Biology Reference
In-Depth Information
To speed up data acquisition, NMR spectrosco-
pists have taken advantage of multiple receivers
for parallel acquisition of two or more nuclei and
have combined three essential standard 2D
NMR pulse sequences into a single experiment
called PANACEA (parallel acquisition NMR,
an all-in-one combination of experimental appli-
cations). 34 The three combined experiments are
the HSQC, HMBC, and INADEQUATE. The
INADEQUATE experiment can trace out the
complete one-bond carbon e carbon connectivity
for an entire molecule, and the chain is only
broken by another heteroatom. 35 However, it is
extremely insensitive for samples at natural
abundance because it requires pairs of adjacent
13 C nuclei. More sensitive proton-detected
versions of the INADEQUATE experiments
have been proposed, but the carbon connectivity
may be broken by quaternary carbons, and the
fragment must have a protonated carbon. 36,37
A quick introduction with practical details on
how NMR experiments are used to determine
skeletal connectivity, relative stereochemistry,
and structure veri
detailed explanations of chemical shifts and
substituent effects, scalar couplings, and many
other pertinent aspects of NMR structure eluci-
dation can be found in topics by Lambert et al.
and Crews et al. 39,40
When using NMR spectroscopy for meta-
bolic pro
ling, the biological sample is usually
a complex mixture. The 1 H NMR spectra of
human serum 41 and urine 42 are shown in
Figures 3 and 4 , respectively, with observable
metabolites marked by identi
able chemical
shift positions of various metabolites. In
Figure 3 , imidazole was added during the
sample preparation stage to serve as a pH indi-
cator. Two-dimensional NMR experiments
(TOCSY, HSQC, and HMBC), described previ-
ously, are collected on select samples using
high
field NMR spectrometers for metabolite
con
cation. The increasing
availability of free web-based and commercial
databases containing searchable NMR chemical
shifts of metabolite
rmation and identi
standards have made
metabolite identi
cation much easier and faster.
Table 3 contains a listing of small molecule
NMR web-based databases free to the public
cation can be found in the
mini-review by Kwan and Huang. 38 More
11
17
16
22
24 28
24
32
1.2
1.1
1.0
0.9
3
3
1
4
1.0
0.8
0.6
x 3
8
36
9
35
5
33
18
20
SP
4
21,29
13 8
26
2
14
21
0.4
0.2
0.0
13
8
6
10
14
7
19
27
6
14
15
11
30
8
12
25
31
23
8.5
8.0
7.5
7.0
6.5
6.0
5.5
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
FIGURE 3 Typical 500 MHz 1 H NMR spectrum of healthy human serum. Numbers indicate the following metabolites:
1, imidazole; 2, urea; 3, D-glucose; 4, L-lactic acid; 5, glycerol; 6, L-glutamine; 7, L-alanine; 8, DSS; 9, glycine; 10, L-glutamic
acid; 11, L-valine; 12, L-proline; 13, L-lysine; 14, L-histidine; 15, L-threonine; 16, propylene glycol; 17, L-leucine; 18, L-tyrosine;
19, L-phenylalanine; 20, methanol; 21,creatinine; 22, 3-hydroxybutyric acid; 23, ornithine; 24, L-isoleucine; 25, citric acid; 26,
acetic acid; 27, carnitine; 28, 2-hydroxybutyric acid; 29, creatine; 30, betaine; 31, formic acid; 32, isopropyl alcohol; 33, pyruvic
acid; 34, choline; 35, acetone; 36, glycerol. (Reprinted from reference #41.)
Search WWH ::




Custom Search