1. Solving Unknown Structures Using NMR
Organic Structure Analysis, Crews, Rodriguez and Jaspars

2. 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. 
SOLUTION
Organic Structure Analysis, Crews, Rodriguez and Jaspars

3. USING 1H NMR DATA UNKNOWN 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

4. 13C NMR DATA UNKNOWN B t t t q q d d q s s A B C D E F GHI J
Organic Structure Analysis, Crews, Rodriguez and Jaspars

5. MOLECULAR FORMULA DETERMINATION
UNKNOWN B
s d t q
(C) + (CH) + (CH2) + (CH3) = C H
= Da
The M+. appears at 154 m/z, so there is
a mass difference of Da (= )
Therefore molecular formula = C H O ( dbe)
Organic Structure Analysis, Crews, Rodriguez and Jaspars

6. 1H NMR DATA UNKNOWN B Integrals: ONLY 17 H! 5H 3H 3H 3H 2H H
Organic Structure Analysis, Crews, Rodriguez and Jaspars

7. SUBSTRUCTURES UNKNOWN B MF = C H O
Organic Structure Analysis, Crews, Rodriguez and Jaspars

8. WORKING STRUCTURES UNKNOWN B
Organic Structure Analysis, Crews, Rodriguez and Jaspars

9. MASS SPECTRAL FRAGMENTATION
UNKNOWN B
Fragments at:
121, 93, 71, 55 and 39 m/z
Organic Structure Analysis, Crews, Rodriguez and Jaspars

10. USING MASS SPECTRAL DATA
UNKNOWN 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 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

11. 13C NMR DATA UNKNOWN G t t q d d d t A B C D E FG
Organic Structure Analysis, Crews, Rodriguez and Jaspars

12. MOLECULAR FORMULA DETERMINATION
UNKNOWN G
s d t q
(C) + (CH) + (CH2) + (CH3) = C H
= Da
The M+. appears at 128 m/z, so there is
a mass difference of Da (= )
Therefore molecular formula = C H O ( dbe)
Organic Structure Analysis, Crews, Rodriguez and Jaspars

13. 1H NMR DATA UNKNOWN G Integrals: 12 H Total 3H 2H H H H H H H H
Organic Structure Analysis, Crews, Rodriguez and Jaspars

14. SUBSTRUCTURES UNKNOWN G MF = C H O
Organic Structure Analysis, Crews, Rodriguez and Jaspars

15. WORKING STRUCTURES UNKNOWN G 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

16. USING COSY DATA UNKNOWN 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

17. 13C NMR DATA UNKNOWN H d t t d s A B C D E
Organic Structure Analysis, Crews, Rodriguez and Jaspars

18. MOLECULAR FORMULA DETERMINATION
UNKNOWN H
s d t q
(C) + (CH) + (CH2) + (CH3) = C H
= Da
The MW is 131, so there is
a mass difference of Da (= )
Therefore molecular formula = C H N O ( dbe)
Organic Structure Analysis, Crews, Rodriguez and Jaspars

19. 1H NMR DATA UNKNOWN H Integrals: D2O so no XH (OH, NH) ONLY 6 H! H H H
b c d d’ e e’
Organic Structure Analysis, Crews, Rodriguez and Jaspars

20. SUBSTRUCTURES UNKNOWN H MF = C H N O
Organic Structure Analysis, Crews, Rodriguez and Jaspars

21. 1H – 1H COSY NMR SPECTRUM UNKNOWN H b c d d’ e e’
Organic Structure Analysis, Crews, Rodriguez and Jaspars

22. 1H – 1H COSY NMR DATA UNKNOWN H
Combine your substructures using COSY data
Organic Structure Analysis, Crews, Rodriguez and Jaspars

23. SUBTRUCTURES UNKNOWN H MF = C H N O Working structures
Consider stereochemistry:
MF = C H N O
Organic Structure Analysis, Crews, Rodriguez and Jaspars

24. ASSIGNING NMR DATA TO A KNOWN STRUCTURE
GUAIAZULENE
Expect:
(C)
(CH)
(CH2)
(CH3)
MF = C H
Organic Structure Analysis, Crews, Rodriguez and Jaspars

25. (C) + (CH) + (CH2) + (CH3) = C H
13C NMR DATA
GUAIAZULENE
(C) + (CH) + (CH2) + (CH3) = C H qq LM d J d H d K
ddd
EFG q N q O s B s D s I s A s C
Organic Structure Analysis, Crews, Rodriguez and Jaspars

26. HSQC NMR DATA GUAIAZULENE n o lm k O O N N LM LM K K
Organic Structure Analysis, Crews, Rodriguez and Jaspars

27. HSQC NMR DATA GUAIAZULENE J H G F E
Organic Structure Analysis, Crews, Rodriguez and Jaspars

28. Label spectrum according to HSQC:
1H NMR DATA
GUAIAZULENE
Label spectrum according to HSQC: 3H l 3H m 3H n 3H o H H H H H H k
Organic Structure Analysis, Crews, Rodriguez and Jaspars

29. We will need expansions:
HMBC NMR DATA
GUAIAZULENE
We will need expansions:
Organic Structure Analysis, Crews, Rodriguez and Jaspars

30. HMBC NMR DATA GUAIAZULENE g e f j h I-g I-j G-f F-g C-h D-j C-g D-e
CDE B A
I-g
I-j
G-f
F-g
C-h
D-j
C-g
D-e
B-h
A-f
Organic Structure Analysis, Crews, Rodriguez and Jaspars

31. HMBC NMR DATA GUAIAZULENE g e f j h N-h K-g K-f O N LM K
Organic Structure Analysis, Crews, Rodriguez and Jaspars

32. HMBC NMR DATA GUAIAZULENE n o lm LM-lm K-lm H-n I-o C-n D-o B-lm A-n
Organic Structure Analysis, Crews, Rodriguez and Jaspars

33. HMBC NMR DATA GUAIAZULENE ‘Obvious’ assignments:
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
‘Obvious’ assignments:
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)
Organic Structure Analysis, Crews, Rodriguez and Jaspars

34. HMBC NMR DATA GUAIAZULENE Signal for f is a dd long-range coupling
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
Signal for f is a dd
long-range coupling
to remaining proton g
Organic Structure Analysis, Crews, Rodriguez and Jaspars

35. HMBC NMR DATA GUAIAZULENE HMBC data can’t decide positions of E, J
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
HMBC data can’t decide positions of E, J
HMBC data can’t decide positions of D, I
How do we decide?
Organic Structure Analysis, Crews, Rodriguez and Jaspars

36. FINALISING THE ASSIGNMENTS
GUAIAZULENE
Organic Structure Analysis, Crews, Rodriguez and Jaspars

37. Complex Peptide from a Cyanobacterium
(600 MHz in MeOH-d4)

38. HSQC-TOCSY Spectra Mixing time 30-180 ms 3-7 bonds
Organic Structure Analysis, Crews, Rodriguez and Jaspars

39. HSQC-TOCSY-spectra of fraction C
Homo-Tyrosine
(600 MHz in MeOH-d4)

40. HSQC-TOCSY-spectra of fraction C
Homo-Phenylalanine
(600 MHz in MeOH-d4)

41. HSQC-TOCSY-spectra of fraction C
Phenylalanine
(600 MHz in MeOH-d4)

42. HSQC-TOCSY-spectra of fraction C
Methionine
(600 MHz in MeOH-d4)

43. HSQC-TOCSY-spectra of fraction C
Lysine
(600 MHz in MeOH-d4)

44. HSQC-TOCSY-spectra of fraction C
Valine
(600 MHz in MeOH-d4)

45. HMBC-spectra of fraction C
Phenylalanine
(600 MHz in MeOH-d4)

46. HMBC-spectra of fraction C
Homo-Phenylalanine
(600 MHz in MeOH-d4)

47. HMBC-spectra of fraction C
Homo-Tyrosine
(600 MHz in MeOH-d4)

48. HMBC-spectra of fraction C
Methionine
(600 MHz in MeOH-d4)

49. Amino acid residues of fraction C
Determination of the amino acid sequence
HMBC-correlation between one carbonyl and
the a- and b-hydrogen of an amino acid residue

50. Amino acid residues of fraction C
Determination of the amino acid sequence
HMBC-correlation between one carbonyl and
the a- and b-hydrogen of an amino acid residue
Correlation between this carbonyl and
the a-hydrogen of the connected amino acid
Chemical shift of the second carbonyl is determined

51. Amino acid residues of fraction C
Determination of the amino acid sequence
HMBC-correlation between one carbonyl and
the a- and b-hydrogen of an amino acid residue
Correlation between this carbonyl and
the a-hydrogen of the connected amino acid
Chemical shift of the second carbonyl is determined
Repetition of this operation should
allow completion of the sequence

52. Sequence determination
13C-spectra of fraction C
(150 MHz in MeOH-d4)

53. Sequence determination
Homo-Tyrosine

54. Sequence determination
Homo-Tyrosine

55. Sequence determination
Methionine

56. Sequence determination
Methionine

57. Sequence determination
Lysine

58. Sequence determination
Lysine

59. Sequence determination
Lysine

60. Sequence determination
Phenylalanine

61. Sequence determination
Valine

62. Sequence determination
Valine

63. Sequence determination
Homo-Phenylalanine

64. Sequence determination
Homo-Phenylalanine

65. Structure elucidation
Structure of polypeptide C
C47H63N7O9S
negative mode
calculated:
found:

66. Structure elucidation
Structure of polypeptide B
C47H61N7O11
negative mode
calculated:
found:

67. Structure elucidation
Structure of polypeptide A
C47H63N7O10S
negative mode
calculated:
found:

68. 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,

69. Dermacozine 1H NMR ( DMSO, 600MHz ) Dermacozine A 21 12 21 4 18 3 19
18` 2 9 8 8
18` 20 2 4 16 3
19` 20 9
19` 16
Dermacozine A 16 12
1H NMR ( DMSO, 600MHz )

70. 2D TOCSY-spectra ( DMSO, 600MHz ) 18 9 19 8 20 2 4 3 8 9 18 20 19 3 416 2 4 3 8 9 18 20 19 3 16 4 2 16
( DMSO, 600MHz )

71. 13C - spectra ( DMSO, 150MHz ) Dermacozine A 21 11 13 4 6 14 4a 5a 18
19` 3 17 19 19 7 18
18` 20 2 8 7 5a
10a 8 1 3 9 14 4
10a 9a
18` 20 17 4a 2 1 9
19` 15
Dermacozine A 21 15 13 11 6 9a
( DMSO, 150MHz )

72. HMBC- spectra 18 9 8 2 4 16 3 6 14 1
10a 5a 9a 15
( DMSO,600MHz )

73. 15N HSQC- spectra 21 12 16 16
N- 5
N-16
N-12
( DMSO,600MHz )