Synthesis of isotopically labelled phytoestrogen standards · elucidation of metabolic pathways ......
Transcript of Synthesis of isotopically labelled phytoestrogen standards · elucidation of metabolic pathways ......
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
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
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
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
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