S. Rasku, K. W a¨ h a¨ l a¨ / Tetrahedron 56 (2000) 913–916
915
as compared to the intense singlets in the undeuterated
compounds. Proton and carbon assignments were also
confirmed by GHMBC (gradient selected heteronuclear
multiple bond correlation) techniques. The order of
exchange of hydrogens was assigned by monitoring the
progress of different deuteration and dedeuteration experi-
ments by NMR. Thus, the reactivity order for apigenin
filtered and washed with water or extracted with EtOAc,
the extracts washed with water (until neutral), dried
(Na SO ) and evaporated.
2
4
0
0
[3,6,8,3 ,5 -D ]-Apigenin 5 {5,7-dihydroxy-2-(4-hydroxy-
5
phenyl-3,5-D )-4H-1-benzopyran-4-one-3,6,8-D }. Recrystal-
2
3
lization from aqueous EtOH gave yellow crystals, mp 345–
0
0
0
0
0
0
0
1
is 8,6Ͼ3Ͼ3 ,5 ӷ2 ,6 , for luteolin 2 8,6Ͼ3Ͼ2 ,5 ,6 ,
346ЊC (345ЊC for D -apigenin); l
(EtOH)/nm 269 (e 27
0
max
0
0
0
0
for quercetin
8
3
8Ͼ6Ͼ2 ,5 Ͼ6 and for fisetin
4
500), 335 (30 200); dH [(CD ) SO] 7.93 (2H, s, 2 - and
3 2
0
0
0
0
Ͼ2 ,5 ,6 ӷ6,5.
6 -H), 10.4 and 10.8 (1H, br, -OH), Ϫ2.0 (1H, br, 5-OH);
D
D
D
dC [(CD ) SO] 93.9 (C-8) , 98.8 (C-6) , 102.5 (C-3) ,
3 2
0 0 0 0
D
The isotopic purity of the products (Table 1) was deter-
mined from the mass spectra operating in the negative ion
electrospray mode since normal electron impact ionization
mass spectra did not give reliable results for all deuterated
flavones. It appears that some kind of deuterium scrambling
between two molecules can take place in EI/MS before the
ionization.
103.7 (C-4a), 115.6 (C-3 , -5 ) , 121.1 (C-1 ), 128.3 (C-2 ,
0
0
-6 ), 157.2 (C-8a), 161.1 (C-4 ), 161.4 (C-5), 163.7 (C-2),
ϩ
164.0 (C-7), 181.7 (C-4); m/z (EI) 275 (M , 100%), 247
(13), 155 (20), 123 (19), 121 (13), (Found: M , 275.0834.
ϩ
C H D O requires M, 275.0842).
15
5
5
5
0
0
[3,3 ,5 -D ]-Apigenin
6
{5,7-dihydroxy-2-(4-hydroxy-
3
phenyl-3,5-D )-4H-1-benzopyran-4-one-3-D}. Recrystal-
lization from aqueous EtOH gave yellow crystals, mp
2
The new polydeuterated flavonoids are stable, isotopically
and chemically pure and thus can be reliably used as
reference compounds in quantification or in metabolic
studies.
345–346ЊC (345ЊC for D -apigenin); l
(EtOH)/nm 269
0
max
(e 27 000), 335 (31 800); d [(CD ) SO] 6.21 (1H, s, 6-H),
H
3 2
0
0
6.50 (1H, s, 8-H), 7.93 (2H, s, 2 - and 6 -H), 9.3 and 9.7 (1H,
br, -OH), Ϫ2.0 (1H, br, 5-OH); d [(CD ) SO] 93.9 (C-8),
C
3 2
D
0 0
D
9
8.8 (C-6), 102.8 (C-3) , 103.7 (C-4a), 115.7 (C-3 , -5 ) ,
121.1 (C-1 ), 128.3 (C-2 , -6 ), 157.3 (C-8a), 161.0 (C-4 ),
0
0
0
0
Experimental
General
1
61.4 (C-5), 163.7 (C-2), 164.1 (C-7), 181.7 (C-4); m/z (EI)
ϩ
273 (M , 100%), 245 (12), 153 (20), 123 (10), 121 (8),
ϩ
(Found: M , 273.0716. C H D O requires M, 273.0717).
15 7 3 5
1
13
All compounds were characterized by H, C and 2D
GHMBC) NMR, LRMS and HRMS and were homogenous
0
0
0
(
[3,6,8,2 ,5 ,6 -D ]-Luteolin 7 {5,7-dihydroxy-2-(3,4-di-
6
by TLC. Melting points were determined in open capillary
tubes with an Electrothermal apparatus, and are uncor-
rected. NMR spectra were recorded on a Varian GEMINI
hydroxyphenyl-2,5,6-D )-4H-1-benzopyran-4-one-3,6,8-
3
D }. Recrystallization from aqueous i-propanol gave yellow
3
crystals, mp 322ЊC (325ЊC for D -luteolin); l (EtOH)/nm
0
max
D
2
000 and Varian Inova 300 WB spectrometers. SiMe was
256 (e 28 600), 352 (18 300); d [D -acetone] 94.7 (C-8) ,
C 6
D D D
4
0
99.5 (C-6) , 104.5 (C-3) , 105.4 (C-4a), 113.9 (C-2 ) ,
D D
used as an internal standard. Chemical shifts are given in d
1
3
0
0
0
0
and J values in Hz. In the C NMR spectra the shifts given
for the C-D triplets are those corresponding to the central
peaks and are marked by ‘D’. Mass spectra were obtained
with a JEOL JMS SX102 mass spectrometer operating at
116.3 (C-5 ) , 120.1 (C-6 ) , 123.7 (C-1 ), 146.5 (C-3 ),
0
150.1 (C-4 ), 158.8 (C-8a), 163.4 (C-5), 164.9 (C-7),
165.2 (C-2), 183.1 (C-4); m/z (EI) 292 (M , 100%), 264
(20), 155 (28), 138 (17), (Found: M , 292.0844.
ϩ
ϩ
7
0 eV. Samples were introduced by a direct inlet probe.
C H D O requires M, 292.0854).
15
4
6
6
Isotopic purities are calculated from the negative ion
0
0
0
electrospray LC-MS obtained with Micromass Quattro II,
[3,2 ,5 ,6 -D ]-Luteolin 8 {5,7-dihydroxy-2-(3,4-dihydroxy-
4
using 20 ml Rheodyne loop and MeOH–H O (1:1) eluent.
phenyl-2,5,6-D )-4H-1-benzopyran-4-one-3-D}. Recrystal-
2
3
The UV spectra were recorded with a CARY 5E UV-VIS-
NIR spectophotometer. TLC was conducted with Merck
lization from aqueous i-propanol gave yellow crystals, mp
321ЊC (325ЊC for D -luteolin); l
(EtOH)/nm 255 (e
0
max
silica gel 60 F254 plates. D O (99.9 at.%) and quercetin
20 600), 352 (18 800); dH [D -acetone] 6.26 (1H, s, 6-H),
2
6
was obtained from Sigma, apigenin (mp 345ЊC) and luteolin
6.53 (1H, s, 8-H); d [D -acetone] 94.6 (C-8), 99.6 (C-6),
C
6
D
0 0
103.9 (C-3) , 105.3 (C-4a), 113.8 (C-2 ) , 116.3 (C-5 ) ,
D
D
D
(mp 325–327ЊC) from Indofine and fisetin (mp Ͼ330ЊC)
0
0
0
0
from Aldrich.
120.1 (C-6 ) , 123.1 (C-1 ), 146.3 (C-3 ), 149.9 (C-4 ),
58.6 (C-8a), 163.2 (C-5), 164.7 (C-7), 165.0 (C-2), 182.9
(C-4); m/z (EI) 290 (M , 100%), 262 (13), 153 (20), 138
1
ϩ
General procedure for deuteration of flavonoids
1
ϩ
(Method A)
(10), (Found: M , 290.0732. C H D O requires M,
15
6
4
6
2
90.0728).
Temperature for all flavone deuterations was 55ЊC. Reaction
times are in the Table 1.
0
0
0
[6,8,2 ,5 ,6 -D ]-Quercetin 9 {3,5,7-trihydroxy-2-(3,4-di-
5
hydroxyphenyl-2,5,6-D )-4H-1-benzopyran-4-one-6,8-
3
General procedure for dedeuteration of labile
deuteriums (Method B)
D }. Recrystallization from aqueous EtOH gave yellow
2
16
crystals, mp 310ЊC (311–313ЊC for D
EtOH)/nm 256 (e 20 900), 374 (22 800); d [D -acetone]
-quercetin) ; lmax
0
(
C
6
D
D
0 0
94.5 (C-8) , 99.0 (C-6) , 104.2 (C-4a), 115.9 (C-2 , -5 ) ,
D
D
Flavonoid (0.1 g) containing labile deuterium atoms was
0
0
0
refluxed in 0.5% CH COCl/MeOH (10 ml) for 20 min.
121.4 (C-6 ) ,123.6 (C-1 ) 136.8 (C-3), 145.8 (C-3 ), 147.0
3
0
The mixture was then poured into ice water, the product
(C-2), 148.4 (C-4 ), 157.8 (C-8a), 162.3 (C-5), 165.0 (C-7),