Stratégia hľadania štruktúry neznámej zlúčeniny pomocou NMR - príklad rastlinných ...

... Why Just NMR? – Because there is hardly another technique that is so informative for many different types of applications, and because there is no other technique that provides so much fun.

Richard R. Ernst: Nobelova cena 1991: FT NMR

STRATÉGIA HĽADANIA ŠTRUKTÚRY NEZNÁMEJ ZLÚČENINY POMOCOU NMR - príklad rastlinných

metabolitov.

Mária Vilkováa, Ján Imricha, Miroslav Repčákb, Veronika Porackáb

aLaboratórium NMR spektroskopie, Ústav chémie, Prírodovedecká fakulta, Univerzita Pavla Jozefa Šafárika, Moyzesova 11, 040 01 Košice

bKatedra botaniky, Ústav biologických a ekologických vied, Prírodovedecká fakulta, Univerzita Pavla Jozefa Šafárika, Moyzesova 11, 040 01 Košice

Varian VNMRS 600 NMR spektrometer:

• gradientová sonda PFG OneNMR (dutina 5 mm)

• 1H NMR (599.877 MHz), 13C NMR (150.855 MHz)

• interný štandard: tetrametylsilán (TMS, 0 ppm)

• deuterované rozpúšťadla: metanol (CD3OD) alebo dimetylsulfoxid (DMSO)

• chemické posuny - stupnica [ppm]

• interakčné konštanty - J [Hz]

• protón-protón a protón-uhlík korelácie - gradientové selektívne homonukleárne a heteronukleárne experimenty s adiabatickými pulzmi

NMR spektroskopia

VNMRS 600 NMR spektrometer(vpredu konzola a RF inteface, vzadu magnet).

Sonda OneNMR.Vzorka v NMR kyvetev rotore (vpravo).

Prierez magnetom.

VZORKA 1: zmes rastlinných metabolitov

1H a 13C NMR spektrum

Obrázok 1. 1H NMR (600 MHz, CD3OD) spektrum (8,50 – 3,00 ppm) zmesi metabolitov.

Obrázok 2. 13C NMR (150 MHz, CD3OD) spektrum (175,0 – 40,0 ppm) zmesi metabolitov.

VZORKA 1: zmes rastlinných metabolitov

1H a 13C NMR spektrum

Obrázok 1. 1H NMR (600 MHz, CD3OD) spektrum (8,50 – 3,00 ppm) vzorky 1.

Obrázok 2. 13C NMR (150 MHz, CD3OD) spektrum (175,0 – 40,0 ppm) vzorky 1.

Obrázok 3. Spektrum DEPT (150 MHz, CD3OD) spektrum (175,0 – 40,0 ppm) vzorky 1.

[13C,1H]-HSQC

Obrázok 4. HSQC (CD3OD) spektrum zmesimetabolitov (vpravo oblasť aromatických protónov a uhlíkov).

H

H

H H

H

HH

(1H) (13C) [ppm] [ppm]8.02 141.17.86 146.17.67 132.77.55 129.97.24 138.37.22 114.47.08 119.66.85 102.76.79 102.56.64 109.96.56 108.66.40 116.56.29 114.65.84 119.3

[13C,1H]-HSQC

H

H

H H

H

HH

(1H) (13C) [ppm] [ppm]4.96 102.54.94 103.64.91 102.8

Obrázok 4. HSQC (CD3OD) spektrum zmesimetabolitov (vpravo oblasť anomérnych protónov a uhlíkov).

[13C,1H]-HSQC

H

H

H H

H

HH

(1H) (13C) [ppm] [ppm]3.90 62.63.69 62.5

62.43.82 56.03.80 55.93.56 74.93.48 78.5

78.13.43 71.4

71.2

Obrázok 4. HSQC (CD3OD) spektrum zmesimetabolitov (vpravo oblasť protónov a uhlíkov patriacich cukorným zložkám).

[1H,1H]-COSY

H

H

H H

H

HH

H

H

H

6.79(1.8)

6.56(1.8, 9.0)

7.67(9.0)

102.5

108.6

132.7

H

H5.84(12.6)

7.24(14.4)

138.8

119.3

H

H

H

6.85(1.8)

6.64(1.8, 9.0)

7.56(9.0)

102.5

109.9

132.7

H

H

6.40(16.2)

8.02(15.6)

141.2

116.5

H

H

7.08(7.8)

7.22(9.6)

114.4

119.6

H

H

7.86(9.6)

6.29(9.6)

114.6

146.1

O

OH

OH

OH

OOH

H4.96(7.8)

102.5

O

OH

OH

OH

OOH

H4.94(7.8)

103.6

O

OH

OH

OH

OOH

H4.91(7.8)

102.8Obrázok 5. COSY spektrum (CD3OD, 8,50 – 3,00 ppm) zmesi metabolitov (vpravo oblasť aromatických protónov a protónov na násobných väzbách).

[13C,1H]-HMBC

H

H

H H

H

HH

163.17.67 (132.7) 158.2

138.3

7.24 (138.8) 158.2 132.7

163.16.79 (102.5) 158.2

119.1 108.6

6.56 (108.6) 119.1 102.5

5.84 (119.3) 171.0 119.1

H

H

H

6.79(1.8)

6.56(1.8, 9.0)

7.67(9.0)

102.5

108.6

132.7H

H5.84(12.6)

7.24(14.4)

138.8

119.3

Obrázok 6. HMBC spektrum (CD3OD) zmesi metabolitov (vpravo prenosy magnetizácie z protónov na uhlíky).

[13C,1H]-HMBC

H

H

H H

H

HH

163.17.67 (132.7) 158.2

138.3 (7.24)

7.24 (138.8) 158.2 132.7 (7.67)

163.16.79 (102.5) 158.2

119.1 108.6 (6.56)

6.56 (108.6) 119.1 102.5 (6.79)

5.84 (119.3) 171.0 (C=O) 119.1

H

H

H

6.79(1.8)

6.56(1.8, 9.0)

7.67(9.0)

102.5

108.6

132.7H

H5.84(12.6)

7.24(14.4)

138.8

119.3

H

H

H

H

H

6.79(1.8)

6.56(1.8, 9.0)

7.67(9.0)

102.5

108.6

132.75.84(12.6)

7.24(14.4)

138.8

119.3


[13C,1H]-HMBC

H

H

H H

H

HH

163.17.67 (132.7) 158.2

138.3 (7.24)

7.24 (138.8) 158.2 132.7 (7.67)

163.16.79 (102.5) 158.2

119.1 108.6 (6.56)

6.56 (108.6) 119.1 102.5 (6.79)

5.84 (119.3) 171.0 (C=O) 119.1

H

H

H

6.79(1.8)

6.56(1.8, 9.0)

7.67(9.0)

102.5

108.6

132.7H

H5.84(12.6)

7.24(14.4)

138.8

119.3

H

H

H

H

H

6.79(1.8)

6.56(1.8, 9.0)

7.67(9.0)

102.5

108.6

132.7

7.24(14.4)

138.8

119.3

158.2163.1


[13C,1H]-HMBC

H

H

H H

H

HH

163.17.67 (132.7) 158.2

138.3 (7.24)

7.24 (138.8) 158.2 132.7 (7.67)

163.16.79 (102.5) 158.2

119.1 108.6 (6.56)

6.56 (108.6) 119.1 102.5 (6.79)

5.84 (119.3) 171.0 (C=O) 119.1H

H

H

6.79(1.8)

6.56(1.8, 9.0)

7.67(9.0)

102.5

108.6

132.7H

H5.84(12.6)

7.24(14.4)

138.8

119.3

H

H

H

H

H

6.79(1.8)

6.56(1.8, 9.0)

7.67(9.0)

102.5

108.6

132.7

7.24(14.4)

138.8

119.3

158.2163.1

H

H

H

H

H

OCOOH

O

6.79(1.8)

6.56(1.8, 9.0)

7.67(9.0)

102.5

108.6

132.7

7.24(14.4)

138.8

119.3

158.2163.1

119.1

171.0

??? ???

Obrázok 6. HMBC spektrum (CD3OD) zmesi metabolitov.

[13C,1H]-HMBC

171.98.02 (141.1) 159.0

129.9 117.0

164.07.55 (132.7) 159.0

141.1

164.36.85 (102.7) 159.0

118.1 109.9

6.64 (109.9) 118.1 102.7

6.40 (116.5) 171.9 118.1

H

H

H H

H

HH

H

H

H

H

H

COOH

O O

6.85(1.8)

6.64(1.8, 9.0)

7.56(9.0)

102.7

109.9

132.7

6.40(16.2)

8.02(15.6)

141.2

116.5

??? ???

164.3

171.9

118.1

159.0

H

H

6.40(16.2)

8.02(15.6)

141.2

116.5

H

H

H

6.85(1.8)

6.64(1.8, 9.0)

7.56(9.0)

102.7

109.9

132.7


[13C,1H]-HMBC

H

H

H H

H

HH

162.87.86 (146.1) 144.4

119.6 116.5 (sl.)

149.77.22 (114.4) 135.9

119.6 116.5

149.77.08 (119.6) 146.1

144.4

6.29 (114.6) 162.8 116.5

H

H

7.08(7.8)

7.22(9.6)

114.4

119.6

H

H

7.86(9.6)

6.29(9.6)

114.6

146.1

H

H H

H

7.86(9.6)

6.29(9.6)

114.6

146.1

7.08(7.8)

7.22(9.6)

114.4

119.6


[13C,1H]-HMBC

H

H

H H

H

HH

162.87.86 (146.1) 144.4

119.6 116.5 (sl.)

149.77.22 (114.4) 135.9

119.6 116.5

149.77.08 (119.6) 146.1

144.4

6.29 (114.6) 162.8 116.5

H

H

7.08(7.8)

7.22(9.6)

114.4

119.6

H

H

7.86(9.6)

6.29(9.6)

114.6

146.1

H

H H

H

7.86(9.6)

6.29(9.6)

114.6

146.1

7.08(7.8)

7.22(9.6)

114.4

119.6

144.4

116.5


[13C,1H]-HMBC

H

H

H H

H

HH

162.87.86 (146.1) 144.4

119.6 116.5 (sl.)

149.77.22 (114.4) 135.9

119.6 116.5

149.77.08 (119.6) 146.1

144.4

6.29 (114.6) 162.8 116.5

H

H

7.08(7.8)

7.22(9.6)

114.4

119.6

H

H

7.86(9.6)

6.29(9.6)

114.6

146.1

H

H H

H

O

7.86(9.6)

6.29(9.6)

114.6

146.1

7.08(7.8)

7.22(9.6)

114.4

119.6

144.4

116.5

162.8


[13C,1H]-HMBC

H

H

H H

H

HH

162.87.86 (146.1) 144.4

119.6 116.5 (sl.)

149.77.22 (114.4) 135.9

119.6 116.5

149.77.08 (119.6) 146.1

144.4

6.29 (114.6) 162.8 116.5

H

H

7.08(7.8)

7.22(9.6)

114.4

119.6

H

H

7.86(9.6)

6.29(9.6)

114.6

146.1

H

H H

H

O

7.86(9.6)

6.29(9.6)

114.6

146.1

7.08(7.8)

7.22(9.6)

114.4

119.6

144.4

116.5

162.8

H

H H

H

O OO

OH

7.86(9.6)

6.29(9.6)

114.6

146.1

7.08(7.8)

7.22(9.6)

114.4

119.6

144.4

116.5

162.8??? 149.7

135.9



[13C,1H]-HMBC

H

H

H H

H

HH

Anomérne protóny4.96 (102.5) 159.0 4.94 (103.6) 158.24.91 (102.8) 149.7

Metylové skupiny3.80 (56.0) 164.33.71 (56.2) 163.1

H

H

H

H

H

OCOOH

O

6.79(1.8)

6.56(1.8, 9.0)

7.67(9.0)

102.5

108.6

132.7

7.24(14.4)

138.8

119.3

158.2163.1

119.1

171.0

??? ???

H

H

H

H

H

COOH

O O

6.85(1.8)

6.64(1.8, 9.0)

7.56(9.0)

102.7

109.9

132.7

6.40(16.2)

8.02(15.6)

141.2

116.5

??? ???

164.3

171.9

118.1

159.0

H

H H

H

O OO

OH

7.86(9.6)

6.29(9.6)

114.6

146.1

7.08(7.8)

7.22(9.6)

114.4

119.6

144.4

116.5

162.8??? 149.7

135.9

[13C,1H]-HMBC

H

H

H H

H

HH



O

OH

OH

OH

O

OH

H

H

H

H

H

H

OCOOH

CH3

6.79(1.8)

6.56(1.8, 9.0)

7.67(9.0)

102.5

108.6

132.7

7.24(14.4)

138.8

119.3

158.2163.1

119.1

171.0

56.23.71

4.94(7.8)

103.6

H

H

H

H

H

COOH

O O

6.85(1.8)

6.64(1.8, 9.0)

7.56(9.0)

102.7

109.9

132.7

6.40(16.2)

8.02(15.6)

141.2

116.5

??? ???

164.3

171.9

118.1

159.0


H

H H

H

O OO

OH

7.86(9.6)

6.29(9.6)

114.6

146.1

7.08(7.8)

7.22(9.6)

114.4

119.6

144.4

116.5

162.8??? 149.7

135.9

[13C,1H]-HMBC

H

H

H H

H

HH



O

OH

OH

OH

O

OH

H

H

H

H

H

H

OCOOH

CH3

6.79(1.8)

6.56(1.8, 9.0)

7.67(9.0)

102.5

108.6

132.7

7.24(14.4)

138.8

119.3

158.2163.1

119.1

171.0

56.23.71

4.94(7.8)

103.6

H

H H

H

O OO

OH

7.86(9.6)

6.29(9.6)

114.6

146.1

7.08(7.8)

7.22(9.6)

114.4

119.6

144.4

116.5

162.8??? 149.7

135.9


O

OH

OH

OH

O

OH

H

H

H

H

H

H

COOH

O

CH3

6.85(1.8)

6.64(1.8, 9.0)

7.56(9.0)

102.7

109.9

132.7

6.40(16.2)

8.02(15.6)

141.2

116.5

164.3

171.9

118.1

159.0

4.96(7.8)

102.5

[13C,1H]-HMBC

H

H

H H

H

HH



O

OH

OH

OH

O

OH

H

H

H

H

H

H

OCOOH

CH3

6.79(1.8)

6.56(1.8, 9.0)

7.67(9.0)

102.5

108.6

132.7

7.24(14.4)

138.8

119.3

158.2163.1

119.1

171.0

56.23.71

4.94(7.8)

103.6

O

OH

OH

OH

O

OH

H

H

H H

H

O O

OH

7.86(9.6)

6.29(9.6)

114.6

146.1

7.08(7.8)

7.22(9.6)

114.4

119.6

144.4

116.5

162.8149.7

135.9

4.91(7.8)102.8

O

OH

OH

OH

O

OH

H

H

H

H

H

H

COOH

O

CH3

6.85(1.8)

6.64(1.8, 9.0)

7.56(9.0)

102.7

109.9

132.7

6.40(16.2)

8.02(15.6)

141.2

116.5

164.3

171.9

118.1

159.0

4.96(7.8)

102.5

Doc. RNDr. Ján Imrich, CSc. Ing. Jan Pelnař, CSc.

Mgr. Branislav Horváth, PhD. RNDr. Marianna Prokaiová

Prof. RNDr. Miroslav Repčák, DrSc.RNDr. Veronika Poracká

POĎAKOVANIE

ĎAKUJEM ZA POZORNOSŤ!

Stratégia hľadania štruktúry neznámej zlúčeniny pomocou NMR - príklad rastlinných ...

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Transcript of Stratégia hľadania štruktúry neznámej zlúčeniny pomocou NMR - príklad rastlinných ...