NMR in natural product research General setup for all Bruker NMR ...

NMR in natural product research 

Extract 

Flash 

chromatography 

HPLC 

Concentration 

Structural elucidation in natural 

product research 

Dr. Till Kühn 

SPE 

Evaporation 

Fate of a plant / animal / cell in NP research 

⇒ a fraction of mg of a (hopefully) pure compound 

2 

NMR Standard parameter-set: 

rpar … 

PROTON 

C13CPD, C13DEPT135 

High Res. MS, Elemental Analysis 

(MW and DBE) 

1 

H NMR 

( 1 H shifts + integrals: some functional groups) 

13 

C NMR, DEPT 

( 13 C shifts, multiplicity: some functional groups and features) 

HSQCEDETGPSI (TS) 

INVIEDETGPSISW (XW) 

COSYGPSW, COSYDFGPPHSW 

HMBCGP (TS) 

INV4GPLPLRNDSW (XW) 

MLEVPHSW 

HSQCGP + h2bcctetl3 

(DQF)COSY + (ed)HSQC 

(fragments, 13 C multiplicity) 

HMBC + TOCSY (+ HSQC-TOCSY + H2BC) 

(connecting the dots, 13 C info) 

General setup for all Bruker NMR experiments 

• put the sample into magnet 

• Make sure you use the correct filling height: 

• 5mm probe: > 4cm (> 550ul in a 5mm tube) 

• 1.7mm probe: > 2cm (> 30ul in a 1.7mm tube 

• 1mm probe: > 1cm (> 5ul in a 1mm tube) 

• for 5mm tubes: use depth gauge to position sample tube in 

spinner 

ROESYPHSW + roesyph.2 

for up to 400MHz 

NOESYPHSW 

for 500MHZ and up 

3 

ROESY / NOESY 

(stereochemistry) 

4 

1

General setup for all Bruker NMR experiments 

Important-1: define the current probe (edhead) 

Type edhead after each probe change, or try it out at any time 

• read the desired parameter set: rpar PARAMETERSET all 

• possibly adjust desired parameters (ns, td, o1p, o2p, sw) 

• read the probe dependant parameters: getprosol 

• lock on the solvent: lock 

• match and tune the probe: wobb or atma 

• shim: topshim, gradshim or shim manually 

• start the experiment: xaua or rga (except for DQF COSY) and zg 

• process the data: 

• automatically: xaup or 

• manually: 1D: efp, apk, abs 

2D: xfb, correct the phase and abs1, abs2 

5 

6 

Important-2: have a complete edprosol table! 

PROTON Channel, standard hard pulses: 

here the probe appears, which is 

defined as the “current probe” by edhead! 


CARBON Channel, standard hard pulses: 

⇒ Important: always run edhead 

after a probe change! 

7 

8 

2


CARBON Channel, standard soft pulses (=shaped pulses): 


Global parameters: Gradients, trim pulses: 

calculate 

calculate 

9 

10 

Even setup a list of experiments in IconNMR 

Dr. Vatcharin’s compound: Instrumentation 

normal experiments (N) 

composite experiments (C) 

a preparation experiment 

is done before the 2D 

11 

• ca. 0.6mg (NMR quantification) HPLC purified compound 

• dissolved in 30ul MeOD 

• all experiments in a 1.7mm MicroProbe 

• on a 400MHz instrument 

• 1D Proton (PROTON) 

• 1D 13 C (C13CPD) and 1D 13 C-Dept-90 (C13DEPT90) 

• 2D edited-HSQC (HSQCEDETGP) 

• 2D COSY (COSYGPSW) and DQF-COSY (COSYGPDFPHSW) 

• 2D HMBC (HMBCGP) and 

2D H2BC (HSQCETGP and type: pulprog h2bccteal3) 

• 2D ROESY (ROESYPHSW) 

• Total experiment time: less than a weekend 

12 

3

Advantages of small volume NMR 

• High mass-sensitivity (4 x sensitivity = 16 times faster): 

13 

• 1.7mm: 1 H sensitivity = 2 x 5mm BBI, 13 C sensitivity = 2 x BBO 

• 1mm: 1H sensitivity = 4 x 5mm BBI 

• 1.7mm MicroCryoProbe: 1 H sens. = 14 x BBI, 13 C sens. = 4 x BBO 

• No problems with solvent impurities (high concentration even with 

very small sample amounts) 

• No problems with solvent suppression (no radiation damping etc.) 

• Advantage of working with small sample amounts: cleaner samples! 

• It’s often much easier to get CLEAN samples in small amounts 

• No danger to overload flash chromatography or HPLC columns 

• You might even use analytical HPLC columns and fraction collect 

(better separation on with small sample amounts) 


rpar … 

PROTON 


14 


(MW and DBE) 

1 


( 1 H shifts + integrals: some functional groups) 

13 


( 13 C shifts, multiplicity: some functional groups and features) 




HMBCGP (TS) 


MLEVPHSW 

HSQCGP + h2bcctetl3 



NOESYPHSW 




HMBC + TOCSY (+ HSQC-TOCSY + H2BC) 

(connecting the dots, 13 C info) 

ROESY / NOESY 


Dr. Vatcharin’s compound: HR-MS: C 20 H 28 O 4 

1st. thing in NMR: 1 H shifts + integrals 

• Application of the rule for double-bond equivalents (DBE): 

2a 

+ 2 + d − b 2*20 + 2 − 28 

CaHbOc 

N DBE = 

= 

= 7 

d 

2 

2 

• 7 double bond equivalents (DBE): 

• 1 double bond or 1 ring closure = 1 DBE 

• 1 triple bond = 2 DBE’s 

⇒ 7 double bonds or 6 rings and 1 db or 5 rings and 2 db… 

15 

Source: 

16 

E. Breitmaier: 

Structure Elucidation by NMR in Organic Chemistry, John Wiley & Sons 

4

Dr. Vatcharin’s compound: 1 H information 

PROTON 

No Phenol OH and no COOH: 

• no aromatic protons and 

• not broad enough 

Aldehyde 

roof effect: 

AB spin system 

Alkene-CH 

AB Spin System 

3 x Methyl 

Setup a list of identified groups 

• Molecular formula: C 20 

H 28 

O 4 

• Identified groups: 

• Alkene C=C-H 6.08 ppm 

• Aldehyde CHO 9.73 ppm 

• 3 Methyl groups CH 3 

1.04 ppm 

CH 3 

0.98 ppm 

CH 3 

0.82 ppm 

• Additional information: 

• AB spin sytem 

• No aromatics 

17 

Solvent: MeOD 

18 

Dr. Vatcharin’s compound: 1 H information 

PROTON 

COSYGPSW 

Setup a list of identified groups 

Isopropyl group 

coupling to 2 of the Methyls 

• Molecular formula: C 20 

H 28 

O 4 

• Identified groups: 

• Alkene C=C-H 6.08 ppm 

• Aldehyde CHO 9.73 ppm 

• 3 Methyl groups CH 3 

1.04 ppm 

CH 3 

0.98 ppm 

CH 3 

0.82 ppm 

• isopropyl group (2.34 ppm) coupling to the first 2 Me’s 

Additional information: 

• AB spin sytem 

• No aromatics 

19 

20 

5

13 

C information: chemical shift + multiplicity 

1 

J CH correlation: multiplicity edited HSQC 

HSQCEDETGP 

DEPT-135 type info in an inverse experiment 

aldehyde (folded) 

H 2 

C-O- 

Source: 

21 

ppm 

200 150 100 50 0 

E. Breitmaier: 

Structure Elucidation by NMR in Organic Chemistry, John Wiley & Sons 

22 

alkene 

1 

J CH correlation: multiplicity edited HSQC 

HSQCEDETGP 

integral: 2H’s integral: 7H’s 

Long range correlation: HMBC 

HMBCGP 

Isopropyl 

4 x CH 2 

-groups 

3 Me-groups 

4 x CH 

Solvent 

info from edited HSQC: 

2 different C’s 

aldehyde 

23 

24 

6

13 

C and group mapping: HSQC + HMBC 

HSQCEDETGP + HMBCGP 

13 

C Mapping: HSQC + HMBC (aliphatic 13 C’s) 


C-3 

alkene 

C-2 

quaternary 

alkene 

quaternary carbon C-6 

CH-9 

CH-8 

CH-7 

1 H integration: 1H rather than 3H 

Solvent 

aliphatic 

CHO-1 

CH 2 

-5 

quaternary carbon C-4 

O bound C’s 

25 

26 

13 

C Mapping: HSQC + HMBC (aliphatic 13 C’s) 


13 

C and group mapping: HSQC + HMBC 


CH 3 

-18 

C-3 

alkene 

CH 3 

-16 

folded aldehyde 

CH 3 

-17 

C-2 

quaternary 

alkene 

CH 2 

-12 

CH-14 

CH 2 

-13 

CH 2 

-15 

CH-11 

CH 2 

-10 

real peak- 

COOH-19 

CHO-1 

13 

C count: 18… but: C 20 

H 28 

O 4 

possibly overlooked something 

27 

13 

C count STILL only: 19… but: C 20 

H 28 

O 4 

possibly overlooked 2 peaks close together = 13 C observe 

28 

7

Dr. Vatcharin’s compound: 13 C chemical shift 

C13CPD: 1 night on 600ug on a 400MHz 1.7mm probe 

Aldehyde (no ketone, see HSQC) 

COOH 

does not show up 

well - too broad! 

Alkene (ed-HSQC) 

too big for junk 

possibly quart. C 

(see HMBC) 

the 3 Methyl 

groups (HSQC) 

Dr. Vatcharin’s compound: 13 C chemical shift 

C13CPD: 1 night on 600ug on a 400MHz 1.7mm probe 

C-20 

C-4 

CH 2 

-5 

CH 2 

-5 C-6 

(we know these are two from ed-HSQC) 

CH 2 

-12 

CH-11 

CH-8 CH-9 

CH 

CH-7 

2 

-10 

29 

Solvent: MeOD 

30 

Number (arbitrary) 

13 C shift 

1 J CH Attached 1 H shift 

Group type 

Number (arbitrary) 

13 C shift 

1 J CH Attached 1 H shift 

Group type 

1 

206.0 

9.75 

-CHO (Aldehyde) 

1 

206.0 

9.75 

-CHO (Aldehyde) 

2 

148.0 

-- 

Alkene Cq=C 

2 

148.0 

-- 

Alkene Cq=C 

3 

130.5 

6.08 

Alkene C=C-H 

3 

130.5 

6.08 

Alkene C=C-H 

4 

74.0 

-- 

Cq 

4 

74.0 

-- 

Cq 

5 

66.5 

3.90, 3.50 

-H 2 C-O (geminal AB) 

5 

66.5 

3.90, 3.50 

-H 2 C-O (geminal AB) 

6 

59.0 

-- 

Cq 

6 

59.0 

-- 

Cq 

7 

46.9 

2.53 

CH 

7 

46.9 

2.53 

CH 

8 

9 

10 

11 

41.3 

41.2 

31.8 

31.1 

List of identified groups = C 20 

H 28 

O 4 

COOH 

2.07 

1.81 

2.05, 1.24 

2.10 

CH 

CH 

CH 2 

CH 

8 

9 

10 

11 

List of identified groups = C 20 

H 28 

O 4 

COOH 

41.3 

2.07 

41.2 

1.81 

Σ Oxygens = 4, OK! 

31.8 

2.05, 1.24 

Σ Protons 31.1 = 27… 2.10 

CH 

CH 

CH 2 

CH 

12 

29.2 

1.91, 1.23 

CH 2 

12 

29.2 

1.91, 1.23 

CH 2 

13 

14 

15 

28.0 

27.5 

26.2 

2.00, 1.72 

2.35 

1.92, 0.98 

CH 2 

CH (isopropyl) 

CH 2 

13 

14 

15 

28.0 

2.00, 1.72 

27.5 

2.35 

⇒1H possibly OH 

26.2 

1.92, 0.98 

CH 2 

CH (isopropyl) 

CH 2 

16 

22.1 

0.97 

CH 3 (isopropyl) 

16 

22.1 

0.97 


17 

20.6 

1.04 


17 

20.6 

1.04 


18 

16.9 

0.82 

CH 3 

18 

16.9 

0.82 

CH 3 

19 

176.5 

-- 

19 

176.5 

-- 

31 20 

66.5 

-- 

Cq 

32 20 

66.5 

-- 

Cq 

8

Identification of geminal CP’s in DQF-COSY 

COSYDQFPHSW: 5h on 600ug on a 400MHz 1.7mm probe (512 td) 

Identification of spin systems TOCSY 

MLEVPHSW: 1h on 600ug on a 400MHz 1.7mm probe 

C 15 H 2 

34 

C 13 H 2 

C 10 C 

H 12 H 2 

2 

HO-C 5 H 2 

C=C 3 H 

C 7 H C 12 H 2 

C 7 H 

isopropyl 

C 14 H C 16 H 3 C 17 H 3 

33 

C 12 H 2 

TopSpin: Peak annotation and linked cursors 

Analysis → Peak picking → Manual peak picking / Arrange windows 

Identification of Fragments: HMBC 

HMBCGP: a long night on 0.6mg on a 400MHz 1.7mm probe 

C=C 3 H 

C 16 H 3 

C 14 H (isopropyl) 

C 7 H 

Cq 6 

C 5 H 

Cq 4 

…only 2 J CH , 3 J CH 

sometimes 4 J CH 

Cq 2 = 

C 19 (COOH) 

35 

36 

9

TopSpin: Peak annotations / linked windows 


TopSpin: Peak annotation and linked cursors 


Also use COSY information 

in overlapped regions 

37 

38 


rpar … 

PROTON 



(MW and DBE) 

1 


(integrals, 1 H info: some functional groups) 

13 


( 13 C info: some functional groups and features) 

Linking the fragments HMBC / H2BC 

Heteronuclear 2-bond correlations 

• enhancement of correlations over two bonds 

• suppressing correlations over more bonds 




HMBCGP (TS) 


MLEVPHSW 



HMBC + TOCSY “new” experiment: 

(connecting the dots, 13 C info) H2BC 



NOESYPHSW 


39 

ROESY / NOESY 


3rd order low-pass J filter for correlating 1 H and 13 C nuclei 

via 1 J CH and 1 H- 1 H couplings. No 2 J CH involved! 

Nils T. 40Nyberg, Jens Ø. Duus, Ole W. Sorensen J. Am. Chem. Soc. 127 6154-6155 (2005) 

10

2-Bond correlation – so what 

HSQCGP: and change pulseprogram to: h2bccteal3 

C 14 H (isopropyl) 

C 14 H-C 17 H 

C 14 H-C 16 H 

covalent bond! 

• Overlay HMBC / H2BC 

• Distinguish between 

2 bond and 3 bond CP 

• Observe 2 bond CP 

even when they are 

missing in HMBC 

(vanishing 2 J CH ) 

2-Bond correlation – so what 

HSQCGP: and change pulseprogram to: h2bccteal3 

C 12 H 

covalent bond! 

• Step through HSQC and 

H2BC overlaid 

like through a COSY 

• Start from HSQC CP and 

use H2BC CP as relay 

points 

• No correlation to 

quaternary carbons 

C 7 H 

C 7 H-C 12 H 



Some fragments of C 20 H 28 O 4 

Putting together the fragments of C 20 H 28 O 4 

O 

OH 

19 

* 

* 

OH 

5 

20 

4 

* 

* 

13 

* 

9 

* 

8 

11 

CH 3 

18 

15 

10 

• Application of the rule for double-bond equivalents (DBE): 

2a 

+ 2 + d − b 2*20 + 2 − 28 

CaHbOc 

N DBE = 

= 

= 7 

d 

2 

2 

H 316 C 

14 

H 317 C 

* 

3 

2 

* 

* 

6 

* 

CH 3 

12 

1 

O 

H 

* 

7 * 

* * 

• 3 DBE’s are used by: CHO, COOH and C=C 

⇒ 4 ring closures are left for C 20 H 28 O 4 

43 

44 

11

The structure of C 20 H 28 O 4 : 

… after quite some detective work … 


rpar … 

PROTON 


(MW and DBE) 

1 


(integrals, 1 H info: some functional groups) 

OH 

OH 

5 

20 

13 

H 

9 

11 

CH 3 

18 


13 


( 13 C info: some functional groups and features) 

45 

H C 3 

16 

14 

H C 3 

17 

19 

O 

2 

3 

4 

7 

6 

12 

8 

H 

1 

O 

H 

15 

10 




HMBCGP (TS) 


MLEVPHSW 



NOESYPHSW 


46 



HMBC + TOCSY “new” experiment: 

(connecting the dots, 13 C info) H2BC 

ROESY / NOESY 


ROESY / NOESY for stereochemistry 

cross peak 

intensity 

with respect 

to 

diagonal peak 

-0.2 

1.0 ω 0 τ 1.0 

c 

ω 0 τ c 

molecule size: ← smaller larger → ← smaller larger → 

temperature: ← higher lower → ← higher lower → 

47 

ω 0 

τ c 

NOESY 

0.5 

= spectrometer frequency 

-0.2 

ROESY 

positive 

= rotational correlation time 

(proportional to molecular size, temperature dependent) 

negative 

ROESY spectrum for stereochemistry 

ROESYPHSW: 4h on ca. 0.6mg on a 400MHz 1.7mm MicroProbe 

48 

C 18 H3 --- C 10 Ha 

the CH 3 group C 18 and 

Ha on C 10 are on the same side 

… 

C 18 H3 --- C 10 Ha 

12

The absolute Stereochemistry of C 20 H 28 O 4 : 

Some HMBC correlations in C 20 H 28 O 4 : 

OH 

OH 

5 

20 

13 

H 

9 

11 

CH 3 

18 

OH 

OH 

5 

20 

13 

H 

9 

11 

CH 3 

18 

H C 3 

16 

14 

H C 3 

17 

19 

O 

2 

3 

4 

7 

6 

12 

8 

H 

1 

O 

H 

15 

10 

H C 3 

16 

14 

H C 3 

17 

19 

O 

2 

3 

4 

7 

6 

12 

8 

H 

1 

O 

H 

15 

10 

also 

4 

J CH couplings 

are observable! 

49 

50 

Thank you very much … 

Dr. Vatcharin 

Lukas Oberer 

Sandra Loss, Matthias Pelzing 

www.bruker-biospin.com 

51 

52 

13

NMR in natural product research General setup for all Bruker NMR ...

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