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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

(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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Sequence determination Homo-Tyrosine

Sequence determination Homo-Tyrosine

Sequence determination Methionine

Sequence determination Methionine

Sequence determination Lysine

Sequence determination Lysine

Sequence determination Lysine

Sequence determination Phenylalanine

Sequence determination Valine

Sequence determination Valine

Sequence determination Homo-Phenylalanine

Sequence determination Homo-Phenylalanine

Structure elucidation Structure of polypeptide C C47H63N7O9S negative mode calculated: 900.4330 found: 900.4320

Structure elucidation Structure of polypeptide B C47H61N7O11 negative mode calculated: 898.4351 found: 898.4348

Structure elucidation Structure of polypeptide A C47H63N7O10S negative mode calculated: 916.4279 found: 916.4312

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

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 )

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

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 )

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

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