Biology Reference
In-Depth Information
and carbon atoms is compared to a molecular
formula obtained from mass spectrometry or
elemental analysis, and the degree of unsatura-
tion (DU) is determined using the following
equation:
type of functional group or what heteroatom
is nearby or directly attached. For example,
the carbon chemical shifts of primary methyl
groups (-CCH
3
) are in the ~5 to 30 ppm and
e
OCH
3
groups are in the ~50 to 60 ppm range.
In the multiplicity-edited HSQC experiment,
the CH and CH
3
groups have opposite phase
to CH
2
groups.
26
The phase difference along
with the carbon chemical shifts is used to
distinguish the three groups from one another.
Two- and three-bond
1
H-
13
C correlations
are obtained from heteronuclear multiple-
quantum correlation (HMBC)
27
data and are
particularly useful for connecting structural
fragments. Correlations to quaternary carbons
are observable in HMBC data. HMBC correla-
tions are dependent on the existence of
n
J
CH
scalar couplings, which are in the 2 to 15 Hz
range. Four- and
1
where C is the number of carbon atoms, H is the
number of hydrogen atoms, X is the number of
heteroatoms with valence 1 (i.e., halogens), and
N is the number of heteroatoms with valence 3
(i.e., nitrogen). This formula to determine the
sum of rings and multiple bonds was deter-
mined by Badertscher and coworkers.
21
Oxygen
and other divalent atoms do not contribute to the
degree of unsaturation. A ring or a double bond
is one degree of unsaturation, and a triple bond
is counted as two degrees of unsaturation. Using
this rule, benzene (C
6
H
6
) would have 4 degrees
of unsaturation, which accounts for 1 ring and
3 double bonds. The equation was derived
from fully saturated hydrocarbons (C
n
H
2n
þ
2
);
therefore, it cannot be used for stable ionic or
radical species.
Proton
e
proton connectivity (
1
H-
1
H) is deter-
mined from 2D correlation spectroscopy
(COSY)
22
and total correlation spectroscopy
(TOCSY)
23
data. Cross-peaks in the COSY spec-
trum come from directly coupled protons that
are usually two or three bonds apart. In the
TOCSY experiment, proton magnetization is
relayed along pairs of coupled protons, allowing
the detection of all protons in isolated spin
systems; not all protons in the spin system need
to be mutually coupled. Both 1D and 2D versions
of the TOCSY experiment are very useful for sepa-
rating peaks from crowded regions. In cases in
which peak overlap is severe, selective excitation
in 1D or 2D experiments is required.
24
One-bond proton
e
carbon (
1
H-
13
C) connec-
tivity is determined from heteronuclear single-
quantum correlation (HSQC)
25
data, which
provides the chemical shifts of all protonated
carbons and their directly attached proton(s).
The carbon chemical shift will
DU
¼
C
H
=
2
X
=
2
þ
N
=
2
þ
five-bond (long-range) correla-
tions are sometimes observed but are often too
weak to be detected because the
4,5
J
C,H
coupling
value is very small.
Relative spatial information can be obtained
from the three-bond scalar coupling (
3
J
H,H
and
3
J
H,C
) value, which is dependent on dihedral
angle and follows the Karplus equation.
28
There
is a null near 90
and the maxima are at 0
and
180
. Qualitative or quantitative distances be-
tween two protons are determined from nuclear
Overhauser effect spcectroscopy (NOESY)
29
and
rotating frame Overhauser effect spcectroscopy
(ROESY)
30,31
data. NOESY and ROESY peak
intensity have an r
e
6
and r
e
3
distance depen-
dence, respectively. Actual distances in ang-
stroms are calculated from multiple 1D or 2D
experiments acquired with increasing length
for the mixing period.
32
The experiments mentioned thus far are
common NMR experiments employed in struc-
ture determination. Many variants of these
experiments exist, some for select applications;
most are improvements and are too numerous
to list. For the interested reader, Berger and
Braun
s wprl contains over 200 NMR experi-
ments for small molecule NMR spectroscopy.
33
'
indicate the
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