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 13 C NMR spectrum. Calculate double bond equivalents. Determine functional groups from IR, 1 H and 13 C NMR Compare 1 H 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 

USING 1 H 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 1 H and 13 C data to determine the structure of the compound

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

Organic Structure Analysis, Crews, Rodriguez and Jaspars MOLECULAR FORMULA DETERMINATION UNKNOWN B (C) 2 + (CH) 2 + (CH 2 ) 3 + (CH 3 ) 3 = C 10 H 17 s d t q BEBE ACAC DFIDFI GHJGHJ = 137 Da The M +. appears at 154 m/z, so there is a mass difference of 17 Da (= OH) Therefore molecular formula = C 10 H 18 O (2 dbe)

Organic Structure Analysis, Crews, Rodriguez and Jaspars 1 H NMR DATA UNKNOWN B Integrals: H 3H2H 3H 5H ONLY 17 H!

Organic Structure Analysis, Crews, Rodriguez and Jaspars SUBSTRUCTURES UNKNOWN B  C 125 d, 132 s  H 5.05 t & Me groups at  H 1.5  C 112 t, 146 d  H 5.00 dd, 5.15 dd, 5.85 dd  C 73 s  H 1.05 s MF = C 10 H 18 O !

Organic Structure Analysis, Crews, Rodriguez and Jaspars WORKING STRUCTURES UNKNOWN B A – Ha should be ddt B – Ha should be ddt C – Ha should be dd

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 cm -1 Use the 1 H and 13 C NMR and MS data to determine the structure of the compound

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

Organic Structure Analysis, Crews, Rodriguez and Jaspars MOLECULAR FORMULA DETERMINATION UNKNOWN G (C) 0 + (CH) 3 + (CH 2 ) 3 + (CH 3 ) 1 = C 7 H 12 s d t q ABCABC DEFDEF G = 96 Da The M +. appears at 128 m/z, so there is a mass difference of 32 Da (= O 2 ) Therefore molecular formula = C 7 H 12 O 2 (2 dbe)

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

Organic Structure Analysis, Crews, Rodriguez and Jaspars SUBSTRUCTURES UNKNOWN G  C 140 d, 101 d  H 6.15 d, 4.70 m  C 96 d  H 4.9 t  C 64 t 4 Oxygens in substructures but only 2 in MF MF = C 7 H 12 O 2 !

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

Organic Structure Analysis, Crews, Rodriguez and Jaspars MOLECULAR FORMULA DETERMINATION UNKNOWN H (C) 1 + (CH) 2 + (CH 2 ) 2 + (CH 3 ) 0 = C 5 H 6 s d t q A BCBC DEDE = 66 Da The MW is 131, so there is a mass difference of 65 Da (= NO 3 H 3 ) Therefore molecular formula = C 5 H 9 NO 3 (2 dbe)

Organic Structure Analysis, Crews, Rodriguez and Jaspars 1 H NMR DATA UNKNOWN H Integrals: H ONLY 6 H! H 2H HH D 2 O so no XH (OH, NH) b c d d’ e e’

Organic Structure Analysis, Crews, Rodriguez and Jaspars SUBSTRUCTURES UNKNOWN H  C 176 s IR band at 3400  C 70 d  H 4.6 m  C 60 d  H 3.9 m MF = C 5 H 9 NO 3 !

Organic Structure Analysis, Crews, Rodriguez and Jaspars 1 H – 1 H COSY NMR SPECTRUM UNKNOWN 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 1 H – 1 H COSY NMR DATA UNKNOWN H c-e/e’b-e/e’b-d/d’ Also 4-bond correlation d-e’ Diastereotopic pairs d-d’ and e-e’

Organic Structure Analysis, Crews, Rodriguez and Jaspars SUBTRUCTURES UNKNOWN H MF = C 5 H 9 NO 3 Working structures:

Organic Structure Analysis, Crews, Rodriguez and Jaspars ASSIGNING NMR DATA TO A KNOWN STRUCTURE GUAIAZULENE MF = C 15 H 18 Expect: (C) 5 (CH) 6 (CH 2 ) 0 (CH 3 ) 4

Organic Structure Analysis, Crews, Rodriguez and Jaspars 13 C NMR DATA GUAIAZULENE sAsA sBsB dJdJ sDsD ddd EFG sCsC dHdH dKdK qq LM qNqN qOqO sIsI (C) 5 + (CH) 6 + (CH 2 ) 0 + (CH 3 ) 4 = C 15 H 18

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

Organic Structure Analysis, Crews, Rodriguez and Jaspars HSQC NMR DATA GUAIAZULENE E E e FF f G G g H H h J J j

Organic Structure Analysis, Crews, Rodriguez and Jaspars 1 H NMR DATA GUAIAZULENE HgHg HeHe HfHf HjHj HhHh HkHk 3H n 3H o 3H l 3H m Label spectrum according to HSQC:

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

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

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

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

Organic Structure Analysis, Crews, Rodriguez and Jaspars HMBC NMR DATA GUAIAZULENE CarbonProton Af, n Bh, l/m Cg, h, n De, j, o E Fg Gf Hn Ig, j, o J Kf, g, l/m Lm Ml Nh O 1 H- 1 H 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: K L M N/O O/N E/J J/E F/H H/F

Organic Structure Analysis, Crews, Rodriguez and Jaspars HMBC NMR DATA GUAIAZULENE CarbonProton Af, n Bh, l/m Cg, h, n De, j, o E Fg Gf Hn Ig, j, o J Kf, g, l/m Lm Ml Nh O K L M N/O O/N E/J J/E H F G Signal for f is a dd long-range coupling to remaining proton g

Organic Structure Analysis, Crews, Rodriguez and Jaspars HMBC NMR DATA GUAIAZULENE CarbonProton Af, n Bh, l/m Cg, h, n De, j, o E Fg Gf Hn Ig, j, o J Kf, g, l/m Lm Ml Nh O K L M E/J J/E H F G B A N O C 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 K L M E J H F G B A O C N NOE 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 (600 MHz in MeOH-d 4 )

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

(600 MHz in MeOH-d 4 ) Homo-Tyrosine HSQC-TOCSY-spectra of fraction C

(600 MHz in MeOH-d 4 ) Homo-Phenylalanine

(600 MHz in MeOH-d 4 ) Phenylalanine HSQC-TOCSY-spectra of fraction C

(600 MHz in MeOH-d 4 ) Methionine HSQC-TOCSY-spectra of fraction C

(600 MHz in MeOH-d 4 ) Lysine HSQC-TOCSY-spectra of fraction C

(600 MHz in MeOH-d 4 ) Valine HSQC-TOCSY-spectra of fraction C

HMBC-spectra of fraction C (600 MHz in MeOH-d 4 ) Phenylalanine

HMBC-spectra of fraction C (600 MHz in MeOH-d 4 ) Homo-Phenylalanine

HMBC-spectra of fraction C (600 MHz in MeOH-d 4 ) Homo-Tyrosine

HMBC-spectra of fraction C (600 MHz in MeOH-d 4 ) Methionine

Amino acid residues of fraction C Determination of the amino acid sequence HMBC-correlation between one carbonyl and the  - and  -hydrogen of an amino acid residue

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 Amino acid residues of fraction C Determination of the amino acid sequence

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 Amino acid residues of fraction C Determination of the amino acid sequence

Sequence determination 13 C-spectra of fraction C (150 MHz in MeOH-d 4 )

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 Sequence determination

Structure elucidation Structure of polypeptide C negative mode calculated: found: C 47 H 63 N 7 O 9 S

Structure elucidation Structure of polypeptide B negative mode calculated: found: C 47 H 61 N 7 O 11

Structure elucidation Structure of polypeptide A negative mode calculated: found: C 47 H 63 N 7 O 10 S

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,

1 H NMR ( DMSO, 600MHz ) ` 19` ` ` Dermacozine A Dermacozine

( DMSO, 600MHz ) D TOCSY-spectra

( DMSO, 150MHz ) Dermacozine A a 18 18` 19` ` 18 18` a 5a a C - spectra

a a 4 10a HMBC- spectra ( DMSO,600MHz )

15 N HSQC- spectra N-12 N-16 N- 5