Exercise plus Solution – Quick overview
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1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0 ppm
1
H
10.83
6.10
6.03
5.61
1.73
1.82
1.83
1.78
1.79
1.83
1.74
Inte-
gral
692.50
685.40
587.32
580.31
2366.31
2359.73
2353.18
2350.09
2343.67
2337.07
2264.00
2257.44
2254.68
2250.82
2248.10
2244.31
2241.55
2234.90
C
13
H
22
N
4
O
4
1
H NMR spectrum
recorded at 500.13 MHz
Problem of the Month:
May 2021
The sample in this problem is an oligopeptide.
Note that one of the amino acid residues is not one of the 21 naturally occurring
eukaryotic amino acids.
Deduce the constitution and assign all nuclei (proton, carbon and nitrogen).
1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0 ppm
1
H
10.83
6.10
6.03
5.61
1.73
1.82
1.83
1.78
1.79
1.83
1.74
Inte-
gral
4111.88
4102.29
4080.93
4071.62
3804.25
3795.57
528.74
522.23
515.49
508.91
1869.86
1862.79
1855.81
1854.22
1847.27
1840.04
2013.42
2006.31
1999.07
1991.88
C
13
H
22
N
4
O
4
1
H NMR spectrum
recorded at 500.13 MHz
This is the same proton spectrum as on the previous slide,
but with different peaks expanded.
You should keep a close eye on the number of double bond equivalents.
53.97
50.40
44.84
44.62
30.20
18.89
18.14
17.47
16.63
172.16
171.92
171.53
171.41
~
~
~
~
20253035404550
ppm
170175
13
C
13
C{
1
H} NMR spectrum
recorded at 125.80{500.13} MHz
16.5
17.0
17.5
18.0
18.5
19.0
19.5
13
C
1.01.11.21.31.4
1
H
ppm
30
35
40
45
50
55
13
C
3.03.23.63.84.04.24.44.64.8
1
H
ppm
~
~
1
H/
13
C-HSQC
recorded at 500.13/125.80 MHz
1.02.03.04.05.06.07.08.0 1
H
ppm
1
2
3
4
5
6
7
8
1
H
~ ~
~ ~
1
H/
1
H-COSY
recorded at 500.13 MHz
1.02.03.04.05.06.07.08.0 1
H
ppm
~ ~
173.0
171.2
171.4
171.6
171.8
172.0
172.2
172.4
172.8
13
C
part of
1
H/
13
C-HMBC
recorded at 500.13/125.80 MHz
119 .8
121.1
122.4
117
118
119
120
121
122
123
125
15
N
7.57.67.77.88.08.18.28.38.4
1
H
ppm
119.8
121.1
122.4
124.3
117
118
119
120
121
122
123
125
15
N
0.61.01.42.22.63.03.4
1
H
ppm
1
H/
15
N-HSQC
recorded at 500.13/50.70 MHz
1
H/
15
N-HMBC
recorded at
500.13/50.70 MHz
7.67.77.88.08.18.28.3
3.7
3.8
3.9
4.0
4.1
4.3
4.4
4.5
4.6
4.7
4.8
1
H
1
H
3.7
3.8
3.9
4.0
4.1
4.3
4.4
4.5
4.6
4.7
4.8
1
H
3.73.83.94.04.14.34.44.54.64.74.8
1
H
ppm
this is an off diagonal part
of the NOESY, don‘t look
for diagonal peaks
parts of
1
H/
1
H-NOESY
recorded at 500.13 MHz
(true NOESY peaks are displayed in red)
1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0 ppm
1
H
10.83
6.10
6.03
5.61
1.73
1.82
1.83
1.78
1.79
1.83
1.74
Inte-
gral
Basics
Integration, double bond
equivalents
sum of measured integrals: 41.09
number of protons: 22
proportionality coefficient: 0.535
measured integrals * coefficient: 0.93 : 0.98 : 0.96 : 0.95 : 0.98 : 0.97 : 0.93 : 3.00 : 3.23 : 3.27 : 5.80
double bond equivalents: 5
C
13
H
22
N
4
O
4
1 31 1 1 1 11 3 63
53.97
50.40
44.84
44.62
30.20
18.89
18.14
17.47
16.63
172.16
171.92
171.53
171.41
~
~
~
~
20253035404550
ppm
170175
13
C
Basics
Number of amino acids
C
13
H
22
N
4
O
4
number of >C=O groups: 4 (171.41, 171.53, 171.92, 172.16 ppm)
Please note:
all oxygen atoms are used now, no –OH group is possible.
double bond equivalents: 5
double bond equivalents: 5
11 9.8
121.1
122.4
117
118
119
120
121
122
123
125
15
N
7.57.67.77.88.08.18.28.38.4
1
H
ppm
119.8
121.1
122.4
124.3
117
118
119
120
121
122
123
125
15
N
0.61.01.42.22.63.03.4
1
H
ppm
Basics
Number of amino acids
1
H/
15
N-HSQC
recorded at 500.13/50.70 MHz
1
H/
15
N-HMBC
recorded at
500.13/50.70 MHz
number of >C=O groups: 4 (171.41, 171.53, 171.92, 172.16 ppm)
Please note:
all oxygen atoms are used now, no –OH group is possible.
double bond equivalents: 5
→ no –OH possible
number of >NH groups (HSQC): 3 (-NH protons between 7.5 and 8.5 ppm)
number of N atoms (from HMBC): 4 (one nitrogen atom is not directly bonded to H)
1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0 ppm
1
H
10.83
6.10
6.03
5.61
1.73
1.82
1.83
1.78
1.79
1.83
1.74
Inte-
gral
C
13
H
22
N
4
O
4
1 31 1 1 1 11 3 63
Basics
XH
n
groups
number of >C=O groups: 4 (171.41, 171.53, 171.92, 172.16 ppm)
double bond equivalents: 5
→ no –OH possible
number of >NH groups (HSQC): 3 (-NH protons between 7.5 and 8.5 ppm)
number of N atoms (from HMBC): 4 (one nitrogen atom is not directly bonded to H)
Finally three H are directly bonded to nitrogen and all other proton
signals belong to CH
n
groups.
> NH
> CH
n
53.97
50.40
44.84
44.62
30.20
18.89
18.14
17.47
16.63
172.16
171.92
171.53
171.41
~
~
~
~
20253035404550
ppm
170175
13
C
C
13
H
22
N
4
O
4
Basics
Double bond equivalents
revisited
number of >C=O groups: 4 (171.41, 171.53, 171.92, 172.16 ppm)
double bond equivalents: 5
→ no –OH possible
number of >NH groups (HSQC): 3 (-NH protons between 7.5 and 8.5 ppm)
number of N atoms (from HMBC): 4 (one nitrogen atom is not directly bonded to H)
There are no sp
2
hybridized carbon atoms other
than the carbonyl groups.
Were is the fifth double bond equivalent?
1.02.03.04.05.06.07.08.0
1
H
ppm
1
2
3
4
5
6
7
8
1
H
~ ~
~ ~
Amino acids
We figured out:
• the three doublets with chemical shifts larger
than 7 ppm belong to NH protons.
• all other protons belong to CH
n
groups
If we start in a COSY from a NH proton and follow the
coupling path as long as possible we will find chains of
the kind
– NH – CH
n
– CH
n
– CH
n
–
Because there are three NH protons we should end with
three of such chains.
To help us keep an overview, let‘s label the signals of one
spin system after the other and remove all signals
already assigned.
1.02.03.04.05.06.07.08.0
1
H
ppm
1
2
3
4
5
6
7
8
1
H
~ ~
~ ~
Amino acids
7.60
3.71
1.17
7.60
3.71
1.17
1 1 3Integral
The protons with the chemical shifts of 7.60 ppm and
3.71 pm are neighbours. Please remember, in this
case chemical shifts large than 7 ppm mean NH and
everything else CH
n
. In our case according to the
integral n is 1.
The next pair of neighbours are the protons with
the chemical shifts of 3.71 ppm and 1.17 ppm.
Please don‘t forget the integral of 3 for the signal
at 1.17 ppm.
C
N
H
H
3.71
7.60
H
3
C
1.17
1.02.03.04.05.06.07.08.0 1
H
ppm
1
2
3
4
5
6
7
8
1
H
~ ~
~ ~
Amino acids
All cross peaks belonging to this amino acid fragment, as
well as the corresponding projections, were deleted from
the COSY. This is not a manipulation of the spectrum, but
was done in the graphics program to make the further
evaluation easier.
In case of a printed spectrum, you can simply cross out
already evaluated parts using a pencil.
C
N
H
H
3
C H
3.71
1.17
7.60
C
N
H
H
3
C H
3.71
1.17
7.60
1.02.03.04.05.06.07.08.0 1
H
ppm
1
2
3
4
5
6
7
8
1
H
~ ~
~ ~
1 1 3Integral
8.15
4.50
1.02
8.15
4.50
1.02
Amino acids
1.02
1.05
1.05
Two signals at 1.02 and 1.05 ppm are very similar. They are
easily distinguishable in the one-dimensional proton
spectrum, but not in the projections of the COSY. If you
take an expansion of the one-dimensional proton
spectrum to better recognize the crowded part of the
projections, you see that the upper right of the two cross
peaks leads to the doublet at 1.02 ppm and the lower left
cross peak leads to the doublet at 1.05 ppm.
C
N
H
H
3
C H
3.71
1.17
7.60
1.02.03.04.05.06.07.08.0 1
H
ppm
1
2
3
4
5
6
7
8
1
H
~ ~
~ ~
1 1 3Integral
8.15
4.50
1.02
8.15
4.50
1.02
Amino acids
1.02
1.05
CH
3
1.02
N
C
H
H
8.15
4.50
The further procedure is the
same as for the first amino
acid residue. There is a
connection between the
protons with the chemical
shifts of 8.15 ppm and 4.50
ppm.
The next connection
is between the
atoms with the
chemical shifts of
4.50 ppm and 1.02
ppm.
C
N
H
H
3
C H
3.71
1.17
7.60
1.02.03.04.05.06.07.08.0 1
H
ppm
1
2
3
4
5
6
7
8
1
H
~ ~
~ ~
1 1 3Integral
8.15
4.50
1.02
8.15
4.50
1.02
Amino acids
1.02
1.05
CH
3
1.02
N
C
H
H
8.15
4.50
The further procedure is the
same as for the first amino
acid residue. There is a
connection between the
protons with the chemical
shifts of 8.15 ppm and 4.50
ppm.
The next connection
is between the
atoms with the
chmical shifts of 4.50
ppm and 1.02 ppm.
CH
3
1.02
N
C
H
H
8.15
4.50
C
N
H
H
3
C H
3.71
1.17
7.60
1.02.03.04.05.06.07.08.0 1
H
ppm
1
2
3
4
5
6
7
8
1
H
~ ~
~ ~
Amino acids
H
3
C
1.05
C
N
H
H
8.21
4.70
1 1 3Integral
8.21
4.70
1.05
8.21
4.70
1.05
8.21 ppm → 4.70 ppm
4.70 ppm → 1.05ppm
C
N
H
H
3
C H
3.71
1.17
7.60
CH
3
1.02
N
C
H
H
8.15
4.50
1.02.03.04.05.06.07.08.0
1
H
ppm
1
2
3
4
5
6
7
8
1
H
~ ~
~ ~
Amino acids
H
3
C
1.05
C
N
H
H
8.21
4.70
C
N
H
H
3
C H
3.71
1.17
7.60
CH
3
1.02
N
C
H
H
8.15
4.50
C
CH
3
H
1.38
4.00
4.00
1.38
1 3Integral
4.00
1.38
There is only one remaining
correlation in the COSY.
Intermediate inventory
H
3
C
1.05
C
N
H
H
8.21
4.70
C
N
H
H
3
C H
3.71
1.17
7.60
CH
3
1.02
N
C
H
H
8.15
4.50
C
CH
3
H
1.38
4.00
• four carbonyl groups (C
4
O
4
)
• four double bond equivalents
• for CHN fragments (C
8
H
19
N
3
)
• five double bond equivalents
• molecular formula C
13
H
22
N
4
O
4
• one double bond equivalent
• CH
3
N
we have
we need
missing
• all fragments together (C
12
H
19
N
3
O
4
)
Intermediate inventory
H
3
C
1.05
C
N
H
H
8.21
4.70
C
N
H
H
3
C H
3.71
1.17
7.60
CH
3
1.02
N
C
H
H
8.15
4.50
C
CH
3
H
1.38
4.00
Even though there is no spectroscopic evidence yet, we can
speculate a bit about these missing pieces of the puzzle at
this point.
The last fragment extracted will probably also be an amino
acid. An additional >NH group is clearly not present, but
what about an >NCH
3
group?
We would then have assigned all atoms, but only
accounted for four double bond equivalents. The fifth
double bond equivalent should be a ring closure.
After these speculations, let us return to serious NMR
and assign all carbon and nitrogen signals.
• one double bond equivalent
• CH
3
N
missing
• one double bond equivalent
• CH
3
N
missing
16.5
17.0
17.5
18.0
18.5
19.0
19.5
13
C
1.01.11.21.31.4
1
H
ppm
H
3
C
1.05
C
N
H
H
8.21
4.70
C
N
H
H
3
C H
3.71
1.17
7.60
CH
3
1.02
N
C
H
H
8.15
4.50
C
CH
3
H
1.38
4.00
Carbon atom assignment (I)
1.38
1.17
1.05
1.02
16.63
18.14
17.47
18.89
Let us start with the
four methyl groups.
Here is one exemple, which
may be used as a template for
all four methyl groups.
C
N
H
H
3
C H
1.05
8.21
4.70
18.14
16.5
17.0
17.5
18.0
18.5
19.0
19.5
13
C
1.01.11.21.31.4
1
H
ppm
C
N
H
H
3
C H
3.71
1.17
7.60
CH
3
1.02
N
C
H
H
8.15
4.50
C
CH
3
H
1.38
4.00
Carbon atom assignment (I)
1.38
1.17
1.05
1.02
16.63
18.14
17.47
18.89
C
N
H
H
3
C H
1.05
8.21
4.70
18.14
The procedure for the three
other methyl groups is the
same.
C
N
H
H
3
C H
3.71
1.17
7.60
18.89
N
C
H
H CH
3
1.02
8.15
4.50
17.47
C
CH
3
H
1.38
4.00
16.63
30
35
40
45
50
55
13
C
3.03.23.63.84.04.24.44.64.8
1
H
ppm
~
~
C
N
H
H
3
C H
1.05
8.21
4.70
18.14
C
N
H
H
3
C H
3.71
1.17
7.60
18.89
N
C
H
H CH
3
1.02
8.15
4.50
17.47
C
CH
3
H
1.38
4.00
16.63
Carbon atom assignment (II)
3.71
4.50
4.00
4.70
44.84
44.62
50.40
53.97
Assigning the next carbon signals
is the same procedure like in the
case of the methyl groups.
Let us see one exemple again
and continue with the three
others without any comment.
C
N
H
H
3
C H
1.05
8.21
4.70
18.14
44.84
30
35
40
45
50
55
13
C
3.03.23.63.84.04.24.44.64.8
1
H
ppm
~
~
C
N
H
H
3
C H
3.71
1.17
7.60
18.89
N
C
H
H CH
3
1.02
8.15
4.50
17.47
C
CH
3
H
1.38
4.00
16.63
Carbon atom assignment (II)
3.71
4.50
4.00
4.70
44.84
44.62
50.40
53.97
C
N
H
H
3
C H
1.05
8.21
4.70
18.14
44.84
Now let‘s assign the three
remaining carbon atoms
using the same strategy.
C
N
H
H
3
C H
3.71
1.17
7.60
50.40
18.89
N
C
H
H CH
3
1.02
8.15
4.50
17.47
44.62
C
CH
3
H
1.38
4.00
53.97
16.63
11 9.8
121.1
122.4
117
118
119
120
121
122
123
125
15
N
7.57.67.77.88.08.18.28.38.4
1
H
ppm
C
N
H
H
3
C H
1.05
8.21
4.70
18.14
44.84
C
N
H
H
3
C H
3.71
1.17
7.60
50.40
18.89
N
C
H
H CH
3
1.02
8.15
4.50
17.47
44.62
C
CH
3
H
1.38
4.00
53.97
16.63
Nitrogen atom assignment
8.15
8.21
7.60
If we repeat the procedure a last
time, we get the correct assignment
of all protonated nitrogen atoms.
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
H CH
3
119.8
1.02
8.15
4.50
17.47
44.62
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
119.8
121.1
122.4
124.3
117
118
119
120
121
122
123
125
15
N
0.61.01.42.22.63.03.4
1
H
ppm
Nitrogen atom assignment
C
CH
3
H
1.38
4.00
53.97
16.63
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
H CH
3
119.8
1.02
8.15
4.50
17.47
44.62
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
1.38
And our fourth
nitrogen atom?
Excluding very unlike
four bond correlations,
there is only one
position possible for the
nitrogen atom with the
chemical shift of 124.3
ppm.
N
C
CH
3
H
124.3
1.38
4.00
53.97
16.63
1
H/
15
N-HMBC
30
35
40
45
50
55
13
C
3.03.23.63.84.04.24.44.64.8
1
H
ppm
~
~
A „forgotten“ methyl group
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
H CH
3
119.8
1.02
8.15
4.50
17.47
44.62
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
H
124.3
1.38
4.00
53.97
16.63
≈2.9
30.20
Inte-
gral
3
CH
3
≈2.9
30.20
CH
3
≈2.9
30.20
There was one remaining cross
peak in the HSQC …
This is clearly a methyl group
The proton signal is a
singlet and there is only
one position for the group
to keep the singlet.
Inventory again
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
H CH
3
119.8
1.02
8.15
4.50
17.47
44.62
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
H
124.3
1.38
4.00
53.97
16.63
CH
3
≈2.9
30.20
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
No atom is missing anymore. We have eight open bonds
coming from the carbonyl groups, 8 open bonds at the amino
acid fragments and we still need one double bond equivalent.
The solution is easy: We have to place the carbonyl groups
between the amino acid fragments resulting in a ring. The ring
is the last missing double bond equivalent.
• four carbonyl groups (C
4
O
4
)
• four double bond equivalents
• for amino acid fragments (C
9
H
22
N
34
)
• five double bond equivalents
• molecular formula C
13
H
22
N
4
O
4
we have
we need
• all fragments together (C
13
H
22
N
4
O
4
)
Inventory again
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
H CH
3
119.8
1.02
8.15
4.50
17.47
44.62
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
H
124.3
1.38
4.00
53.97
16.63
CH
3
≈2.9
30.20
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
Finally
CH
C
O
H
N CH
C O
HN
H
C
C
O
N
H
CH
CO
N
Me
Me
Me
Me
Me
Finally we have to end with this molecule.
But what is the order of the amino acids?
No atom is missing anymore. We have eight open bonds
coming from the carbonyl groups, 8 open bonds at the amino
acid fragments and we still need one double bond equivalent.
The solution is easy: We have to place the carbonyl groups
between the amino acid fragments resulting in a ring. The ring
is the last missing double bond equivalent.
• four carbonyl groups (C
4
O
4
)
• four double bond equivalents
• for amino acid fragments (C
9
H
22
N
34
)
• five double bond equivalents
• molecular formula C
13
H
22
N
4
O
4
we have
we need
• all fragments together (C
13
H
22
N
4
O
4
)
1.02.03.04.05.06.07.08.0 1
H
ppm
~ ~
173.0
171.2
171.4
171.6
171.8
172.0
172.2
172.4
172.8
13
C
Carbonyl groups
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
H CH
3
119.8
1.02
8.15
4.50
17.47
44.62
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
171.92
4.50
3.71
According to the HMBC the carbonyl signal at
171.92 ppm is adjacent to two C-H protons:
3.71 ppm
4.50 ppm
O
C
171.92
171.92 ppm / 3.71 ppm
171.92 ppm / 4.50 ppm
1.02.03.04.05.06.07.08.0 1
H
ppm
~ ~
173.0
171.2
171.4
171.6
171.8
172.0
172.2
172.4
172.8
13
C
Carbonyl groups
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
H CH
3
119.8
1.02
8.15
4.50
17.47
44.62
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
171.92
4.50
3.71
O
C
171.92
O
C
171.92
The consequence for the linkage of the
two amino acid building blocks seems
logical.
171.92 ppm / 3.71 ppm
171.92 ppm / 4.50 ppm
1.02.03.04.05.06.07.08.0 1
H
ppm
~ ~
173.0
171.2
171.4
171.6
171.8
172.0
172.2
172.4
172.8
13
C
Carbonyl groups
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
H CH
3
119.8
1.02
8.15
4.50
17.47
44.62
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
171.92
4.50
3.71
O
C
171.92
But what about this cross peak?
1.05
1.02
Because of two very close proton signals
there might be two possibilities:
171.92 ppm / 1.05 ppm
171.92 ppm / 1.02 ppm
4 bonds
even more than 4 bonds
Both possibilities seem to be unlikely.
171.92 ppm / 3.71 ppm
171.92 ppm / 4.50 ppm
171.92 ppm / 1.02(5) ppm
1.02.03.04.05.06.07.08.0 1
H
ppm
~ ~
173.0
171.2
171.4
171.6
171.8
172.0
172.2
172.4
172.8
13
C
Carbonyl groups
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
H CH
3
119.8
1.02
8.15
4.50
17.47
44.62
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
171.92
4.50
3.71
O
C
171.92
1.05
1.02
171.92 ppm / 3.71 ppm
171.92 ppm / 4.50 ppm
171.92 ppm / 1.02(5) ppm
What about switching the two amino acid
fragments?
Let us try!
1.02.03.04.05.06.07.08.0 1
H
ppm
~ ~
173.0
171.2
171.4
171.6
171.8
172.0
172.2
172.4
172.8
13
C
Carbonyl groups
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
171.92
4.50
3.71
O
C
171.92
1.05
1.02
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
N
C
H
H CH
3
119.8
1.02
8.15
4.50
17.47
44.62
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
171.92 ppm / 3.71 ppm
171.92 ppm / 4.50 ppm
171.92 ppm / 1.02(5) ppm
1.02.03.04.05.06.07.08.0 1
H
ppm
~ ~
173.0
171.2
171.4
171.6
171.8
172.0
172.2
172.4
172.8
13
C
Carbonyl groups
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
171.92
4.50
3.71
1.05
1.02
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
171.92 ppm / 3.71 ppm
171.92 ppm / 4.50 ppm
171.92 ppm / 1.02(5) ppm
Let us check our already found three
correlations step by step.
three bonds ✓
1.02.03.04.05.06.07.08.0 1
H
ppm
~ ~
173.0
171.2
171.4
171.6
171.8
172.0
172.2
172.4
172.8
13
C
Carbonyl groups
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
171.92
4.50
3.71
1.05
1.02
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
171.92 ppm / 4.50 ppm
171.92 ppm / 1.02(5) ppm
Let us check our already found three
correlations step by step.
two bonds ✓
1.02.03.04.05.06.07.08.0 1
H
ppm
~ ~
173.0
171.2
171.4
171.6
171.8
172.0
172.2
172.4
172.8
13
C
Carbonyl groups
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
171.92
4.50
3.71
1.05
1.02
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
171.92 ppm / 1.02(5) ppm
Let us check our already found three
correlations step by step.
three bonds ✓
1.02.03.04.05.06.07.08.0 1
H
ppm
~ ~
173.0
171.2
171.4
171.6
171.8
172.0
172.2
172.4
172.8
13
C
Carbonyl groups
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
171.92
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
There are further pieces of information that
are consistent with our structure.
7.60
two bonds ✓
1.02.03.04.05.06.07.08.0 1
H
ppm
~ ~
173.0
171.2
171.4
171.6
171.8
172.0
172.2
172.4
172.8
13
C
Carbonyl groups
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
171.92
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
There are further pieces of information that
are consistent with our structure.
8.15
three bonds ✓
1.02.03.04.05.06.07.08.0 1
H
ppm
~ ~
173.0
171.2
171.4
171.6
171.8
172.0
172.2
172.4
172.8
13
C
Carbonyl groups
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
The secret to connecting all of the amino acid
fragments in the right way using the carbonyl
groups seems to be contained in this area of
the HMBC.
1.02.03.04.05.06.07.08.0 1
H
ppm
~ ~
173.0
171.2
171.4
171.6
171.8
172.0
172.2
172.4
172.8
13
C
Carbonyl groups
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
We already used two of the cross peaks. Let us
extract the chemical shift pairs of the
remaining six cross peaks step by step.
1.02.03.04.05.06.07.08.0 1
H
ppm
~ ~
173.0
171.2
171.4
171.6
171.8
172.0
172.2
172.4
172.8
13
C
Carbonyl groups
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
172.16
4.70
172.16 / 4.70
1.02.03.04.05.06.07.08.0 1
H
ppm
~ ~
173.0
171.2
171.4
171.6
171.8
172.0
172.2
172.4
172.8
13
C
Carbonyl groups
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
172.16
3.71
172.16 / 4.70
172.16 / 3.71
1.02.03.04.05.06.07.08.0 1
H
ppm
~ ~
173.0
171.2
171.4
171.6
171.8
172.0
172.2
172.4
172.8
13
C
Carbonyl groups
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
171.53
4.50
172.16 / 4.70
172.16 / 3.71
171.53 / 4.50
1.02.03.04.05.06.07.08.0 1
H
ppm
~ ~
173.0
171.2
171.4
171.6
171.8
172.0
172.2
172.4
172.8
13
C
Carbonyl groups
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
171.53
4.00
172.16 / 4.70
172.16 / 3.71
171.53 / 4.50
171.53 / 4.00
1.02.03.04.05.06.07.08.0 1
H
ppm
~ ~
173.0
171.2
171.4
171.6
171.8
172.0
172.2
172.4
172.8
13
C
Carbonyl groups
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
171.14
4.70
172.16 / 4.70
172.16 / 3.71
171.53 / 4.50
171.53 / 4.00
171.14 / 4.70
1.02.03.04.05.06.07.08.0 1
H
ppm
~ ~
173.0
171.2
171.4
171.6
171.8
172.0
172.2
172.4
172.8
13
C
Carbonyl groups
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
171.14
4.00
172.16 / 4.70
172.16 / 3.71
171.53 / 4.50
171.53 / 4.00
171.14 / 4.70
171.14 / 4.00
We don‘t need any further information from
the HMBC. There are still three unassigned
carbonyl groups. Let‘s use these six
correlations to complete this assigment.
Carbonyl groups
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
172.16 / 4.70
172.16 / 3.71
171.53 / 4.50
171.53 / 4.00
171.14 / 4.70
171.14 / 4.00
O
C
172.16
172.16 / 4.70
172.16 / 3.71
Carbonyl groups
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
171.53 / 4.50
171.53 / 4.00
171.14 / 4.70
171.14 / 4.00
O
C
172.16
172.16 / 4.70
172.16 / 3.71
O
C
171.53
171.53 / 4.50
171.53 / 4.00
Carbonyl groups
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
171.14 / 4.70
171.14 / 4.00
O
C
172.16
172.16 / 4.70
172.16 / 3.71
O
C
171.53
171.53 / 4.50
171.53 / 4.00
O
C
171.14
171.14 / 4.70
171.14 / 4.00
Carbonyl groups
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
O
C
172.16
172.16 / 4.70
172.16 / 3.71
O
C
171.53
171.53 / 4.50
171.53 / 4.00
O
C
171.14
171.14 / 4.70
171.14 / 4.00
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
Apparently one carbonyl group is next to the
already known dipeptide fragment.
Carbonyl groups
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
O
C
172.16
172.16 / 4.70
O
C
171.53
171.53 / 4.50
171.53 / 4.00
O
C
171.14
171.14 / 4.70
171.14 / 4.00
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
The next correlation is over three bonds, as soon
as we attach the carbonyl atom with the chemical
shift of 172.16 ppm and the nitrogen with the
chemcial shift of 121.1 ppm together.
Carbonyl groups
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
O
C
172.16
172.16 / 4.70
O
C
171.53
171.53 / 4.50
171.53 / 4.00
O
C
171.14
171.14 / 4.70
171.14 / 4.00
C
N
H
H
3
C H
122.4
3.71
1.17
7.60
50.40
18.89
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
The next coorelation is over three bonds, as soon
as we attach the carbonyl atom with the chemical
shift of 172.16 ppm and the nitrogen with the
chemcial shift of 121.1 ppm together.
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
172.16 / 4.70
C
N
H
H
3
C H
121.1
1.05
8.21
4.70
18.14
44.84
Carbonyl groups
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
O
C
171.53
171.53 / 4.50
171.53 / 4.00
O
C
171.14
171.14 / 4.70
171.14 / 4.00
C
C
O
N
C
H
N
H
H
3
C H
H
3
C H
122.4
121.1
3.71
1.17
7.60
1.05
8.21
4.70
50.40
172.16
18.14
18.89
44.84
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
Let us continue.
Carbonyl groups
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
O
C
171.53
171.53 / 4.50
171.53 / 4.00
O
C
171.14
171.14 / 4.70
171.14 / 4.00
C
C
O
N
C
H
N
H
H
3
C H
H
3
C H
122.4
121.1
3.71
1.17
7.60
1.05
8.21
4.70
50.40
172.16
18.14
18.89
44.84
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
Let us continue.
O
C
171.53
Carbonyl groups
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
171.53 / 4.00
O
C
171.14
171.14 / 4.70
171.14 / 4.00
C
C
O
N
C
H
N
H
H
3
C H
H
3
C H
122.4
121.1
3.71
1.17
7.60
1.05
8.21
4.70
50.40
172.16
18.14
18.89
44.84
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
There is one more correlation.
O
C
171.53
Carbonyl groups
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
171.53 / 4.00
O
C
171.14
171.14 / 4.70
171.14 / 4.00
C
C
O
N
C
H
N
H
H
3
C H
H
3
C H
122.4
121.1
3.71
1.17
7.60
1.05
8.21
4.70
50.40
172.16
18.14
18.89
44.84
N
C
H
C
O
H CH
3
119.8
1.02
8.15
4.50
17.47
171.92
44.62
There is one more correlation.
O
C
171.53
N
C
CH
3
CH
3
H
124.3
≈2.9
1.38
4.00
53.97
16.63
30.20
Carbonyl groups
O
C
171.14
171.14 / 4.70
171.14 / 4.00
C
C
O
N
C
H
N
H
H
3
C H
H
3
C H
122.4
121.1
3.71
1.17
7.60
1.05
8.21
4.70
50.40
172.16
18.14
18.89
44.84
N
C
O
N
C
CH
3
C
CH
3
H
H
C
O
H CH
3
124.3
119.8
≈2.9
1.02
8.15
1.38
4.00
4.50
171.53
171.92
44.62
53.97
16.63
30.20
There is only one way to insert the last carbonyl group and to close
the ring.
Nevertheless let us check, whether the HMBC signals found earlier are
in coincidence with this ring closure.
Carbonyl groups
O
C
171.14
171.14 / 4.70
171.14 / 4.00
C
C
O
N
C
H
N
H
H
3
C H
H
3
C H
122.4
121.1
3.71
1.17
7.60
1.05
8.21
4.70
50.40
172.16
18.14
18.89
44.84
N
C
O
N
C
CH
3
C
CH
3
H
H
C
O
H CH
3
124.3
119.8
≈2.9
1.02
8.15
1.38
4.00
4.50
171.53
171.92
44.62
53.97
16.63
30.20
O
C
171.14
The last unassigned HMBC cross peak becomes explained by ring closure
between the two open ends of this linear molecule.
Carbonyl groups
171.14 / 4.00
C
C
O
N
C
H
N
H
H
3
C H
H
3
C H
122.4
121.1
3.71
1.17
7.60
1.05
8.21
4.70
50.40
172.16
18.14
18.89
44.84
N
C
O
N
C
CH
3
C
CH
3
H
H
C
O
H CH
3
124.3
119.8
≈2.9
1.02
8.15
1.38
4.00
4.50
171.53
171.92
44.62
53.97
16.63
30.20
C
(S)
O
C
O
N
C
C
H
N
H
H
3
C H
H
3
C H
122.4
121.1
3.71
1.17
7.60
1.05
8.21
4.70
50.40
172.16
18.14
18.89
44.84
N
C
O
N
C
CH
3
C
CH
3
H
H
C
O
H CH
3
124.3
119.8
≈2.9
1.02
8.15
1.38
4.00
4.50
17.47
171.53
171.92
44.62
53.97
16.63
30.20
171.14
And now let us beautify everything a little bit.
121.1
N
122.4
N
119.8
N
124.3
N
44.84
C
50.40
C
44.62
C
53.97
C
O
171.14
C
O
172.16
C
O
171.92
C
O
171.53
C
8.21
H
7.60
H
8.15
H
≈2.9
30.20
CH
3
4.70
H
3.71
H
4.50
H
4.00
H
H
3
C
H
3
C
CH
3
1.38
16.63
CH
3
1.17
1.05
18.14
18.89
1.02
17.47
Carbonyl groups
121.1
N
122.4
N
119.8
N
124.3
N
44.84
C
50.40
C
44.62
C
53.97
C
O
171.14
C
O
172.16
C
O
171.92
C
O
171.53
C
8.21
H
7.60
H
8.15
H
≈2.9
30.20
CH
3
4.70
H
3.71
H
4.50
H
4.00
H
H
3
C
H
3
C
CH
3
1.38
16.63
CH
3
Carbonyl groups
1.17
1.05
18.14
18.89
1.02
17.47
121.1
N
122.4
N
119.8
N
124.3
N
44.84
C
50.40
C
44.62
C
53.97
C
O
171.14
C
O
172.16
C
O
171.92
C
O
171.53
C
8.21
H
7.60
H
8.15
H
≈2.9
30.20
CH
3
4.70
H
3.71
H
4.50
H
4.00
H
CH
3
1.17
18.89
CH
3
1.02
17.47
CH
3
1.38
16.63
H
3
C
1.05
18.14
Final structure
Only the constitution, but not
the chirality (correctly stated
here) can be derived from the
HMBC/HSQC data.
Contributions
Measurements
Rainer Haeßner
Spectrometer time
TU Munich
Discussions and
native English
language support
Alan M. Kenwright
Nils Schlörer
Compilation
Rainer Haeßner
