrate the utility of this approach for the spectral
assignment of a small molecule and show how the
experiment can be used for identifying compounds in mixtures
of metabolites.
Materials and methods
Jasmonic acid (SigmaAldrich) was dissolved in CDCl3 at
a concentration about 60 mM. The constant-time 2D
HMQC-COSY spectrum of jasmonic acid was recorded at
25 C on a Bruker Avance 500 MHz spectrometer equipped
with a z-gradient triple resonance CPTXO (...truncated)