Synthesis of isotopically labelledphytoestrogen standards

Kristiina Wähälä

Laboratory of Organic Chemistry, Department of Chemistry

P.O. Box 55, FIN-00014 University of Helsinki, Finland

Why label ?

analytical reference standards in mass spectral quantitation

elucidation of metabolic pathways

clarification of NMR spectra and mass spectral fragmentation pathways

Which labels?

Cold Stable Radioactive

H D T

12C 13C 14C

16O 18O

14N 15N

O

OOH

HO D3PO4-BF3/D2O 7 days, 100oC, autoclave

O

OOH

HOD

D

D

DD

D

certain hydrogens may be changed into deuteriumswithin the finished molecular framework

A 13C-label be must be carried over all the way from a13C-label containing starting material in a total synthesis sequence

Synthesis of triply labelled glyciteinHO

O

O

OO

O

13CH3

13C

O

O OH

O

13CH313C

O

HO OH

O 13C13CH3

O

BnO OBn

O 13C13CH3

O

13CHO

BnO

BnO OBn

O 13C13C

O

13CH(OMe)2

OBn

HO OH

O 13C13C

O

13CH(OMe)2

OH

HO O

O 13C13C

OOH

13C

13CN

BnO

I

BnO

+

Zhang and Botting, Tetrahedron 60 (2004) 12211–12216

99 % pure 13C-labeled phenol costs ca. 600 US $ for100 mg

Doubly 13C-labeled acetyl chloride 1000 US $ for 1 gram

99 % pure D2O 1 US $ for 1 gram

OH

HO

alkylresorcinols

OHO

OH

O

O

OOH

HOOH

HO

O

OOH

OH

HO

OH

steroidestrogens

flavonoids &isoflavonoids

lignans

8 a

4 a

6 '

5 '

4 '

3 '

2 '

8

7

6

5

3

2

Coumaronochromone s

Isoflavano ls

2 -Arylbe nzofurans

α-Me t hylde oxybe nzoins

Coume s t ans

Is oflav-3 -e ne s

3 -Arylcoumarins

Isoflavans

Pt e rocarpansRot e noids

Isoflavanone sIs oflavone s

O

O

O O

O

O

O

O

O

O

O O

O

O

O O

O

O

O

OH

O

O O

C

B

A

Skeletal structuresfor the isoflavonoid

classes

phytoestrogenicisoflavonoids

O

O12

3

45

6

7 8 9

1'

2'3'

4'5'

6'

7'8' 9'

Type 1Dibenzylbutyrolactones

OHOH

R

Type 2Dibenzylbutanediols

O

Type 3Dibenzyltetrahydrofurans

Raffaelli, Hoikkala, Leppälä, Wähälä J. Chromatogr. B, 2003.

CHO

OH

Et3N/D2O

100oC, 6 days 87%

CHO

OHD D

K2CO3/MeI

acetone 84%

CHO

OMeD D

BzO OH

O

+

CHO

OMeD D

KOH/EtOH

54%

BzO OH

O

OMeD

D

1. Py/Ac2O2. Tl(NO3)3-3H2O/KOH 73%

BzO O

OOMe

D

D

Pd(OH)2/C

EtOH 93%

HO O

OOMe

D

D

TOTAL YIELD 27%

H. A. M. Al-Ani and P. M. Dewick, J. Chem. Soc. Perkin Trans I (1984) 2831

OBnO

OBn

O

OO

O

O

NaBD4

DMFOO

D

O

D

O

O

DD

SPhPhS

OBn

O

O

DD Br

OBn

n-BuLiTHF

- 78 oC

PhS SPh DD

RaNiEtOH

rf

OHO

OH

O

DD

Kirk et al. J. Chem. Soc. Perkin Trans. I (1985) 35.

O

OOH

HOCF3COOD

O

OOH

HOD

DD

DDaidzein D4-daidzein

O

OOH

HOCF3COOD

O

OOH

HOD

DD

DDihydrodaidzein D4-dihydrodaidzein

O

OH

HONaOD

O

OH

HOD

DD

DEquol D4-equol

D2O

OH

OOH

HOPBr3

OH

OOH

HOD

DD

DO-demethylangolensin

D5-O-demethylangolensin

D

D2O

K. Wähälä et al. J. Chem. Soc. Perkin Trans I (1986) 95H. Adlercreutz et al. Clin. Chim. Acta 199 (1991) 263

OHO

OH

O

PBr3D2O O

HO

OH

O

D

DD

D

D

D

LiAlH4THF

HO

OH

D

DD

D

D

D

OHOH

ISOTOPIC PURITY > 90%ENL END

K. Wähälä et al. J. Chem. Soc. Perkin Trans I (1986) 95

Requirements for the deuterium labeled standard

•No unlabeled species can be present and isotopic purity of the deuterated product should be higher than 90%

•D labels must be stable during the analytical procedure

•A standard should be

- chemically,- isomerically,- isotopically pure

At least three stable D atoms

•Working with polyhydroxy aromatics, it is desirable that the reference compound contains at least three stable D atoms,since the unlabeled compound, TMS derivatised for GC,will show fairly intense m+1 and m+2 ions in its mass spectrumowing to the heavier natural isotopes of the high number of carbon and silicon atoms in the molecule.

Thus several D atoms are required to shift the peaks of the reference compound to higher m/z values of interference from the peaks of the analyte

•D3PO4•BF3•Et2O/D2O•D2O•BF3•CH3COOD/D2O/K2PtCl4

Adlercreutz et al. Clin. Chim. Acta, 199, 1991, 263-278

O

OHOOH

HO

Genistein

CF3COODO

OHOOH

HOD

DD

D6,8,3',4'-D4-genistein

8

6

Synthesis of stable 2’,3’,5’,6’-D4-genistein

Wähälä & Rasku THL, 38 1997, 7287-7290Yield 91%Isotopic purity 94%

stable 2’,3’,5’,6’-D4-genistein

O

OHOOH

HO8

66'

5'

2'3'

D2O

acetone

O

ODOOD

DO

1. D3PO4-BF3/D2O2. H2O

O

OHOOH

HOD

D

DD

DD

CH3COCl

MeOH

O

OHOOH

HOD

D

DD

MeO

HOO

OHOMe

O

Matairesinol

D3PO4-BF3/D2O

20h

MeO

HOO

OHOMe

O

D6-Matairesinol

D

DD

D

D

D

LiAlD4, THF

MeO

HO

OHOMe

D8-secoisolariciresinol

D

DD

D

D

D

OHOH

D D

HClO4, acetoneMeO

HO

OHOMe

D8-anhydrosecoisolariciresinol

D

DD

D

D

D

D D

O

HOO

OH

O

HOO

OH

OOH

HOO

OMe

O

MeO

HOO

OH

O

HO

HOO

O

MeO

MeO

HO

OH

OHOH

HOO

OH

O

MeOO

OH

OMeO

HO

HO

OH

OHOH

D

DD

DD D

D

D

D

D

D

DD

D

DD

D

DD

D

D

DD

D

D

DD

DD

DD

D

DD

D

D

DD

DD

DD

DD

D

D

D

DD

D

DD

DD

D

DD

D

D

D

D

OH

D

D D

DD

D

Deuterated mammalian lignans metabolites

Eija Leppälä and Monika PohjoispääLeppälä & Wähälä JLCR, 47, 2004, 25.

By the combined use of

mass spectra and 1H and13C NMR spectra

it is easy to establish the degree of

deuterationpurity and identity

of a polydeuterated product

HOO

OH

O

2'

4'5'

6'

2

45

6

HOO

OH

O

D

DD

D

D

DD

D

D8-enterolactone

M+ of unlabelled and deuterated2,3-dimethoxy-3’-hydroxylignano-9,9’-lactone

OHO

OO

O

OHO

O

D

DD

D

D

D

D

O

O

M+ of unlabelled and deuterated TMS-ENL

O

O

TMSO

OTMS

O

O

TMSO

OTMS

D

DDD

DD

DD

13C-labelled isoflavononoids

Single label

Botting et al. Tetrahedron 56 (2000) 455–460

13CO

HO O

OH

13CO

HO O

OMe

13CO

HO O

OMe

13CO

HO O

OH

OH

OH

[4-13C]daidzein

[4-13C]formononetin [4-13C]biochanin A

[4-13C]genistein

13C13C13C

O

HO O

OH

MeO

[2,3,4-13C3]glycitein

13C13CO

HO 13C O

OH[3,4,8-13C3]daidzein

13C13C

13CO

O

OHO

OH[6,6a,11a-13C3]coumestrol

13CH13C

HO 13C O

OH

[3,4,8-13C3]equol

Al-Maharik et al. Tetrahedron 60 (2004) 1637–1642

Zhang and Botting, Tetrahedron 60 (2004) 12211–12216 Oldfield et al. Tetrahedron 60 (2004) 1887–1893

Grace at al. Analytical Biochemistry 315 (2003) 114-121

7 steps

13 steps

13C-labelled isoflavonoids

Triply labelled

13C-labelled lignans13C

13CO

13CO

O

O

HO

OH

13C

13C

O

O

HO

OH

13COHOH

13C

O13C

13CO

HH

OO

OO

13C

O13C

13CO

HH

OHO

OHO

O

[7,7',9'-13C3]matairesinol [7,7',9-13C3]secoisolariciresinol

[7,8,9-13C3]sesamin [7,8,9-13C3]medioresinol

Fryatt, T. and Botting, N. P. J. Label. Compd. Radiopharm. 2005; 48: 951-969.

12 steps

13 steps

Haajanen, K. and Botting, N. P. Steroids 2006; 71: 231-239.

Microwave techniques

O

OHO

HO6

86'

5'

2'3'

CF3COO D

O

OHOOH

HO

Daidzein

Genistein

O

OHO

HO

MeO

Glycitein

Microwave irradiation

Deuteration of Isoflavones with CF3COOD

Daidzein 1) refl. 27h,2) fresh CF3COODrefl 8 days

Product6,8,3’,5’-D4-daidzein

Yield/ Isotopic purity75%/ 91%

Daidzein MW 50W, 5h180°C, 12 barRepeated 2x

6,8,3’,5’, 2’,6’-D6-daidzein

89%/ 90%

Genistein 1)refl. 2days2) fresh CF3COODrefl 2 days, 2x

6,8,3’,5’-D4-genistein

75%/ 90 %

Genistein MW 50W, 5h180°C, 12 barRepeated 2x

6,8,3’,5’, 2’,6’-D6-genistein

90%/ 90%

Soidinsalo & Wähälä JLCR, 2006, in press.

Synthesis of glycitein-d6 with CF3COOD in microwave

OHO

OOH

MeO

D

D

D

D

D

D

13C-NMR spectrum of glycitein-d6 ESI+ [M+H] of glycitein-d6

Sulfates

daidzein-d6 R1=D, R2=H, R3=Dgenistein-d4 R1=H, R2=OH, R3=Hglycitein-d6 R1=D, R2=D, R3=OMe

OHO

OOH

D

D

D

D

R3

R2

R1

OO

OO

D

D

D

D

R3

R2

NaO3S

SO3Na

R1

ClSO3H

pyridine

Isotopic purity > 90 %

1H and 13C NMR spectra Varian Gemini 2000 nad Bruker Avance 500 spectrometersLC-MS(ESI+) HP1100 equipped with Mariner ESI-TOF. High-resolution mass spectra Bruker Mikrotof on the negative mode with Tunemix as the internal standards

Soidinsalo & Wähälä, JLCRP, 2006, in press

Yields 85-90 %Isotopic purity > 90 %

O

OOH

HOD

D

D

a)

b)

c)

O

OOH

OD

D

D

O

OHOH

OH

OH

7-O-glucoside

O

OO

OD

D

D

O

OHOH

OH

OH

O

OH OH

HO

HO

O

OO

HOD

D

D O

OH OH

HO

HO

7,4'-O-diglucoside

4'-O-glucoside

D3-daidzein

a) 1. K2CO3, acetonitrile2. 18-crown-6, α-acetobromoglucose3. NaOMe/MeOH

b) 1. t-BuOK, acetonitrile2. 18-crown-6, α-acetobromoglucose3. NaOMe/MeOH

c) 1. Quinoline, Ag2CO32. α-acetobromoglucose3. NaOMe/MeOH

Cl-/[AlCl3]

Cl- Br- I- Cl-/[InCl3][NO3]- [SO4]2-

[CF3COO]-

[CF3SO2]-

[BF4]-

[PF6]-

[(CF3SO2)2N]-

NR4

R3R1R2

PR4

R3R1R2

COO

H3COH

H

NN

R1

R2OH

N

NR1 R2

NO

R

Examples of Ionic Liquids

N

R2

R3R4

R1

NN

R1R2

R3

NR1

R2

SR2

R1 R3

Key Features of Ionic Liquids

Liquid range of 300 °C (-96 - +200 °C) Excellent solvents for organic, inorganic and polymeric materialsCatalysts as well as solventsHighly solvating - low volumes usedNo measurable vapour pressureNon-flammableThermally stable under conditions up to 200 °C Display Brönsted, Lewis and ‘super’ acidityElectric conductivityBiphasic systems possibleLiquid crystalline structures

Ionic Liquids: Ionic Liquids: ““DesignerDesigner”” Solvents for Green SynthesisSolvents for Green Synthesis by Kenneth R. by Kenneth R. SeddonSeddon , , ChemFilesChemFiles, Enabling Technologies, Ionic Liquids, Enabling Technologies, Ionic Liquids, Vol. 5, 2005, Vol. 5, 2005

DCl-D2O and ionic liquid

compound reaction time/min

power/W yield/% Isotopicpurity

D4-daidzein 30 50 95 > 90

D6-daidzein 10 + 10 100 90 >90

D6-matairesinol 30 40 92 > 92

D5-ODMA 15 40 90 > 90

D6-2,4,4’trihydr-oxydeoxybenzoin

30 50 95 > 90

D3-glycitein 30 50 90 > 90

Ullastiina Hakala

Deuteration with ionic liquid

D6-Daidzein

10,00 9,00 8,00 7,00

c:\perch\koe.obs

HO O

OOH

D

D

D

DD

D

Conclusion

Deuterated and C-13 labeled isoflavonoids and lignans

can be used as internal standads in

quatitation of phytoestrogens

Acknowledgements

Dr. S. Rasku

Dr. P. Kiuru

Dr. A. Salakka

Dr. P. Lewis

U. Hakala

E. Leppälä

M. Pohjoispää

B. Raffaelli

O. Soidinsalo

T. Jokela

S. Heinonen

H. Kanerva

V. Johansson

Finnish Academy

University Research Funds

Emil Aaltonen Foundation

Antti and Jenny Wihuri

Foundation

PHYTOHEALTH EU-project

Dr Johanna Lampe, Fred

Hutchinson Cancer

Research Center , US

University of Helsinki Š Department of Chemistry

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