solving unknown structures using nmr organic structure analysis, crews, rodriguez and jaspars
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Solving Unknown Structures Using NMR
Organic Structure Analysis, Crews, Rodriguez and Jaspars
Six Simple Steps for Successful Structure Solution
• Get molecular formula. Use combustion analysis, mass spectrum and/or 13C NMR spectrum. Calculate double bond equivalents.
• Determine functional groups from IR, 1H and 13C NMR • Compare 1H integrals to number of H’s in the MF. • Determine coupling constants (J’s) for all multiplets. • Use information from 3. and 4. to construct spin systems
(substructures) • Assemble substructures in all possible ways, taking account of
dbe and functional groups. Make sure the integrals and coupling patterns agree with the proposed structure.
Organic Structure Analysis, Crews, Rodriguez and Jaspars
Organic Structure Analysis, Crews, Rodriguez and Jaspars
USING 1H NMR DATAUNKNOWN B
• A compound shows an M+. in the EIMS at 154 m/z• Further fragments are at 121, 93, 71, 55 and 39 m/z• The IR shows bands at 3400 cm-1 (broad) & 1450 cm-1
Use the 1H and 13C data to determine the structure of the compound
Organic Structure Analysis, Crews, Rodriguez and Jaspars
d
s
d
t
s
tq
q
t
q
13C NMR DATAUNKNOWN B
A B C D E F GHI J
Organic Structure Analysis, Crews, Rodriguez and Jaspars
MOLECULAR FORMULA DETERMINATIONUNKNOWN B
(C)2 + (CH)2 + (CH2)3 + (CH3)3 = C10H17
s d t q
BE
AC
DFI
GHJ
= 137 Da
The M+. appears at 154 m/z, so there isa mass difference of 17 Da (= OH)
Therefore molecular formula = C10H18O (2 dbe)
Organic Structure Analysis, Crews, Rodriguez and Jaspars
1H NMR DATAUNKNOWN B
Integrals:
H
3H 2H3H 3H
5HONLY 17 H!
Organic Structure Analysis, Crews, Rodriguez and Jaspars
SUBSTRUCTURESUNKNOWN B
H3C
H
CH3
A
C 125 d, 132 sH 5.05 t&Me groups at H 1.5
H
H
H
B
C 112 t, 146 dH 5.00 dd, 5.15 dd, 5.85 dd
H3C OH
C
C 73 sH 1.05 s
H H H H
D'D
MF = C10H18O !
Organic Structure Analysis, Crews, Rodriguez and Jaspars
WORKING STRUCTURESUNKNOWN B
OH
Ha
Ha
Ha
OH
OH
A – Ha should be ddt
B – Ha should be ddt
C – Ha should be dd
Organic Structure Analysis, Crews, Rodriguez and Jaspars
MASS SPECTRAL FRAGMENTATIONUNKNOWN B
OH
Ha
O+mass spectral fragmentation
1m/z 99
H
HaOH
+O
O+
mass spectral fragmentation
2
m/z 113
m/z 85
H
H
Ha
OH +O
mass spectral fragmentation
m/z 71H
3
Fragments at: 121, 93, 71, 55 and 39 m/z
Organic Structure Analysis, Crews, Rodriguez and Jaspars
USING MASS SPECTRAL DATAUNKNOWN G
• A compound shows an M+. in the EIMS at 128 m/z• Further fragments are at 99, 83, 72 and 57 m/z• The IR shows bands at 1680 cm-1 (strong) &
bands at 1400 - 1500 cm-1
Use the 1H and 13C NMR and MS data to determine the structure of the compound
Organic Structure Analysis, Crews, Rodriguez and Jaspars
13C NMR DATAUNKNOWN G
A B C D E FG
d dd t
t t q
Organic Structure Analysis, Crews, Rodriguez and Jaspars
MOLECULAR FORMULA DETERMINATIONUNKNOWN G
(C)0 + (CH)3 + (CH2)3 + (CH3)1 = C7H12
s d t qABC
DEF
G
= 96 Da
The M+. appears at 128 m/z, so there isa mass difference of 32 Da (= O2)
Therefore molecular formula = C7H12O2 (2 dbe)
Organic Structure Analysis, Crews, Rodriguez and Jaspars
1H NMR DATAUNKNOWN G
Integrals:
HH
H H
3H
H H2H
H
12 H Total
Organic Structure Analysis, Crews, Rodriguez and Jaspars
SUBSTRUCTURES UNKNOWN G
C 140 d, 101 dH 6.15 d, 4.70 m
C 96 dH 4.9 tO
H
H
O
O
H
O
H H
C 64 tMe
4 Oxygens in substructures but only 2 in MF
MF = C7H12O2 !
Organic Structure Analysis, Crews, Rodriguez and Jaspars
WORKING STRUCTURES UNKNOWN G
O
O
O OO O
36*
62 20*
15
64
26
10*64
15
O O O
O O+.
+
m/z 83
m/z 99
+.O O
O O
m/z 72m/z 56
+.
13C Shift additivity data
MS Fragmentation
Retro Diels-Alder
Fragments are at 99, 83, 72 and 57 m/z
Organic Structure Analysis, Crews, Rodriguez and Jaspars
USING COSY DATAUNKNOWN H
• A compound shows an [M + H]+ in the FAB MS at 132 m/z• MW = 131 (Odd) therefore odd number of nitrogens• A further fragment is at 86 m/z• The IR shows bands at 3400cm-1 (broad) & 1640 cm-1 (broad)
Use the NMR data to determine the structure of the compound
Organic Structure Analysis, Crews, Rodriguez and Jaspars
d
s
d
t
t
13C NMR DATAUNKNOWN H
A B C D E
Organic Structure Analysis, Crews, Rodriguez and Jaspars
MOLECULAR FORMULA DETERMINATIONUNKNOWN H
(C)1 + (CH)2 + (CH2)2 + (CH3)0 = C5H6
s d t q
A BC
DE
= 66 Da
The MW is 131, so there isa mass difference of 65 Da (= NO3H3)
Therefore molecular formula = C5H9NO3 (2 dbe)
Organic Structure Analysis, Crews, Rodriguez and Jaspars
1H NMR DATAUNKNOWN H
Integrals:
HONLY 6 H!
H2H H H
D2O so noXH (OH, NH)
b c d d’ e e’
Organic Structure Analysis, Crews, Rodriguez and Jaspars
SUBSTRUCTURESUNKNOWN H
C 176 sIR band at 3400
C 70 dH 4.6 m
C 60 dH 3.9 m
MF = C5H9NO3 !
OH
O
A
OH
B
C
HN
ED
Organic Structure Analysis, Crews, Rodriguez and Jaspars
1H – 1H COSY NMR SPECTRUMUNKNOWN H
b c d d’ e e’
b-e/e’
c-e/e’
d-d’
d-e’
e-e’
b-d/d’
Organic Structure Analysis, Crews, Rodriguez and Jaspars
1H – 1H COSY NMR DATAUNKNOWN H
c
c-e/e’
e e'
b-e/e’
OHb
b-d/d’
d d'
Also 4-bond correlation d-e’Diastereotopic pairs d-d’ and e-e’
Organic Structure Analysis, Crews, Rodriguez and Jaspars
SUBTRUCTURESUNKNOWN H
c
e e'
OHb
d d'
HN
OH
O
MF = C5H9NO3
HN COOH
HO
Hc
Hb
HN COOH
HO Hb
Hc
1 2
Working structures:
12 % NOENO NOE
Organic Structure Analysis, Crews, Rodriguez and Jaspars
ASSIGNING NMR DATA TO A KNOWN STRUCTUREGUAIAZULENE
10 12
3
4
56
8
9
11
12
1314
15
7
MF = C15H18
Expect:(C)5
(CH)6
(CH2)0
(CH3)4
Organic Structure Analysis, Crews, Rodriguez and Jaspars
13C NMR DATAGUAIAZULENE
sA
sB
dJ
sD
dddEFG
sC
dH
dK
qqLM
qN
qO
sI
(C)5 + (CH)6 + (CH2)0 + (CH3)4 = C15H18
Organic Structure Analysis, Crews, Rodriguez and Jaspars
HSQC NMR DATAGUAIAZULENE
KK
k
LMLM
lm
NN
n
OO
o
Organic Structure Analysis, Crews, Rodriguez and Jaspars
HSQC NMR DATAGUAIAZULENE
EE
e
FF
f
GG
g
HH
h
JJ
j
Organic Structure Analysis, Crews, Rodriguez and Jaspars
1H NMR DATAGUAIAZULENE
Hg
He
Hf
Hj
Hh
Hk
3Hn
3Ho
3Hl
3Hm
Label spectrum according to HSQC:
Organic Structure Analysis, Crews, Rodriguez and Jaspars
HMBC NMR DATAGUAIAZULENE
We will need expansions:
Organic Structure Analysis, Crews, Rodriguez and Jaspars
HMBC NMR DATAGUAIAZULENE
g e f j h
HI
GFCDEB
A
C-gF-g
I-g
D-e
A-f
G-fD-j
I-j
B-h
C-h
Organic Structure Analysis, Crews, Rodriguez and Jaspars
HMBC NMR DATAGUAIAZULENE
g e f j h
O
NLM
KK-g K-f
N-h
Organic Structure Analysis, Crews, Rodriguez and Jaspars
HMBC NMR DATAGUAIAZULENE
n o lm
LM-lmK-lm
B-lm
I-oH-n
D-oC-n
A-n
Organic Structure Analysis, Crews, Rodriguez and Jaspars
HMBC NMR DATAGUAIAZULENE
Carbon Proton
A f, n
B h, l/m
C g, h, n
D e, j, o
E
F g
G f
H n
I g, j, o
J
K f, g, l/m
L m
M l
N h
O
1H-1H COSY data indicates that e and j are adjacent (J(e-j) = 4 Hz) as are f and h (J(f-h) = 11 Hz)
‘Obvious’ assignments:
KL
M
N/O
O/N
E/J
J/E F/H
H/F
Organic Structure Analysis, Crews, Rodriguez and Jaspars
HMBC NMR DATAGUAIAZULENE
Carbon Proton
A f, n
B h, l/m
C g, h, n
D e, j, o
E
F g
G f
H n
I g, j, o
J
K f, g, l/m
L m
M l
N h
O
KL
M
N/O
O/N
E/J
J/E H
F
G
Signal for f is a ddlong-range couplingto remaining proton g
Organic Structure Analysis, Crews, Rodriguez and Jaspars
HMBC NMR DATAGUAIAZULENE
Carbon Proton
A f, n
B h, l/m
C g, h, n
D e, j, o
E
F g
G f
H n
I g, j, o
J
K f, g, l/m
L m
M l
N h
O
KL
M
E/J
J/E H
F
G B
A
N
O
C
HMBC data can’t decide positions of E, JHMBC data can’t decide positions of D, I
How do we decide?
Organic Structure Analysis, Crews, Rodriguez and Jaspars
FINALISING THE ASSIGNMENTSGUAIAZULENE
KL
M
E
J H
F
G B
A
O
C
NNOE
Placing D (134 ppm) here
D
puts it in a similar environment to C (136 ppm)
This puts I (125 ppm) here
I
Complex Peptide from a Cyanobacterium
ppm (t1)1.02.03.04.05.06.07.0
0
50000000
100000000
2.00
10.06
0.250.950.91
0.96
2.60
2.02
0.991.081.93
3.92
3.99
2.893.943.92
1.15
3.49
1.520.97
6.060.81
1.98
(600 MHz in MeOH-d4)
HSQC-TOCSY Spectra
Organic Structure Analysis, Crews, Rodriguez and Jaspars
A B C D E F
ab
c
d
d'
ef
C
H
Direct correlations (C-H)
Indirect (long range) correlations
Mixing time 30-180 ms3-7 bonds
H
C C C C C C
H H H H H
C
H
A B C D E F G
a b c d e f g
(600 MHz in MeOH-d4)
ppm (t2)1.001.502.002.503.003.504.004.50
50.0
55.0
60.0
ppm (t1)
Homo-Tyrosine
HSQC-TOCSY-spectra of fraction C
-HTyr4.78
HN
O
OH
HMBC
HSQC-TOCSY-spectra of fraction C
(600 MHz in MeOH-d4)
ppm (t2)1.001.502.002.503.003.504.004.50
50.0
55.0
60.0
ppm (t1)
-HPhe4.75
HN
OHMBC
Homo-Phenylalanine
(600 MHz in MeOH-d4)
ppm (t2)1.001.502.002.503.003.504.004.50
50.0
55.0
60.0
ppm (t1)
Phenylalanine
HSQC-TOCSY-spectra of fraction C
-Phe4.55
HN
OHMBC
(600 MHz in MeOH-d4)
ppm (t2)1.001.502.002.503.003.504.004.50
50.0
55.0
60.0
ppm (t1)
Methionine
HSQC-TOCSY-spectra of fraction C
-Met4.54
HN
O
S
HMBC
(600 MHz in MeOH-d4)
ppm (t2)1.001.502.002.503.003.504.004.50
50.0
55.0
60.0
ppm (t1)
Lysine
HSQC-TOCSY-spectra of fraction C
-Lys3.67
-Lys2.92
-Lys4.06
HN
O
HN
(600 MHz in MeOH-d4)
ppm (t2)1.001.502.002.503.003.504.004.50
50.0
55.0
60.0
ppm (t1)
Valine
HSQC-TOCSY-spectra of fraction C
-Val3.93
HN
O
HMBC-spectra of fraction C
(600 MHz in MeOH-d4)
ppm (t2)2.302.402.502.602.702.802.903.003.103.20
130.0
135.0
140.0
ppm (t1)
HN
O
-Phe3.14
-Phe3.00
130.5
138.3
Phenylalanine
HMBC-spectra of fraction C
(600 MHz in MeOH-d4)
ppm (t2)2.302.402.502.602.702.802.903.003.103.20
130.0
135.0
140.0
ppm (t1)
Homo-Phenylalanine
HN
O
-HPhe2.88
-HPhe2.73
142.3
129.8
HMBC-spectra of fraction C
(600 MHz in MeOH-d4)
ppm (t2)2.302.402.502.602.702.802.903.003.103.20
130.0
135.0
140.0
ppm (t1)
Homo-Tyrosine -HTyr2.36
HN
O
OH
130.0
133.1
HMBC-spectra of fraction C
(600 MHz in MeOH-d4)
Methionine
ppm (t2)1.801.902.002.102.202.302.40
10.0
15.0
20.0
ppm (t1)
HN
O
S
-Met2.34
15.1
Amino acid residues of fraction C
HN
O
HN
O
HN
O
OH
HN
O
SHN
O
HN
HN
O
Phe HPhe Tyr
Met Lys Val
Determination of the amino acid sequence
HMBC-correlation between one carbonyl and the - and -hydrogen of an amino acid residue
HN
O
R
HN
O
HN
O
HN
O
OH
HN
O
SHN
O
HN
HN
O
Phe HPhe Tyr
Met Lys Val
HMBC-correlation between one carbonyl and the - and -hydrogen of an amino acid residue
Correlation between this carbonyl and the -hydrogen of the connected amino acid
Chemical shift of the second carbonyl is determined
HN
O
R
NH
O
R'
Amino acid residues of fraction C
Determination of the amino acid sequence
HN
O
HN
O
HN
O
OH
HN
O
SHN
O
HN
HN
O
Phe HPhe Tyr
Met Lys Val
HMBC-correlation between one carbonyl and the - and -hydrogen of an amino acid residue
Correlation between this carbonyl and the -hydrogen of the connected amino acid
Repetition of this operation should allow completion of the sequence
Chemical shift of the second carbonyl is determined
HN
O
R
NH
O
R'NH
O
R''
Amino acid residues of fraction C
Determination of the amino acid sequence
Sequence determination
13C-spectra of fraction C
ppm (t1)172.0173.0174.0175.0176.0
0
5000000
10000000175.
892
175.
819
174.
921
174.
843
174.
411
172.
001
(150 MHz in MeOH-d4)
A175.9
B175.8
C174.9
D174.8
E174.4
F172.0
Sequence determination
Homo-Tyrosine
ppm (t2)2.002.503.003.504.004.50
160.0
165.0
170.0
175.0
ppm (t1)
-HTyr4.78
-HTyr2.18
-HTyr1.84
N
O
HO
F
F172.0
Homo-Tyrosine
ppm (t2)2.002.503.003.504.004.50
160.0
165.0
170.0
175.0
ppm (t1)
-Met4.54
N
HN
OO
SMe
HO
F
Sequence determination
-HTyr4.78
-HTyr2.18
-HTyr1.84
F172.0
Methionine
ppm (t2)2.002.503.003.504.004.50
160.0
165.0
170.0
175.0
ppm (t1)
-Met4.54
-Met2.18
-Met1.93
HN
O
SMe
E
E174.4
Sequence determination
Methionine
ppm (t2)2.002.503.003.504.004.50
160.0
165.0
170.0
175.0
ppm (t1)
-Lys3.67
-Lys2.92
HN NH
O
O
HN
SMe
E
Sequence determination
-Met4.54
-Met2.18
-Met1.93
E174.4
Lysine
ppm (t2)2.002.503.003.504.004.50
160.0
165.0
170.0
175.0
ppm (t1)
-Lys1.86
-Lys1.67
-Lys4.06
B175.8
NH
OHNB
Sequence determination
Lysine
ppm (t2)2.002.503.003.504.004.50
160.0
165.0
170.0
175.0
ppm (t1)
-Val3.93
NH
NH
O
O
HNB
Sequence determination
-Lys1.86
-Lys1.67
-Lys4.06
B175.8
Lysine
ppm (t2)2.002.503.003.504.004.50
160.0
165.0
170.0
175.0
ppm (t1)
-Phe4.55
U159.5
NH
OHN
O
HN
O
B U
Sequence determination
-Lys4.06
B175.8
Phenylalanine
ppm (t2)2.002.503.003.504.004.50
160.0
165.0
170.0
175.0
ppm (t1)
A175.9
-Phe3.14
-Phe3.00
-Phe4.55
HN
O A
Sequence determination
Valine
ppm (t2)2.002.503.003.504.004.50
160.0
165.0
170.0
175.0
ppm (t1)
NH
OD
-Val3.93
-Val1.99
D174.8
Sequence determination
Valine
ppm (t2)2.002.503.003.504.004.50
160.0
165.0
170.0
175.0
ppm (t1)
-HPhe4.75
HN
NH
OO
D
Sequence determination
-Val3.93
-Val1.99
D174.8
Homo-Phenylalanine
ppm (t2)2.002.503.003.504.004.50
160.0
165.0
170.0
175.0
ppm (t1)
HN
O
C
Sequence determination
-HPhe4.75
-HPhe2.12
-HPhe1.90
C174.9
Homo-Phenylalanine
ppm (t2)2.002.503.003.504.004.50
160.0
165.0
170.0
175.0
ppm (t1)
-HPhe2.12
-HPhe1.90
-HTyr4.75
C174.9
NMe2.72
N
HN
O
O
HO
C
Sequence determination
-HPhe4.75
N
HN
HN
NH
NH
O
OO
OO
HN
O
HN
O OH
SMe
HO
HPhe
HTyr
Met
Val
Lys
Phe
Structure elucidation
Structure of polypeptide C
negative modecalculated: 900.4330 found: 900.4320
C47H63N7O9S
N
HN
HN
NH
NH
O
OO
OO
HN
O
HN
O OH
O
HO
O
HPhe
HTyr
Ser(Ac)
Val
Lys
Phe
Structure elucidation
Structure of polypeptide B
negative modecalculated: 898.4351 found: 898.4348
C47H61N7O11
N
HN
HN
NH
NH
O
OO
OO
HN
O
HN
O OH
HO
SO
HPhe
HTyr
Met(O)
Val
Lys
Phe
Structure elucidation
Structure of polypeptide A
negative modecalculated: 916.4279 found: 916.4312
C47H63N7O10S
Nodulapeptin A
Non-Toxic Peptide isolated from Nodularia spumigena AV1 by Harada and co-workers
Fujii K., Sivonen K., Adachi K., Noguchi K., Sano H., Hirayama K., Suzuki M and Harada K. Tetrahedron Lett. 1997, 38, 5525-5528
HPhe
HTyr
Ser(Ac)
Met(O2)
Lys
Ile
N
HN
HN
NH
NH
O
OO
OO
HN
O
HN
O OH
O
HO
O
SO
O
1H NMR ( DMSO, 600MHz )
N
N
HN
H2N O
O O
22
3
3
4
48
989
18
18`19`
19
201818`
201919`
12
12
16
16
16
21
21
Dermacozine ADermacozine A7 . 1 57 . 2 07 . 2 57 . 3 07 . 3 57 . 4 07 . 4 57 . 5 07 . 5 57 . 6 07 . 6 57 . 7 07 . 7 57 . 8 07 . 8 57 . 9 07 . 9 58 . 0 08 . 0 5
f 1 ( p p m )
1.05
3.04
1.05
2.27
1.07
1.13
1.19
1.00
7.1
7
7.1
9
7.2
7
7.2
9
7.3
0
7.3
9
7.4
0
7.4
1
7.4
5
7.4
6
7.4
7
7.7
5
7.7
6
7.7
7
7.8
4
7.9
3
7.9
5
8.0
0
8.0
2
Dermacozine
( DMSO, 600MHz )
4
3
2
32 4
8
89
9
20
19
18
18
1920
N
N
NO OMe
NO
H
2
3
4
8
9
17
18'
19
19'
20
18
HH
16
16
16
2D TOCSY-spectra
( DMSO, 150MHz )
Dermacozine ADermacozine A
N
N
HN
H2N O
O O21
21
11
11
13
13
15
15
9a
9a
18
18`19`
19
20
1 2 11 2 21 2 31 2 41 2 51 2 61 2 71 2 81 2 91 3 01 3 11 3 21 3 31 3 41 3 51 3 61 3 71 3 81 3 91 4 01 4 11 4 21 4 31 4 41 4 5f 1 ( p p m )
122.
68
124.
81
130.
18
130.
85
131.
19
133.
29
133.
85
134.
35
134.
85
136.
80
136.
97
137.
30
138.
34
142.
14
142.
76
20
1919`
1818`
112
2
3
3
4
4
4a
4a
5a
5a
6
6
7
78
89
9 10a10a
14
14
17
17
13C - spectra
7.07.17.27.37.47.57.67.77.87.98.08.1f2 (ppm)
100
105
110
115
120
125
130
135
140
145
150
155
160
165
170
f1 (
ppm
)
2
15
9
5a
8
14
18
6
9a
4
10a
3
N
N
NO OMe
NO
H
1
2
3
44a 5a
67
8
99a10a
15
11 13
14
17
18'
19
19'
20
18
12
16
21
10
5
HH
1
16
HMBC- spectra
( DMSO,600MHz )
( DMSO,600MHz )
N
N
NO O
Me
NH2O
H
1
2
3
44a 5a
67
8
99a10a
15
11 13
14
17
18'
19
19'
20
18
12
16
21
10
5
15N HSQC- spectra
12 16 16
21
N-12
N-16
N- 5
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