The synthesis of multiply 13C-labelled plant and mammalian lignans as internal standards for LC-MS and GC-MS analysis

Article abstract of DOI:10.1002/jlcr.1007

The syntheses of multiply ¹³C-labelled derivatives of the two mammalian lignans, enterolactone and enterodiol, and two of their plant lignan precursors, secoisolariciresinol and matairesinol, are described. Three ¹³C atoms were incorporated into each lignan using potassium [ ¹³C]cyanide as the source for all of the ¹³C atoms. The compounds were prepared for use as internal standards in the LC-MS and GC-MS analysis of lignans. Copyright

Full text of DOI:10.1002/jlcr.1007

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 951–969.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1007
Research Article
13
The synthesis of multiply C-labelled plant and
mammalian lignans as internal standards for LC-MS
and GC-MS analysis
Tara Fryatt and Nigel P. Botting*
School of Chemistry, University of St. Andrews, St. Andrews, Fife KY16 9ST, UK
Summary
The syntheses of multiply 13C-labelled derivatives of the two mammalian lignans,
enterolactone and enterodiol, and two of their plant lignan precursors, secoisolar-
iciresinol and matairesinol, are described. Three 13C atoms were incorporated into
each lignan using potassium [13C]cyanide as the source for all of the 13C atoms. The
compounds were prepared for use as internal standards in the LC-MS and GC-MS
analysis of lignans. Copyright # 2005 John Wiley & Sons, Ltd.
Key Words: lignans; phytoestrogens; enterolactone; enterodiol; analysis
Introduction
The lignans are a widely distributed group of plant phenols. There has
been increasing interest in these compounds initially as a result of their use
in traditional remedies in many cultures,1 and more recently following
the identification of the lignans enterolactone (1) and enterodiol (2)
(enterolignans) in humans and the establishment of their dietary
origin.2,3 Enterodiol and enterolactone are exclusively formed by mam-
malian gut microflora2 and their excretion in humans and other animals
is positively correlated with the consumption of lignan-rich food.2,3 High
levels of enterolactone in the body have since also been correlated with a
lower risk of developing chronic diseases, such as breast cancer and coronary
heart disease.4–7 Matairesinol (3) and secoisolariciresinol (4) were the
two plant lignans first identified as precursors to the mammalian lignans
(Scheme 1).8
*Correspondence to: Nigel P. Botting, School of Chemistry, University of St. Andrews, St. Andrews, Fife
KY16 9ST, UK. E-mail: npb@st-andrews.ac.uk
Contact/grant sponsor: Food Standards Agency (FSA); Contract/grant number: T05001
Copyright # 2005 John Wiley & Sons, Ltd.
Received 4 August 2005
Revised 10 August 2005
Accepted 12 August 2005

952
T. FRYATT AND N. P. BOTTING
H
H
H
H
H
H3CO
HO
HO
H3CO
HO
HO
OH
OH
OH
OH
O
O
H
H
H
O
O
OCH3
OH
OCH3
OH
OH
OH
1
2
3
4
Scheme 1. Structures of mammalian and plant lignans
In order to better understand the significance of the biological effects of
lignans, accurate analysis is important to establish the exposure of the
population to the plant lignans through their diet and also in epidemiological
studies to investigate the associations between lignan exposure, mammalian
lignan levels and disease. Both gas chromatography-mass spectrometry
(GC-MS)9–11 and liquid chromatography-mass spectrometry (LC-MS)12,13
have been used to quantify the low levels of lignans found in biological
samples. The nature of the internal standard is an important aspect of any
quantitative analytical procedure. For LC-MS and GC-MS the optimum
internal standard is a pure, stable, isotopically labelled analogue of the
analyte, with a sufficiently large enough mass difference to nullify the effect of
natural abundance heavy isotopes in the analyte. This mass difference depends
on the molecular weight of the analyte and for lignan-type structures a
minimum of three extra mass units is necessary.14 A number of deuterated
standards have been used for the analysis of lignans,15 which were prepared
using exchange methods to incorporate the deuterium atoms into the phenol
rings.16,17 However, under the analysis conditions back exchange can occur,
such that the deuteriums are slowly replaced by hydrogen, resulting in both a
reduction in the amount of internal standard and an apparent increase in the
amount of the lignan being analysed.18 In order to circumvent these problems
it was decided to synthesize a series of lignans with three 13C atoms
incorporated into the carbon framework of the molecule as a new generation
of internal standards.
Results and discussion
The synthesis of lignans has attracted much attention and been regularly
reviewed.19,20 For our purposes a racemic synthesis of the trans-a,b-dibenzyl-
g-butyrolactone framework of the target lignans was sufficient. These have
been most commonly synthesized via Michael addition of a dithioacetal
derived carbanion to a butenolide followed by in situ benzylation.21,22
However, this route did not offer an obvious strategy for the introduction
of the required three 13C-atoms and so an alternative, albeit longer, synthetic
route was identified that appeared to be more suitable.23 Scheme 2 shows the
retrosynthetic strategy for [13C3]enterolactone (5).
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 951–969

SYNTHESIS OF MULTIPLY 13C-LABELLED PLANT AND MAMMALIAN LIGNANS 953
13
CH
2
HO
Br
O
13
CH
2
13
O
C
O
C
13
13
13
CH
2
H3CO
CH
2
O
OCH3
7
OH
5
6
Br
13CH2
13
CH
2
H3CO
CO2Et
CO2Et
OTs
OH
K13CN
OCH3
9
6
8
Scheme 2. Proposed retrosynthesis for [7,70,90-13C3]enterolactone
This synthetic route allows two molecules of the same benzyl bromide (6) to
be employed in the construction of the lignan. In our previous studies on the
isotopic labelling of isoflavones,14,24 potassium [13C]cyanide was used to label
the methylene group of a substituted benzyl bromide, via palladium catalysed
cyanation of a suitable aryl iodide25 followed by functional group
interconversion, i.e. hydrolysis, reduction and bromination, to provide the
aryl bromide. One molecule of (6) is used for the synthesis of the key lactone
fragment (7), which is then alkylated with the second molecule of (6), thus
introducing one 13C-atom near the beginning and one near the end of the
synthesis. Previous studies showed that the key alkylation step gives the
required relative stereochemistry, with the two substituents in a trans
orientation.23 Two options were then available for the introduction of the
third 13C-atom. Diethyl [2-13C]malonate can be obtained but is expensive and
would be used very early in the synthesis. A more cost-efficient option was to
use a third molecule of potassium [13C]cyanide in the assembly of the lactone
(7). The synthesis can then be readily modified for all four target molecules.
13C-labelled enterodiol is prepared via reduction of the enterolactone (5) and
by using suitably modified benzyl bromides both 13C-labelled secoisolaricir-
esinol and matairesinol are also accessible.
The synthesis of [7,7,90-13C3]enterolactone (5) thus began with cyanation of
3-methoxyiodobenzene (10) using potassium [13C]cyanide and a palladium (II)
acetate catalyst in DMF under basic conditions (calcium hydroxide)25 to give
the 13C-labelled nitrile (11) in 64% yield (Scheme 3). The 13C atom was clearly
identified by the enhanced signal in the 13C NMR spectrum at 119.6 ppm. The
nitrile (11) was then hydrolysed under alkaline conditions, reduced using
lithium aluminium hydride in THF and converted to the bromide (13). The
benzyl bromide was reacted with the anion derived from diethyl malonate and
the resultant diester reduced to the diol. Tosylation of just one of the two
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 951–969

954
T. FRYATT AND N. P. BOTTING
CH2OTs
CH2OH
13CH2Br
13CN
13CH2OH
13CH2
I
b,c
a
e,f,g
d
OCH3
OCH3
OCH3
OCH3
OCH3
10
11
12
13
14
h,i
13CH2I
O
13
13
13
CH
2
H3CO
C
CH
HO
2
13CH2
O
O
j,
O
13
k
13
C
OCH3
C
13
13
CH
2
CH
O
2
O
OCH3
7
OCH3
OH
5
16
Scheme 3. Synthesis for [7,70,90-13C3]enterolactone(5). Reagents and conditions:
(a) K13CN, Ca(OH)2, Pd(OAc)2, DMF (64%); (b) 2 M NaOH, reflux (87%);
(c) LiAlH4, Et2O (83%); (d) PBr3, Et2O (91%); (e) KOtBu, CH2(CO2Et)2,
glyme (86%); (f) LiAlH4, Et2O (84%); (g) p-TsCl, Et3N, DCM (25%); (h)
K13CN, 18-crown-6, MeCN (82%); (i) 2 M NaOH, reflux (86%); (j) LDA,
THF, ꢀ788C (55%); and (k) BBr3, DCM (68%)
equivalent alcohols was then required, but despite many attempts at
optimization, using unlabelled materials, this was only achieved in 25% yield,
despite claims of a higher yield in the literature.23 The low yields were in part
due to the formation of the di-tosylated derivative (12%) and presence of
unreacted starting material (15%), but also due to difficulties in separating the
three compounds by column chromatography. Nucleophilic substitution of
the tosylate (14) with potassium [13C]cyanide in DMF in the presence of
18-crown-6 and cyclization under basic conditions afforded the key lactone
intermediate (7) in 70% over the two steps. The lactone contains two
13C-atoms, which could be clearly observed in the 13C NMR spectrum as
enhanced signals at 38.9 and 176.9 ppm.
The original plan at this stage was to alkylate the enolate formed by
treatment of (7) with another molecule of the benzyl bromide (6). However, in
test reactions with unlabelled material this reaction would not take place.
Eventually, the problem was solved by using the more reactive iodide
derivative instead of the bromide. Therefore, some of the 13C-labelled benzyl
alcohol (12) was converted to the iodide (15), in quantitative yield, by
treatment with phosphorus triiodide. Reaction of the iodide with the enolate
of the lactone then gave the coupled product (16) in 55% yield and the
[7,70,90-13C3]enterolactone (5) was obtained following cleavage of the two
methyl ethers with boron tribromide. [7,70,9-13C3]Enterodiol (17) was obtained
by direct reduction of the enterolactone with lithium aluminium hydride in
THF.
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SYNTHESIS OF MULTIPLY 13C-LABELLED PLANT AND MAMMALIAN LIGNANS 955
In the final products the three 13C atoms could be observed by 13C NMR
spectroscopy. For enterolactone the three signals are at 35.4, 38.6 and
178.6 ppm, while for enterodiol only two signals are seen at 35.1 and 60.7 ppm
as the molecule is now symmetrical. The rest of the spectral data for the
13C-labelled mammalian lignans were in close agreement with literature data
for the unlabelled compounds.23,26,27 Both products were purified by reverse
phase HPLC before being employed as internal standards.13,26
A modified synthetic route was employed for the 13C-labelled matairesinol
and secoisolariciresinol (Scheme 4). The major difference in this case was that
the starting aryl iodide is not commercially available and had to be prepared.
Monoiodination of guaiacol (18) was investigated and found to give
iodination at the desired 4-position (19). Cyanation with 13C-labelled cyanide
O
13
C
13CH2
13CH2Br
I
O
a,b
c,d,e,f
l
g to k
OCH3
OCH3
OCH3
OCH3
OBn
OH
OBn
OBn
18
19
20
21
H3CO
HO
13CH2
H3CO
BnO
13CH2
m
O
O
13
C
13
C
13CH2
13CH2
O
O
22
23
OCH3
OCH3
OH
13CH2
OBn
n,m
H3CO
HO
13
CH OH
2
CH OH
2
13CH2
24
OCH3
OH
Scheme 4. Synthesis of [7,70,90-13C3]matairesinol(23) and [7,70,9-13C3]secoiso-
lariciresinol(24). Reactions and conditions: (a) NaOH, I2, MeOH (78%); (b)
BnCl, KOH, EtOH (71%); (c) K13CN, Ca(OH)2, Pd(OAc)2, DMF (51%); (d)
2 M NaOH, reflux (91%); (e) LiAlH4, Et2O (91%); (f) TMSBr, Et2O (77%);
(g) KOtBu, CH2(CO2Et)2, glyme (87%); (h) LiAlH4, Et2O (68%); (i) p-TsCl,
Et3N, DCM (30%); (j) K13CN, 18-crown-6, MeCN (65%); (k) 2 M NaOH,
reflux (57%); (l) LDA, THF, ꢀ788C, then (20) (46%); (m) H2, Pd/C (60%); and
(n) LiAlH4, THF (32%)
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956
T. FRYATT AND N. P. BOTTING
under our normal conditions followed by hydrolysis, reduction and bromina-
tion as before gave the benzyl alcohol (20). This labelled starting material
could then be taken through the synthesis to provide the two lignans with three
13C-atoms incorporated. In this series the key alkylation of the lactone
intermediate (21) worked in reasonable yield using the benzyl bromide (20),
meaning that it was not necessary to prepare the iodide as before.
Both labelled lignans were purified by reverse phase HPLC to produce high
purity material suitable for use as an internal standard. As before the
incorporation of the 13C atoms was confirmed by the enhanced signals in the
13C NMR spectra and by the expected increase in the molecular weight as
determined by mass spectrometry. In the 13C NMR spectrum for
[7,70,90-13C3]matairesinol (23) the three 13C atoms were observed as enhanced
signals at 35.6, 39.1 and 181.9 ppm and for the [7,70,9-13C3] secoisolariciresinol
(24) two signals were observed at 36.3 and 61.4 ppm. The rest of the spectral
data were in good agreement with literature data for the unlabelled plant
lignans.28
All four of the 13C-labelled lignans have now been successfully employed as
internal standards in both GC-MS29 and LC-MS13 analysis. The use of these
standards has resulting in improved reproducibility of analysis which has
proven to be important in large scale epidemiological studies such as the
European Investigation of Cancer and Nutrition (EPIC) large numbers of
samples.30
General
Melting points were determined in open capillary tubes and are uncorrected.
1H and 13C NMR spectra were recorded at 300 and 75.46 MHz, respectively,
using Bruker Avance 500 and Varian Gemini 2000 spectrometers. The
chloroform peaks (7.27 ppm for 1H, 77.00 ppm for 13C) were used as
references. The EI and CI mass spectra were obtained on a VG Autospec
mass spectrometer, and the ESI and APCI mass spectra on a Micromass LCT
mass spectrometer. Diethyl ether and tetrahydrofuran were distilled over
sodium and dichloromethane over calcium hydride. HPLC purification by was
carried out on a Waters 600 Multisolvent Delivery System using a
150 ꢁ 4.60 mm Kingsorb 3m C-18 column eluted with a 1:1 acetonitrile/water
mixture at a flow rate of 0.3 ml/min.
3-Methoxybenzo[13C]nitrile (11)
Potassium [13C]cyanide (0.5 g, 7.4 mmol) was added to a solution of
3-iodoanisole (1.73 g, 7.4 mmol), palladium acetate (0.25 g, 1.13 mmol) and
calcium hydroxide (0.28 g, 3.7 mmol) in dry DMF (50 ml) and the mixture was
heated under reflux for 24 h. After cooling the DMF was removed at reduced
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SYNTHESIS OF MULTIPLY 13C-LABELLED PLANT AND MAMMALIAN LIGNANS 957
pressure. Water (50 ml) was added and the mixture extracted with diethyl ether
(3 ꢁ 50 ml). The combined organic phases were dried (MgSO4) and concen-
trated at reduced pressure. The crude product was purified by column
chromatography (silica; light petroleum/diethyl ether (95:5)) to give the title
compound 31 as a yellow oil (0.62 g, 64%), (HRMS found M+, 134.0565.
C7 13CH7NO requires 134.0561); nmax (neat)/cmꢀ1 2179 (13CN) and 1579
(C ¼ C); dH (300 MHz; CDCl3) 3.82 (3 H, s, OMe), 7.11–7.14 (2 H, m, H-2, 4),
7.23–7.25 (1 H, m, H-6) and 7.33–7.37 (1 H, m, H-5); dC (75 MHz; CDCl3) 55.5
(OMe), 113.4 (d, J ¼ 56, C-1), 116.9 (C-2), 118.8 (enhanced, 13CN),
119.4 (C-4), 124.6 (C-6), 130.4 (C-5) and 159.8 (C-3); m/z (EI) 134 (M+,
100%), 104 (55), 91 (43), 77 (13), 63 (17) and 50 (9).
3-Methoxy[carboxy-13C]benzoic acid
A solution of 3-methoxybenzo[13C]nitrile (0.55 g, 4.1 mmol) in 2 M sodium
hydroxide solution (20 ml) was heated under reflux for 18 h. After cooling the
mixture was acidified to pH 1 with 1 M hydrochloric acid. The mixture was
extracted with diethyl ether (3 ꢁ 20 ml). The combined organic phases were
dried (MgSO4) and concentrated at reduced pressure to give the title compound
as a white solid (0.55 g, 87%), mp 94–978C (lit.32 98–1018C); (Found C, 62.5;
H, 4.9. C7 13CH8O4 requires C, 62.7; H, 5.3%); (HRMS found M+, 153.0515.
C7 13CH8O4 requires 153.0506); nmax (nujol)/cmꢀ1 1657 (13C ¼ O) and 1581
(C ¼ C); dH (300 MHz; CDCl3) 3.87 (3 H, s, OMe), 7.14–7.18 (1 H, m, H-4),
7.39 (1 H, t, J ¼ 7:8, H-5), 7.61–7.64 (1 H, m, H-2) and 7.69–7.73 (1 H, m,
H-6); dC (75 MHz; CDCl3) 172.4 (enhanced, 13CO2H); m/z (EI) 153 (M+,
100%), 136 (26), 107 (13), 91 (12), 81 (11), 77 (14) and 63 (14).
3-Methoxy[a-13C]benzyl alcohol (12)
A solution of 3-methoxy[carboxy-13C]benzoic acid (0.55 g, 3.6 mmol) in dry
diethyl ether (45 ml) was added to a suspension of lithium aluminium hydride
(0.41 g, 10.7 mmol) in dry diethyl ether (20 ml) at 08C and the mixture was
stirred at room temperature for 48 h. The mixture was cooled to 08C and was
quenched with water (30 ml) and 2 M sodium hydroxide solution (30 ml) and
silica was added. The mixture was filtered through celite and was washed with
dichloromethane. The phases were separated and the organic phase was dried
(MgSO4) and concentrated at reduced pressure to give the title compound as a
colourless oil (0.41 g, 83%); (HRMS found M+, 139.0716. C7 13CH10O2
requires 139.0714); nmax(neat)/cmꢀ1 3349 (OH) and 1602 (C ¼ C); dH
(300 MHz; CDCl3) 3.81 (3 H, s, OMe), 4.67 (2 H, dd, J ¼ 142:8, 5.7, 13CH2),
6.82–6.85 (1 H, m, H-2), 6.90–6.95 (2 H, m, H-4, 6) and 7.26–7.30 (1 H, m,
H-5); dC (75 MHz; CDCl3) 65.3 (enhanced, 13CH2); m/z (EI) 139 (M+, 100%),
138 (27), 122 (14), 109 (63), 94 (21) and 77 (35).
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958
T. FRYATT AND N. P. BOTTING
3-Methoxy[a-13C]benzyl bromide (13)
Phosphorus tribromide (7.2 ml) was added to a solution of 3-methoxy[a-13C]-
benzyl alcohol (9.53 g, 68.6 mmol) in dry diethyl ether (50 ml) at 08C and the
mixture was stirred at room temperature overnight. The reaction mixture was
poured into ice water and extracted with diethyl ether (3 ꢁ 75 ml). The
combined organic phases were dried (MgSO4) and concentrated at reduced
pressure to give the title compound as a colourless oil (12.6 g, 91%). This
compound was used without further purification.
Diethyl 2-(30-methoxy[a-13C]benzyl)malonate
Potassium tbutoxide (7.0 g, 62.3 mmol) was added to a solution of
diethyl malonate (9.5 ml, 62.3 mmol) in 1,2-dimethoxyethane (140 ml) and
the mixture was stirred at room temperature for 30 min 3-Methoxy[a-13C]-
benzyl bromide (12.6 g, 62.3 mmol) was added and the mixture was heated
under reflux for 1 h. After cooling, water was added and the 1,2-
dimethoxyethane was removed at reduced pressure. The residue was extracted
with diethyl ether (3 ꢁ 100 ml) and ethyl acetate (3 ꢁ 100 ml). The combined
organic phases were dried (MgSO4) and concentrated at reduced pressure. The
crude product was purified by column chromatography (silica; light
petroleum/diethyl ether (4:1) elution) to give the title compound as a yellow
oil (15.1 g, 86%); (HRMS found M+, 281.1337. C14 13CH20O5 requires
281.1344); dH (300 MHz; CDCl3) 1.21 (3 H, t, J ¼ 7:2, CO2CH2CH3), 1.28
(3 H, t, J ¼ 7:2, CO2CH2CH3), 3.19 (2 H, dd, J ¼ 132, 8.1, 13CH2), 3.60–3.67
(1 H, m, CH), 3.77 (3 H, s, OMe), 4.11–4.24 (4 H, m, CO2CH2CH3), 6.74–6.81
(3 H, m, H-20,40,60) and 7.15–7.21 (1 H, m, H-50); dC (75 MHz; CDCl3) 34.6
(enhanced, 13CH2); m/z (EI) 281 (M+, 79%), 236 (12), 207 (100), 162 (69), 135
(19) and 122 (15).
2-ð30-Methoxy½a-13CꢂbenzylÞpropane-1; 3-diol
A Solution of diethyl 2-(30-methoxy[a-13C]benzyl)malonate (15.1 g, 53.6 mmol)
in dry diethyl ether (45 ml) was added to a suspension of lithium aluminium
hydride (5.3 g, 137.8 mmol) in dry diethyl ether (130 ml) at 08C and the mixture
was stirred at room temperature overnight. The reaction was cooled to
08C and was quenched with 1 M sulphuric acid. The mixture was extracted
with diethyl ether (3 ꢁ 120 ml) and the organic phases were dried (MgSO4)
and concentrated at reduced pressure to give the title compound as a white
solid (8.85 g, 84%), mp 73–778C (from diethyl ether) (lit.23 81–828C); (Found
C, 67.1; H, 8.4. C10 13CH16O3 requires C, 67.0; H, 8.2%); (HRMS found
M+, 197.1139. C10 13CH16O3 requires 197.1132.); dH (300 MHz; CDCl3)
2.02–209 (1 H, m, CH), 2.59 (2 H, dd, J ¼ 130:5, 7.5, 13CH2), 3.62–3.75
(2 H, m, CH2OH), 3.76–3.81 (5 H, m, OMe, CH2OH), 6.73–6.79 (3 H, m,
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SYNTHESIS OF MULTIPLY 13C-LABELLED PLANT AND MAMMALIAN LIGNANS 959
H-20,40,60) and 7.17–7.22 (1 H, m, H-50); dC (75 MHz; CDCl3) 34.2 (enhanced,
13CH2), 55.2 (OMe), 65.6 (CH2OH), 111.5 (C-40), 114.9 (C-20), 121.5 (C-60)
and 129.6 (C-50); m/z (EI) 197 (M+, 25%), 148 (10), 135 (8), 123 (100) and
92 (9).
2-(30-Methoxy[a-13C]benzyl)-3-O-tosyl propanol (14)
Triethylamine (4.2 ml, 31.3 mmol) was added to a solution of 2-(30-
methoxy[a-13C]benzyl)propane-1,3-diol (8.83 g, 44.8 mmol) and p-toluenesul-
phonyl chloride (5.97 g, 31.3 mmol) in dry dichloromethane (200 ml) and
the mixture was stirred at room temperature overnight. The mixture was
washed with 1 M hydrochloric acid (2 ꢁ 120 ml), dried (MgSO4) and
concentrated at reduced pressure. The crude product was purified by column
chromatography (silica; dichloromethane/ethyl acetate) to give the title
compound as a yellow oil (3.99 g, 25%); (HRMS found M+, 351.1212. C17
13
CH22SO5 requires 351.1221); dH (300 MHz; CDCl3) 2.03–2.09 (1 H, m, CH),
2.59 (2 H, ddd, J ¼ 127:8, 7.5, 3.3, 13CH2), 2.43 (3 H, s, Me), 3.54–3.66
(2 H, m, CH2OH), 3.81 (3 H, s, OMe), 3.98–4.10 (2 H, m, CH2OTs), 6.64–6.74
(3 H, m, H-20,40,60), 7.14 (1 H, t, J ¼ 8:4, H-50), 7.32 (2 H, d, J ¼ 7:8, ArH)
and 7.76 (2 H, d, J ¼ 7:8, ArH); dC (75 MHz; CDCl3) 21.6 (CH3), 33.2
(enhanced, 13CH2), 42.5 (d, J ¼ 34, CH), 55.1 (OMe), 61.4 (CH2OTs), 69.6
(CH2OH), 111.8 (C-40), 114.8 (C-20), 121.4 (C-60), 128.0 (CH), 130.0 (CH); m/z
(EI) 351 (M+, 27%), 281 (10), 179 (23), 161 (21), 148 (42), 135 (28), 123 (100)
and 91 (52).
2-(30-Methoxy[a-13C]benzyl)-3-[cyano-13C]cyanopropanol
Potassium [13C]cyanide (0.76 g, 11.4 mmol) was added to a solution of 2-(30-
methoxy[a-13C]benzyl)-3-O-tosyl propanol (3.99 g, 11.4 mmol) and 18-crown-6
(3.02 g, 11.4 mmol) in acetonitrile (120 ml) and the mixture was heated under
reflux for 48 h. After cooling, the mixture was filtered through a pad of silica
and washed with copious amounts of diethyl ether and ethyl acetate. The
combined organic washings were concentrated at reduced pressure. The crude
product was purified by column chromatography (silica; dichloromethane/
ethyl acetate (4:1)) to give the title compound as a brown oil (1.94 g, 82%);
(HRMS found M+, 207.1162. C10 13C2H15NO2 requires 207.1169.); dH
(300 MHz; CDCl3) 2.18–2.25 (1 H, m, CH), 2.40–2.50 (2 H, m,
C10 13C2H15NO2), 2.72 (2 H, dddd, J ¼ 127:5, 38.7, 13.8, 6.6, 13CH2), 3.58–
3.78 (2 H, m, CH2OH), 3.79 (3 H, s, OMe), 6.73–6.81 (3 H, m, H-20,40,60) and
7.23 (1 H, t, J ¼ 8:1, H-50); dC (75 MHz; CDCl3) 36.4 (enhanced, 13CH2), 55.2
(OMe), 63.5 (CH2OH), 112.0 (C-40), 114.8 (C-20), 118.7 (enhanced, 13CN) and
121.5 (C-60); m/z (EI) 207 (M+, 20%), 148 (6), 123 (100), 122 (32), 108 (9), 92
(18) and 79 (9).
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960
4-(30-Methoxy[a-13C]benzyl)dihydro-2(3H)-[carbonyl-13C]furanone (7)
solution of 2-(30-methoxy[a-13C]benzyl)-3-[cyano-13C]cyanopropanol
T. FRYATT AND N. P. BOTTING
A
(1.94 g, 9.38 mmol) in 2 M sodium hydroxide (65 ml) were heated under reflux
for 24 h. After cooling, the mixture was acidified to pH 1 with 6 M
hydrochloric acid. The acidic aqueous phase was extracted with diethyl ether
(2 ꢁ 75 ml). The combined organic phases were dried (MgSO4) and concen-
trated at reduced pressure. The crude product was purified by column
chromatography (silica; dichloromethane) to give the title compound as a
yellow oil (1.67 g, 86%); (HRMS found M+, 208.1016. C10 13C2H14O3
requires 208.1010); nmax (neat)/cmꢀ1 1722 (C ¼ O) and 1600 (C ¼ C); dH
(300 MHz; CDCl3) 2.24–2.34 (1 H, m, H-4), 2.51–2.99 (2 H, m, 13CH2), 2.56–
2.66 (1 H, m, H-3a), 2.83–2.94 (1 H, m, H-3b), 3.80 (3 H, s, OMe), 4.00–4.13
(1 H, m, H-5a), 4.29–4.37 (1 H, m, H-5b), 6.68–6.80 (3 H, m, H-20,40,60) and
7.23 (1 H, t, J ¼ 8:1, H-50); dC (75 MHz; CDCl3) 38.9 (enhanced, 13CH2), 55.2
(OMe), 72.6 (CH2O), 111.9 (C-40), 114.7 (C-20), 121.0 (C-60), 129.9 (C-50), and
176.9 (enhanced, 13C ¼ O); m/z (EI) 208 (M+, 39%), 148 (6), 123 (100), 122
(40), 108 (8), 92 (18), 84 (12) and 78 (10).
3-Methoxy[a-13C]benzyl iodide (15)
Phosphorus triiodide (8.88 g, 21.5 mmol) was added to a solution of 3-
methoxy[a-13C]benzyl alcohol (1.76 g, 12.7 mmol) in dry diethyl ether (40 ml)
at 08C and the mixture was stirred at room temperature overnight. The
reaction mixture was poured into ice water and the mixture was extracted with
diethyl ether (3 ꢁ 75 ml). The combined organic phases were dried (MgSO4)
and concentrated at reduced pressure to give the title compound as a brown
solid (2.9 g, 92%), which was used without further purification. dH (300 MHz;
CDCl3) 3.79 (3 H, s, OMe), 4.41 (2 H, d, J ¼ 152:1, 13CH2I), 6.78 (1 H, dd,
J ¼ 8:1, 2.4, H-4), 6.88–6.91 (1 H, m, H-2), 6.96 (1 H, dd, J ¼ 8:1, 2.4, H-6)
and 7.20 (1 H, t, J ¼ 8:1, H-5).
3; 4-bis-(30-Methoxy[a-13C]benzyl)dihydro-2(3H)-[carbonyl-13C]furanone (16)
This reaction must be carried out under a continuous flow of argon gas. All
glassware and syringes must be oven dried overnight and THF must be freshly
dried. LDA (2 M in hexane/THF/ethylbenzene; 3.1 ml, 6.08 mmol) was added
to a solution a solution of 4-(30-methoxy[a-13C]benzyl)dihydro-2(3 H)-[carbo-
nyl-13C]furanone (0.96 g, 4.62 mmol) in dry THF (22 ml) at ꢀ788C and the
mixture was left to stir for 3 h. A precooled solution of 3-methoxy[a-13C]ben-
zyl iodide (1.53 g, 6.2 mmol) (20 min in an ice bath) in dry THF (14 ml) was
added and the solution was stirred at ꢀ788C for 4 h and allowed to warm to
room temperature overnight. The reaction was quenched with brine and the
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SYNTHESIS OF MULTIPLY 13C-LABELLED PLANT AND MAMMALIAN LIGNANS 961
mixture was extracted with diethyl ether (3 ꢁ 50 ml). The combined organic
phases were dried (MgSO4) and concentrated at reduced pressure. The crude
product was purified by column chromatography (silica; light petroleum/
diethyl ether) to give the title compound as a yellow oil (0.85 g, 55%); (Found
M+, 329.1625. C17 13C3H22O4 requires 329.1618); nmax (neat)/cmꢀ1 1724
(C ¼ O) and 1601 (C ¼ C); dH (300 MHz; CDCl3) 2.48–2.86 (4 H, m, CH,
13CH2), 3.07–3.29 (2 H, m, 13CH2), 3.75 (3 H, s, OMe), 3.79 (3 H, s, OMe), 3.81
(1 H, m, H-5a), 4.06–4.13 (1 H, m, H-5b), 6.50–6.52 (1 H, m, H-20), 6.56–6.59
(1 H, m, H-20), 6.70–6.78 (4 H, m, H-40, 60), 7.16 (1 H, t, J ¼ 7:8, H-50) and 7.20
(1 H, t, J ¼ 7:8, H-50); dC (75 MHz; CDCl3) 35.3 (enhanced, 13CH2), 38.5
(enhanced, 13CH2) and 178.4 (enhanced, 13C ¼ O); m/z (EI) 329 (M+, 64%),
279 (5), 207 (15), 160 (6), 149 (15), 123 (100), 122 (38), 92 (17) and 69 (9).
[7,70,90- 13C3]Enterolactone (5)
Boron tribromide (1.0 M in dichloromethane; 21 ml, 21 mmol) was added to a
solution of 3,4-bis-(30-methoxy[a-13C]benzyl)dihydro-2(3 H)-[carbonyl-13C]fur-
anone (0.85 g, 2.59 mmol) in dry dichloromethane (80 ml) and the mixture was
stirred for 2.5 h at room temperature. Water (50 ml) and diethyl ether (50 ml)
were added and the phases were separated. The aqueous phase was extracted
with diethyl ether (3 ꢁ 75 ml). The combined organic phases were dried
(MgSO4) and concentrated at reduced pressure. The crude product was
purified by column chromatography (silica; dichloromethane/ethyl acetate
(3:1)) to give the title compound as a white solid (0.53 g, 68%), which was
further purified by reverse phase HPLC. (Found C, 69.88; H, 5.63. C15 13C3
H18O4:0:5H2O requires C, 69.66; H, 5.85%); (HRMS found M+., 301.1315.
C15 13C3H18O4 requires 301.1305); nmax (nujol)/cmꢀ1 3392 (OH), 1703
(13C ¼ O) and 1589 (C ¼ C); dH (300 MHz; d6-acetone) 2.42–2.58 (2 H, m,
H-8 and 80), 2.64–3.20 (4 H, m, 13CH2), 3.83–3.91 (1 H, m, H-9a), 3.99–4.08
(1 H, m, H-9b), 6.58–6.79 (6 H, m, H-2,4,6, 20,40 and 60), 7.08 (1 H, t, J ¼ 7:8,
H-5 or H-50), 7.12 (1 H, t, J ¼ 7:8, H-5 or H-50) and 8.27 (2 H, br, ArOH);
dC (75 MHz; d6-acetone) 35.4 (enhanced, 13CH2), 38.6 (enhanced, 13CH2), 71.4
(C-9), 114.4, 114.6 (C-4 and 40), 116.3, 117.0 (C-2 and C-20), 120.5, 121.4 (C-6
and C-60), 130.2, 130.3 (C-5 and C-50), 140.9 (C-1 and C-10), 158.3 (C-3 and
C-30) and 178.6 (enhanced, 13C ¼ O); m/z (EI) 301 (M+, 52%), 193 (24), 134
(26), 109 (100) and 108 (60).
[7,70,9- 13C3]Enterodiol (17)
A solution of [7,70,90-13C3]enterolactone (150 mg, 0.5 mmol) in dry THF (9 ml)
was added to a suspension of lithium aluminium hydride (103 mg, 2.68 mmol)
in dry THF (12 ml) at 08C and the reaction was stirred at room temperature
overnight. The reaction was quenched at 08C with 1 M sulphuric acid and
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962
T. FRYATT AND N. P. BOTTING
extracted with diethyl ether (4 ꢁ 40 ml). The combined organic phases were
dried (MgSO4) and concentrated at reduced pressure. The crude product was
purified by column chromatography (silica; dichloromethane/ethyl acetate
(1:1)) to give the title compound as a white solid (59 mg, 39%), which was
further purified by reverse phase HPLC. mp 174–1768C (lit.23 171–1738C);
(Found C, 69.92; H, 7.18. C15 13C3H22O4:0:1H2O requires C, 70.4; H, 7.22%);
nmax (nujol)/cmꢀ1 3410 (OH), and 1588 (C ¼ C); dH (300 MHz; d4-MeOH)
2.06–2.11 (2 H, m, H-8 and 80), 2.74 (4 H, dd, J ¼ 126:3, 5.1, 13CH2), 3.7 (2 H,
dm, J13C 1H
¼ 140, 13CH2OH), 3.60–3.80 (2 H, m, CH2OH), 6.67–6.74 (6 H, m,
;
H-2, 4, 6, 20, 40 and 60) and 7.15 (2 H, t, J ¼ 7:5, H-5 and H-50); dC (75 MHz;
d4-MeOH) 35.1 (enhanced, 13CH2), 39.2–40.2 (CH), 60.7 (enhanced,
13CH2OH), 112.6 (C-4 and C-40), 115.9 (C-2 and C-20), 120.3 (C-6 and C-
60), 129.1 (C-5 and C-50), 141.0 (d, J ¼ 8, C-1 and C-10) and 157.2 (C-3 and
C-30); m/z (EI) 305 (2%), 256 (9), 108 (100), and 107 (60).
4-Hydroxy-3-methoxyiodobenzene
Iodine (6.8 g, 26.8 mmol) was added portionwise to a solution of guaiacol
(3.3 g, 26.8 mmol) and sodium hydroxide (2.01 g) in methanol (70 ml) keeping
the temperature between ꢀ2 and 18C. The mixture was stirred at 08C for 1.5 h
before being acidified with 5% hydrochloric acid solution to pH 2. The solvent
was removed at reduced pressure and the residue was dissolved in diethyl
ether. The aqueous phase was extracted with diethyl ether (3 ꢁ 100 ml). The
organic phase was washed with 10% sodium thiosulphate solution (2 ꢁ 75 ml),
dried (MgSO4) and concentrated at reduced pressure to give the title compound
as a brown oil (5.22 g, 78%); dH (300 MHz; CDCl3) 3.85 (3 H, s, OMe), 5.55
(1 H, s, OH), 6.70 (1 H, d, J ¼ 8:2, H-5), 6.80–6.95 (1 H, m, H-2) and 7.10–7.18
(1 H, m, H-6).
4-Benzyloxy-3-methoxyiodobenzene (19)
Benzyl chloride (3.7 ml, 32.4 mmol) was added to a solution of 4-hydroxy-3-
methoxyiodobenzene (7.23 g, 29 mmol) and potassium hydroxide (1.8 g,
32.4 mmol) in ethanol (140 ml) and the mixture was heated under reflux
overnight. After cooling the solvent was concentrated at reduced pressure.
Water (100 ml) was added and the mixture was extracted with ethyl acetate
(3 ꢁ 100 ml). The combined organic phases were washed with 2 M potassium
hydroxide solution (5 ꢁ 100 ml), dried (MgSO4) and concentrated at reduced
pressure. The crude product was purified by column chromatography (silica;
light petroleum/ethyl acetate (2:1)) to give the title compound as a yellow oil
(6.98 g, 71%); nmax (neat)/cmꢀ1 1582 (C ¼ C); dH (300 MHz; CDCl3) 3.86 (3 H,
s, OMe), 5.11 (2 H, s, OCH2), 6.62 (1 H, d, J ¼ 8:7, H-5), 6.87–6.91 (1 H, m,
H-2), 7.13–7.17 (1 H, m, H-6) and 7.29–7.43 (5 H, m, ArH); dC (75 MHz;
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SYNTHESIS OF MULTIPLY 13C-LABELLED PLANT AND MAMMALIAN LIGNANS 963
CDCl3) 56.2 (OMe), 71.1 (OCH2), 83.2 (C-1), 112.1 (C-5), 114.4 (C-2), 121.1
(C-6), 127.4 (CH), 128.1 (CH), 128.7 (CH), 136.8 (C-1’), 148.4 (C-4) and 150.7
(C-3); m/z (EI) 340 (M+, 10%), 214 (16), 92 (13), 91 (100) and 65 (12).
4-Benzyloxy-3-methoxybenzo[13C]nitrile
Potassium [13C]cyanide (1.43 g, 21.4 mmol) was added to a solution of 4-
benzyloxy-3-methoxyiodobenzene (7.26 g, 21.4 mmol), palladium acetate
(0.73 g, 3.3 mmol) and calcium hydroxide (0.82 g, 10.7 mmol) in dry DMF
(150 ml) and the mixture was heated under reflux for 15 h. After cooling the
solvent was removed at reduced pressure. Water (120 ml) was added and the
mixture was extracted with ethyl acetate (3 ꢁ 150 ml). The combined organic
phases were dried (MgSO4) and concentrated at reduced pressure. The crude
product was purified by column chromatography (silica; light petroleum/ethyl
acetate (9:1)) to give the title compound as a pale yellow solid (2.61 g, 51%), mp
81–838C (lit.33 73–748C); (Found C, 74.8; H, 5.3; N, 5.3 C14 13CH13NO4
requires C, 74.8; H, 5.45; N, 5.8%); (HRMS found M+, 240.0987. C14
13
CH13NO4 requires 240.0980); nmax (nujol)/cmꢀ1 2169 (13CN); dH (300 MHz;
CDCl3) 3.89 (3 H, s, OMe), 5.19 (2 H, s, OCH2), 6.89 (1 H, d, J ¼ 8:1, H-5),
7.08 (1 H, dd, J ¼ 5:4, 1.8, H-2), 7.17–7.19 (1 H, m, H-6) and 7.31–7.39 (5 H,
m, ArH); dC (75 MHz; CDCl3) 118.8 (enhanced, 13CN); m/z (EI) 240 (M+,
9%), 91 (100), 65 (13) and 59 (6).
4-Benzyloxy-3-methoxy[carbonyl-13C]benzoic acid
A solution of 4-benzyloxy-3-methoxybenzo[13C]nitrile (1.4 g, 5.8 mmol) in 2 M
sodium hydroxide solution (20 ml) was heated under reflux overnight. After
cooling the mixture was acidified to pH 1 with 6 M hydrochloric acid. The
acidic aqueous was extracted with diethyl ether (3 ꢁ 50 ml). The combined
organic phases were dried (MgSO4) and concentrated at reduced pressure to
give the title compound as a yellow solid (1.38 g, 91%); mp 161–1648C (lit.34
168–1698C) (Found C, 69.6; H, 5.2. C14 13CH14O4 requires C, 69.5; H, 5.4%);
(HRMS found M+, 259.0921. C14 13CH14O4 requires 259.0925); dH
(300 MHz; CDCl3) 3.94 (3 H, s, OMe), 5.29 (2 H, s, OCH2), 6.92 (1 H, d,
J ¼ 8:7, H-5), 7.31–7.44 (5 H, m, ArH), 7.59–7.62 (1 H, m, H-6) and 7.66–7.69
(1 H, m, H-2); dC (75 MHz; CDCl3) 171.0 (enhanced, 13CO2H); m/z (EI) 259
(M+, 33%), 91 (100) and 65 (13).
4-Benzyloxy-3-methoxy[a-13C]benzyl alcohol
A solution of 4-benzyloxy-3-methoxy[carboxy-13C]benzoic acid (1.38 g,
5.3 mmol) in dry diethyl ether (50 ml) was added to a suspension of lithium
aluminium hydride (0.61 g, 16 mmol) in dry diethyl ether (50 ml) at 08C and the
mixture was stirred for 48 h at room temperature. The mixture was cooled to
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T. FRYATT AND N. P. BOTTING
08C and was quenched with water (45 ml) and 2 M sodium hydroxide solution
(45 ml) and silica was added. The mixture was filtered through celite and was
washed with dichloromethane. The phases were separated and the organic
phase was dried (MgSO4) and concentrated at reduced pressure. The crude
product was recrystallized from diethyl ether/light petroleum to give the title
compound as a white solid (1.19 g, 91%); mp 70–728C (from diethyl ether)
(lit.35 72–738C); (Found C, 73.5; H, 6.7. C14 13CH16O3 requires C, 73.5; H,
6.6%); (HRMS found M+, 245.1140. C14 13CH16O3 requires 245.1132); dH
(300 MHz; CDCl3) 3.89 (3 H, s, OMe), 4.60 (2 H, dd, J ¼ 142:8, 5.7,
13CH2OH), 5.15 (2 H, s, OCH2), 6.78–6.86 (2 H, m, H-5, 6), 6.94 (1 H, d,
J ¼ 3:3, H-2) and 7.28–7.44 (5 H, m, ArH); dC (75 MHz; CDCl3) 65.3
(enhanced, 13CH2); m/z (EI) 245 (M+, 29%), 91 (100), 84 (14), 65 (10) and
49 (17).
4-Benzyloxy-3-methoxy[a-13C]benzyl bromide (20)
Trimethylsilyl bromide (0.6 ml, 7.4 mmol) was added to a solution of 4-
benzyloxy-3-methoxy[a-13C]benzyl alcohol (1.2 g, 4.9 mmol) in dry diethyl
ether (30 ml) and the mixture was stirred at room temperature for 2.5 h. The
reaction was quenched with water (10 ml) and was extracted with diethyl ether
(2 ꢁ 15 ml). The combined organic phases were washed with water (15 ml),
dried (MgSO4) and concentrated at reduced pressure to give the title compound
as a white solid (1.16 g, 77%) which was used without further purification. dH
(300 MHz; CDCl3) 3.90 (3 H, s, OMe), 4.48 (2 H, d, J ¼ 153:3, 13CH2Br), 5.19
(2 H, s, OCH2), 6.81 (1 H, d, J ¼ 7:5, H-5), 6.52–6.87 (1 H, m, H-6), 6.93 (1 H,
dd, J ¼ 5:4, 1.8, H-2) and 7.29–7.43 (5 H, m, ArH).
Diethyl 2-(40-benzyloxy-30-methoxy[a-13C]benzyl)malonate
t
Potassium butoxide (0.42 g, 3.77 mmol) was added to a solution of diethyl
malonate (0.6 ml, 3.77 mmol) in 1,2-dimethoxyethane (40 ml) and the mixture
was stirred at room temperature for 30 min 4-Benzyloxy-3-methoxy[a-13C]-
benzyl bromide (1.16 g, 3.77 mmol) was added and the mixture was heated
under reflux for 1.5 h. After cooling, water (40 ml) was added and the mixture
was extracted with diethyl ether (3 ꢁ 50 ml) and ethyl acetate (3 ꢁ 50 ml). The
combined organic phases were dried (MgSO4) and concentrated at reduced
pressure. The residue was purified by column chromatography (silica; light
petroleum/diethyl ether (2:1)) to give the title compound as a colourless oil
(1.26 g, 87%); (HRMS found M+, 387.1762. C21 13CH26O6 requires
387.1771); dH (300 MHz; CDCl3) 1.20 (3 H, t, J ¼ 7:2, CO2CH2CH3), 1.27
(3 H, t, J ¼ 7:2, CO2CH2CH3), 3.13 (2 H, dd, J ¼ 131:7, 7.8, 13CH2), 3.56–3.63
(1 H, m, CH), 3.85 (3 H, s, OMe), 4.11–4.24 (4 H, m, CO2CH2CH3), 5.10 (2 H,
s, OCH2), 6.67–6.79 (3 H, m, H-20, 50, 60) and 7.28–7.42 (5 H, m, ArH);
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SYNTHESIS OF MULTIPLY 13C-LABELLED PLANT AND MAMMALIAN LIGNANS 965
dC (75 MHz; CDCl3) 14.0 (CO2CH2CH3), 34.3 (enhanced, 13CH2), 61.5
(CO2CH2CH3), (OCH2), 127.4 (CH), 128.6 (CH); m/z (EI) 387 (M+, 81%),
296 (31), 222 (59), 133 (60), 115 (100) and 91 (83).
2-(40-Benzyloxy-30-methoxy[a-13C]benzyl)propane-1,3-diol
A Solution of diethyl 2-(40-benzyloxy-30-methoxy[a-13C]benzyl)malonate
(1.26 g, 3.3 mmol) in dry diethyl ether (5 ml) was added to a suspension of
lithium aluminium hydride (0.32 g, 8.4 mmol) in dry diethyl ether (10 ml) at
08C and the mixture was stirred at room temperature for 15 h. The reaction
was quenched at 08C with 1 M H2SO4. The mixture was extracted with diethyl
ether (3 ꢁ 20 ml), dried (MgSO4) and concentrated at reduced pressure. The
crude product was recrystallized from diethyl ether to give the title compound
as a white solid (0.67 g, 68%); mp 82–858C (from diethyl ether); (Found C,
71.2; H, 7.5. C17 13CH22O4 requires C, 71.3; H, 7.3%); (HRMS found M+,
303.1562. C17 13CH22O4 requires 303.1551); dH (300 MHz; CDCl3) 1.91 (2 H,
br, OH), 2.01–2.02 (1 H, m, CH), 2.55 (2 H, dd, J ¼ 126:6, 7.5, 13CH2), 3.63–
3.69 (2 H, m, CH2OH), 3.76–3.79 (2 H, m, CH2OH), 3.82 (3 H, s, OMe), 5.11
(2 H, s, OCH2), 6.64–6.74 (2 H, m, H-20, 60), 6.79 (1 H, d, J ¼ 8:1, H-50) and
7.27–7.44 (5 H, m, ArH); dC (75 MHz; CDCl3) 33.9 (enhanced, 13CH2); m/z
(EI) 303 (M+, 5%), 194 (13), 150 (13), 92 (12) and 91 (100).
2-(40-Benzyloxy-30-methoxy[a-13C]benzyl)-3-O-tosyl-propanol
Triethylamine (0.4 ml, 2.98 mmol) was added to a solution of 2-(40-benzyloxy-
30-methoxy[a-13C]benzyl)propane-1,3-diol (1.3 g, 4.3 mmol) and p-toluenesul-
phonyl chloride (0.57 g, 2.98 mmol) in dry dichloromethane (40 ml) and the
mixture was stirred for 24 h at room temperature. The mixture was washed
with 1 M hydrochloric acid (2 ꢁ 30 ml). The organic phase was dried (MgSO4)
and concentrated at reduced pressure. The crude product was purified by
column chromatography (silica; dichloromethane/ethyl acetate) to give the
title compound as a yellow oil (0.59 g, 30%); (HRMS found M+, 457.1630.
C26 13CH28O6S requires 457.1640); dH (300 MHz; CDCl3) 2.06–2.08 (1 H, m,
CH), 2.44 (3H, s, ArCH3), 2.55 (2 H, ddd, J ¼ 127:5, 7.5, 3, 13CH2), 3.52–3.67
(2 H, m, CH2OH), 3.85 (3 H, s, OMe), 3.97–4.13 (2 H, m, CH2OTs), 5.10 (2 H,
s, OCH2Ph), 6.52–6.56 (1 H, m, H-60), 6.66–6.68 (1 H, m, H-20), 6.75 (1 H, d,
J ¼ 8:1, H-50), 7.29–7.44 (7 H, m, ArH) and 7.77 (2 H, d, J ¼ 6:6, ArH); dC
(75 MHz; CDCl3) 33.2 (enhanced, 13CH2); m/z (EI) 457 (M+, 12%), 273 (8),
164 (12), 150 (9), 131 (11), 91 (100) and 69 (19).
2-(40-Benzyloxy-30-methoxy[a-13C]benzyl)-3-[cyano-13C]cyanopropanol
Potassium [13C]cyanide (38 mg, 0.56 mmol) was added to a solution of 2-(40-
benzyloxy-30-methoxy[a-13C]benzyl)-3-O-tosyl-propanol (0.26 g, 0.56 mmol)
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T. FRYATT AND N. P. BOTTING
and 18-crown-6 (0.15 g, 0.56 mmol) in acetonitrile (20 ml) and the mixture was
heated under reflux for 48 h. After cooling the mixture was filtered through a
pad of silica, washed with copious amounts of diethyl ether and ethyl acetate
and concentrated at reduced pressure. The crude product was purified by
column chromatography (silica; light petroleum/diethyl ether (1:1)) to give the
title compound as a yellow oil (0.11 g, 65%); (HRMS found M+, 313.1593.
C17 13C2H21NO3 requires 313.1588); dH (300 MHz; CDCl3) 2.11–2.19 (1 H, m,
CH), 2.31–2.44 (2 H, m, CH2 13CN), 2.46–2.98 (2 H, m, 13CH2), 3.60 (1 H, dt,
J ¼ 10:5, 4.1, CH2OH), 3.73 (1 H, ddd, J ¼ 10:5, 7.1, 2.6, CH2OH), 3.87 (3 H,
s, OMe), 5.12 (2 H, s, OCH2), 6.65–6.73 (2 H, m, H-20, 60), 6.82 (1 H, d,
J ¼ 8:4, H-50) and 7.29–7.44 (5 H, m, ArH); dC (75 MHz; CDCl3) 35.9
(enhanced, 13CH2) and 118.8 (enhanced, 13CN); m/z (EI) 313 (M+, 41%), 222
(7), 204 (5), 164 (10), 132 (5) and 91 (100).
4-(40-Benzyloxy-30-methoxy[a-13C]benzyl)dihydro-2(3H)[carbonyl-13C]fura-
none (21)
A solution of 2-(40-benzyloxy-30-methoxy-[13C]benzyl)-3-cyanopropanol (0.27g,
0.87mmol) in 2 M sodium hydroxide solution (10ml) was heated under reflux
for 24 h. After cooling the mixture was acidified to pH 1 with 6 M hydrochloric
acid. The acidic aqueous layer was extracted with diethyl ether. The combined
organic phases were dried (MgSO4) and concentrated at reduced pressure. The
crude product was purified by column chromatography (silica; light petroleum/
ethyl acetate (1:1) elution) to give the title compound as a white oily solid (0.16 g,
57%); (HRMS found M+, 314.1437. C17 13C2H20O4 requires 314.1428); dH
(300MHz, CDCl3) 2.22–2.82 (1 H, m, H-3a), 2.52–2.64 (1H, m, H-3b), 2.69
(2H, ddd, J ¼ 127:8, 8.1, 3.3, 13CH2), 2.78–2.84 (1H, m, H-4), 3.86 (3H, s,
OMe), 3.84–4.05 (1 H, m, H-5a), 4.27–4.34 (1H, m, H-5b), 5.12 (2H, s, OCH2),
6.59–6.68 (2H, m, H-20, 60), 6.82 (1H, d, J ¼ 8:1, H-50) and 7.29–7.44 (5 H, m,
ArH); dC (75MHz; CDCl3) 38.6 (enhanced, 13CH2) and 176.9 (enhanced,
13C ¼ O); m/z (EI) 314 (M+, 16%), 91 (100) and 65 (6).
3; 4-bis-(40-Benzyloxy-30-methoxy[a-13C]benzyl)dihydro-2(3H)-[carbonyl-13C]
furanone (22)
This reaction must be carried out under a flow of argon gas, balloons do not
work. All glassware and syringes must be oven dried and THF must be freshly
dried. LDA (2.0 M in hexane/THF/ethylbenzene; 1.32 ml, 2.63 mmol) was
added to a solution of 4-(40-benzyloxy-30-methoxy[a-13C]benzyl)dihydro-
2(3H)-[carbonyl-13C]furanone (0.66 g, 2.1 mmol) in dry THF (15 ml) at
–788C and the mixture was stirred for 2.5 h. A precooled solution of
4-benzyloxy-3-methoxy[a-13C]benzyl bromide (0.94 g, 3.04 mmol) (20 min in
an ice bath) in dry THF (6 ml) was added and the mixture was stirred at
ꢀ788C for 5 h. The reaction was quenched at ꢀ788C with brine (15 ml) and
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SYNTHESIS OF MULTIPLY 13C-LABELLED PLANT AND MAMMALIAN LIGNANS 967
allowed to warm to room temperature. The mixture was extracted with diethyl
ether (3 ꢁ 50 ml). The combined organic phases were dried (MgSO4) and
concentrated at reduced pressure. The crude product was purified by column
chromatography (silica; light petroleum/ethyl acetate (2:1) to give the title
compound as a colourless oil (0.52 g, 46%); (HRMS found M+, 541.2467.
C31 13C3H34O6 requires 541.2456); dH (300 MHz; CDCl3) 2.33–2.61 (2 H, m,
H-3, 4), 2.53 (2 H, ddd, J ¼ 163:8, 13.5, 6.3, 13CH2), 2.68–3.15 (2 H, m,
13CH2), 3.80 (3 H, s, OMe), 3.82 (3 H, s, OMe), 3.84–3.88 (1 H, m, H-5a), 4.06–
4.14 (1 H, m, H-5b), 5.11 (4 H, s, OCH2), 6.46–6.57 (3 H, m, H-20, 60), 6.70
(1 H, s, H-20), 6.76 (2 H, t, J ¼ 7:2, H-50) and 7.24–7.42 (10 H, m, ArH); dC
(75 MHz; CDCl3) 34.5 (enhanced, 13CH2), 38.0 (enhanced, 13CH2) and 178.7
(enhanced, 13C ¼ O); m/z (EI) 541 (M+, 6%), 138 (15), 91 (100) and 65 (8).
[7,70,90-13C3]Matairesinol (23)
To a solution of 3,4-bis-(40-benzyloxy-30-methoxy[a-13C]benzyl)dihydro-2(3H)-
[carbonyl-13C]furanone (0.28 g, 0.51 mmol) in ethyl acetate (15 ml) was added to
5% palladium-on-carbon (14 mg) and the mixture was stirred under a hydrogen
atmosphere for 48 h. The mixture was filtered through celite and washed with
ethyl acetate. The organic phase was concentrated at reduced pressure. The
crude product was purified by column chromatography (silica; dichloro-
methane/ethyl acetate) to give the title compound as a white solid (0.11 g, 60%),
which was further purified by reverse phase HPLC. (Found C, 65.9; H, 5.9.
C17 13C3H22O6:0:1H2O requires C, 66.1; H, 6.2%); (HRMS found M+.,
361.1505. C17 13C3H22O6 requires 361.1517); nmax (nujol)/cmꢀ1 3390 (OH),
1702 (13C ¼ O) and 1590 (C ¼ C); dH (300 MHz; d4-MeOH) 2.72 (2 H, dd,
J ¼ 79:8, 5.1, H-8), 2.67–2.71 (1 H, m, H-80), 2.81–2.88 (1 H, m, H-3), 3.02
(1 H, dddd, JCH ¼ 77:1, J3 a23 b ¼ 8:4, J3 23 ¼ 4:2, J3 24 ¼ 1:8, H-300a), 3.07
00
00
00
00
(1 H, dt, JCH ¼ 77:1, J3 a23 b ¼ 8:4, J3 23¼3 24 ¼ 3:6, H-300b), 3.96 (3 H, s,
OMe), 3.97 (3 H, s, OMe), 4.08–4.13 (1 H, m, H-5), 4.32–4.37 (1 H, m, H-5),
6.68–6.71 (1 H, m, H-60), 6.74–6.75 (1 H, m, H-20), 6.76–6.78 (1 H, m, H-60),
6.85–6.86 (1 H, m, H-20), 6.87 (1 H, d, J ¼ 5:1, H-50) and 6.89 (1 H, dd,
J ¼ 8; 10, H-50); dC (75 MHz; d4-MeOH) 35.6 (enhanced, 13CH2-7’), 39.1
(enhanced, 13CH2-7), 42.8 (d, J ¼ 35, C-8), 56.6 (OMe), 73.2 (C-9), 113.5, 114.1
(C-2 and C-20), 116.3, 116.4 (C-5 and C-50), 122.5, 123.3 (C-6 and C-60), 130.7,
131.3 (C-1 and C-10), 146.5, 146.6 (C-4 and C-40), 149.3 (C-3 and C-30) and
181.9 (enhanced, 13C ¼ O); m/z (EI) 361 (M+, 27%), 277 (7), 138 (100), 123
(17) and 95 (18).
00
00
00
00
2, 3-bis-(40-Benzyloxy-30-methoxy-[a-13C]benzyl)[1-13C]butan-1-4-diol
A solution of 3,4-bis-(40-benzyloxy-30-methoxy[a-13C]benzyl)dihydro-2(3H)-
[carbonyl-13C]furanone (0.52 g, 0.96 mmol) in dry THF (21 ml) was added to a
Copyright # 2005 John Wiley & Sons, Ltd.
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968
T. FRYATT AND N. P. BOTTING
suspension of lithium aluminium hydride (0.18 g, 4.85 mmol) in dry THF
(21 ml) at 08C and the mixture was stirred at room temperature overnight. The
mixture was cooled to 08C and was quenched with water (8 ml) and 2 M
sodium hydroxide (8 ml) and silica was added. The resulting mixture was
filtered through celite and the phases were separated. The organic phase was
dried (MgSO4) and concentrated at reduced pressure to give the title compound
(0.52 g, 100%). The crude product was used without purification.
[7,70,9-13C3]Secoisolariciresinol (24)
A solution of 2,3-(40benzyloxy-30-methoxy[a-13C]benzyl)[1-13C]butan-1,4-diol
(0.52 g, 0.96 mmol) in ethyl acetate (30 ml) was added to 5% palladium-on-
carbon (27 mg) and the mixture was stirred under a hydrogen atmosphere for
48 h. The mixture was filtered through celite and washed with ethyl acetate.
The organic phase was concentrated at reduced pressure. The crude product
was purified by column chromatography (silica; ethyl acetate/dichloromethane
(55:45)) to give the title compound as a white solid (0.11 g, 32%), which was
further purified by reverse phase HPLC. (Found C, 65.9; H, 6.9. C17 13C3H26
O6 requires C, 65.7; H, 7.2%); (HRMS found M+., 365.1825. C17 13C3H26O6
requires 365.1830); nmax (nujol)/cmꢀ1 3410 (OH), and 1588 (C ¼ C); dH
(300 MHz; CDCl3) 1.8–1.9 (2 H, m, H-8 and H-80), 2.60 (2 H, Br, 2 ꢁ OH), 2.6
(4H, dm, J13C;1H ¼ 124, 13CH2OH), 3.20–4.0 (4H, m, 2 x CH2OH), 3.80 (6H, s,
OMe), 5.4 (2 H, br, ArOH), 6.50–6.61 (4 H, m, H-2, 20, 6 and 60) and 6.75 (2 H,
d, J ¼ 8, H-50); dC (75 MHz; CDCl3) 36.3 (enhanced, 13CH2), 56.2 (OMe), 61.4
(enhanced, O13CH2); m/z (EI) 365 (M+, 30%), 256 (5), 236 (6), 138 (100), 123
(15), 69 (38), 57 (30) and 55 (29).
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