A convenient synthesis of 4-hydroxy[1-13c]benzoic acid and related ring-labelled phenolic compounds

Article abstract of DOI:10.1055/s-1998-2109

4-Hydroxy[1-¹³C]benzoic acid (5) was synthesized starting from commercially available sodium [2-¹³C]acetate (1) by a 4-step sequence in an overall yield of 55-65%. Key step in this synthesis is the formation of the benzene ring by base-catalyzed condensation of diethyl [2-¹³C]malonate (3) with 4H-pyran-4-one. Acid 5 or its ethyl ester could be converted into 3,4- dihydroxy[1-¹³C]benzoic acid (9), D,L-[1'-¹³C]tyrosine (14) and 1,2,4- trihydroxy[4-¹³C]benzene (19) with high efficiency.

Full text of DOI:10.1055/s-1998-2109

SYNTHESIS
July1998
1047
A Convenient Synthesis of 4-Hydroxy[1-13C]benzoic Acid and Related Ring-Labelled
Phenolic Compounds
Jürgen Beyer, Susanne Lang-Fugmann, Andrea Mühlbauer, Wolfgang Steglich*
Institut für Organische Chemie der Universität München, Karlstrasse 23, D-80333 München, Germany
Fax: +49(89)5902604; E-mail: wos@org.chemie.uni-muenchen.de
Received 24 January 1998
Abstract: 4-Hydroxy[1-13C]benzoic acid (5) was synthesized
starting from commercially available sodium [2-13C]acetate (1) by
a 4-step sequence in an overall yield of 55–65% . Key step in this
synthesis is the formation of the benzene ring by base-catalyzed
condensation of diethyl [2-13C]malonate (3) with 4H-pyran-4-one.
Acid 5 or its ethyl ester could be converted into 3,4-di-
hydroxy[1-13C]benzoic acid (9), D,L-[1′-13C]tyrosine (14) and
1,2,4-trihydroxy[4-13C]benzene (19) with high efficiency.
Key words: 13C-labelled aromatic compounds, 4-hydroxybenzoic
acid, 3,4-dihydroxybenzoic acid, 1,2,4-trihydroxybenzene,
tyrosine
Reagents and conditions: a) (EtO)2SO2, 168°C; b) LiN[Si(CH3)3]2/
THF, –78°C, then ClCO2Et, –78°C for 3 h, then r.t.; c) 4H-pyran-4-
one/KOBu-t/t-BuOH, reflux for 20 h, then 1 N HCl, reflux for 30
min; d) 2 N NaOH, overnight, r.t.
4-Hydroxybenzoic acid is the biosynthetic precursor of
ubiquinones1 and other natural products,2 e.g. arbutin,3 al-
kannin,4 shikonin5 and pentabromopseudilin.6 As part of
Scheme 1
an ongoing project concerning the biosynthesis of bo-
viquinones and related meroterpenoids from mushrooms7
we needed specifically 13C ring-labelled 4-hydroxybenzo-
ic acid, 3,4-dihydroxy-benzoic acid, tyrosine and 1,2,4-
trihydroxybenzene for feeding experiments. Previous
work in this field includes syntheses of 4-hydroxy[1,2-
13C2]benzoic acid and [1,2-13C2]tyrosine from 13C-la-
belled ethylene,8 the synthesis of [3,5-13C2]-9 and [3,4,5-
13C3]tyrosine5 from 13C-labelled acetone and the prepara-
tion of 1,2,4-trihydroxy[1,4-13C2]benzene10 from
Na13CN. These syntheses, however, require many steps
and afford the labelled compounds only in moderate over-
all yields. 4-Hydroxy[2,6-13C2]benzoic acid was recently
obtained by feeding [1-13C]glucose to a recombinant
strain of Klebsiella pneumoniae.11 In this publication we
describe an efficient and straightforward synthesis of
4-hydroxy[1-13C]benzoic acid and compounds derived
therefrom.
cohol, methanol, ethanol, toluene and THF) tested, the re-
action with potassium tert-butoxide in refluxing tert-butyl
alcohol afforded 4 in an optimized yield of 85%. Alka-
line hydrolysis of ester
4
provided the free
4-hydroxy[1-13C]benzoic acid (5) in 55–65% overall
yield.
For the synthesis of 3,4-dihydroxy[1-13C]benzoic acid
(9), the 13C-labelled ester 4 was treated with bromine in
chloroform to yield the monobromo derivative 6 (89%)
accompanied by a small amount of the dibromo com-
pound 7 (3%) (Scheme 2). Both products could be easily
separated by flash chromatography. Hydrolysis of ester 6
Our approach starts from commercially available sodium
[2-13C]acetate (1) which yielded ethyl [2-13C]acetate (2)
on heating in diethyl sulfate (Scheme 1). It is important to
use freshly distilled diethyl sulfate and to perform this re-
action under anhydrous conditions. Otherwise the product
might be contaminated with a significant amount of etha-
nol which cannot be separated from 2 by distillation. Ester
2 can thus be obtained in 88–97% yield.
Deprotonation of 2 with lithium hexamethyldisilazide fol-
lowed by addition of ethyl chloroformate produced di-
ethyl [2-13C]malonate (3) in 90% yield.12 The labelled
malonate 3 was directly transformed into ethyl 4-hy-
droxy[1-13C]benzoate (4) by treatment with 4H-pyran-4-
one in the presence of a suitable base followed by acidic
workup.13 Among several bases (potassium tert-butoxide,
sodium methoxide, sodium ethoxide, potassium hydride,
sodium hydride and DBU) and solvents (tert-butyl al-
Reagents and conditions: a) Br2/CHCl3, 0°C for 10 h, r.t. for 15 h; b)
2 N NaOH, 18 h, r.t., c) 4 N KOH/Cu (activated)/CuSO4•5 H2O, re-
flux for 20 h
Scheme 2

SYNTHESIS
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with 2 N NaOH afforded the acid 8 in quantitative yield.
Replacement of the bromine by a hydroxyl group was
achieved by refluxing 8 in aqueous potassium hydroxide
solution in the presence of activated copper and copper
sulfate.14 This reaction provided 3,4-dihydroxy[1-13C]-
benzoic acid (9) in 87% yield.
D,L-[1′-13C]Tyrosine (14) was prepared from ethyl 4-
hydroxy[1-13C]benzoate (4) by the phthalimidomalonate
procedure (Scheme 3). For this purpose, ester 4 was O-
methylated and the methoxy derivative 10 reduced to ben-
zylic alcohol 11 with LiAlH4 in THF.5 Treatment of 11
with phosphorus tribromide and pyridine in diethyl ether
provided 4-methoxy[1-13C]benzyl bromide (12). The re-
sulting crude material was immediately coupled with so-
dium diethyl phthalimidomalonate in DMF to afford the
4-methoxybenzyl derivative 13 in 98% yield. Heating of
13 in hydrobromic acid furnished D,L-[1′-13C]tyrosine
(14) in 42% overall yield.15
Reagents and conditions: a) MeI/K2CO3/acetone, reflux, 25 h; b)
LiAlH4/THF, 0˚C; PCC/CH2Cl2/mol sieves, 1 h; d) H2O2/MeOH/
H2SO4; e) BBr3/CH2Cl2, –78°C for 1.5 h, r.t. for 1.5 h
Scheme 4
Sodium [2-13C]acetate (99% 13C) was purchased from CAMPRO Sci-
entific BV, 46 422 Emden/Germany. 1H and 13C NMR spectra were
recorded on Bruker ARX 300, Bruker AMX 600 and Varian VXR
400 S using TMS as internal standard. Mass spectra (MS) were re-
corded with Finnigan MAT 90 and MAT 95 Q spectrometers. Sol-
vents were purified and dried according to standard procedures.
Petroleum ether used refers to the fraction with bp 40–60°C. TLC
analyses were carried out on Merck silica gel 60 F254 sheets. Flash
chromatography was performed on silica gel 60 (230-400 mesh) pur-
chased from Merck. Melting points were determined with a Büchi
melting point apparatus and are uncorrected.
Ethyl [2-13C]Acetate (2):
A suspension of sodium [2-13C]acetate (1; 1.00 g, 12.0 mmol) in
freshly distilled diethyl sulfate (5 mL) was slowly heated to 168˚C
while the ester was distilled continuously into a trap cooled with dry
ice; yield: 0.92–1.04 g (88–97%).
Diethyl [2-13C]Malonate (3):
To a cooled (–78˚C) solution of hexamethyldisilazane (3.2 g, 20.0
mmol) in anhyd THF (7 mL) under Ar was added dropwise 1.6 M
BuLi (9.0 mL, 14.4 mmol) in hexane. The mixture was allowed to
warm to r.t. during 30 min before it was cooled again to –78°C. A so-
lution of ethyl [2-13C]acetate (640 mg, 7.18 mmol) in THF (1 mL)
was added dropwise and the mixture was stirred for 30 min at –78°C.
Then ethyl chloroformate (797 mg, 7.35 mmol) was added dropwise
and the stirring was continued at –78°C for 3 h. The mixture was
warmed to r.t. and after addition of 6 N HCl (1.6 mL) in H2O (9.5 mL)
extracted with t-BuOMe (3 × 15 mL). The combined organic layers
were washed with 5% aq NaHCO3 solution (3 x 5 mL) and brine
(5 mL), dried (Na2SO4) and evaporated. Column chromatography (pe-
troleum ether/EtOAc, 3:2) afforded 3 (1.04 g, 90%) as a colorless oil.
Reagents and conditions: a) MeI/K2CO3/acetone, reflux for 20 h; b)
LiAlH4/THF, 0°C; c) PBr3/pyridine/Et2O; d) sodium diethyl phthal-
imidomalonate/DMF, 100 °C for 15 h; e) 48% HBr, reflux for 5 h
Scheme 3
In order to obtain ring-labelled 1,2,4-trihydroxy[4-
13C]benzene (19) we developed a reaction sequence start-
ing from 3,4-dihydroxy[1-13C]benzoic acid (9) (Scheme
4). Treatment of this compound with methyl iodide and
K2CO3 in acetone afforded methyl 3,4-dimethoxy-
[1-13C]benzoate (15) in 88% yield. Reduction of ester 15
to the alcohol 16 with LiAlH4 in THF followed by oxida-
tion with pyridinium chlorochromate in CH2Cl2 led to
3,4-dimethoxy[1-13C]benzaldehyde (17). Acid-catalyzed
oxidation of aldehyde 17 with hydrogen peroxide in meth-
anol afforded 3,4-dimethoxy[1-13C]phenol (18) in 71%
yield.16 After deprotection of 18 with boron tribromide
1,2,4-trihydroxy[4-13C]benzene (19) was obtained in 76%
yield. Starting from 3,4-dihydroxy[1-13C]benzoic acid (9)
the synthesis of 19 was accomplished in five steps in 42%
overall yield.
Ethyl 4-Hydroxy[1-13C]benzoate (4):
4H-Pyran-4-one (2.00 g, 20.8 mmol) and diethyl [2-13C]malonate (3;
2.08 g, 12.9 mmol) were heated at reflux in t-BuOH (35 mL). A solu-
tion of KOBu-t (1.90 g, 16.9 mmol) in t-BuOH (85 mL) was added
dropwise over a 3 h period which resulted in a reddish-brown colored
solution. Then 1 N HCl (27 mL) was added and the mixture refluxed
for another 30 min. The solvent was removed in vacuo, H2O (100 mL)
added and the mixture extracted with t-BuOMe (3 × 50 mL). The or-
ganic phases were washed with H2O (2 × 30 mL) and brine (30 mL),
dried (Na2SO4) and evaporated in vacuo. Column chromatography
(silica gel, petroleum ether/EtOAc, 4:1) yielded 4 (1.84 g, 85%) as a
colorless solid; mp 112–114°C.

SYNTHESIS
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1H NMR (400 MHz, CDCl3): δ = 1.38 (t, J = 7.1 Hz, 3 H, CH3), 4.36
(q, J = 7.1 Hz, 2 H, OCH2), 6.91 (dd, J = 9.0, 7.5 Hz, 2 H, 3-H, 5-H),
7.00 (br s, 1 H, OH), 7.96 (dd, J = 9.0, 0.7 Hz, 2 H, 2-H, 6-H).
13C NMR (100.6 MHz, CDCl3): δ = 14.31 (CH3), 61.09 (OCH2),
115.32 (d, J = 0.5 Hz, C-3, C-5), 122.39 (C-1), 131.94 (d, J = 59.2 Hz,
C-2, C-6), 160.53 (d, J = 8.8 Hz, C-4), 167.30 (d, J = 77.1 Hz, C=O).
1H NMR (300 MHz, CD3COCD3): δ = 7.09 (dd, 1 H, J = 8.5, 8.5 Hz,
H-5), 7.88 (dd, 1 H, J = 8.5, 2.1 Hz, H-6), 8.15 (dd, 1 H, J = 2.1, 1.7
Hz, H-2).
13C NMR (100.6 MHz, CD3COCD3): δ = 110.09 (C-3), 117.04 (C-5),
124.36 (C-1), 131.55 (d, J = 59 Hz, C-6), 135.69 (d, J = 59 Hz, C-2),
158.99 (d, J = 7.0 Hz, C-4), 166.25 (d, J = 75 Hz, C=O).
MS: m/z (%) = 219 (96) [M+, 12C613CH581BrO3], 216.9466 (100) [M+,
12C613CH579BrO3], 202 (79), 173 (14), 93 (14), 64 (29), 63 (13), 59
(12).
4-Hydroxy[1-13C]benzoic Acid (5):
Ester 4 (1.84 g, 11.0 mmol) was stirred in 2 N NaOH (16.5 mL) over-
night at r.t. The mixture was cooled in an ice bath, acidified with 2 N
HCl and extracted with t-BuOMe (3 × 10 mL). The combined organic
layers were washed with 1 N HCl (2 × 5 mL) and dried (MgSO4).
Evaporation of the solvent yielded 5 (1.51 g, 99%) as a colorless sol-
id; mp 212–213°C.
HRMS (EI): m/z Calcd for 12C613CH581BrO3: 218.9435; Found:
218.9437.
3,4-Dihydroxy[1-13C]benzoic Acid (9):
Acid 8 (117 mg, 0.54 mmol), CuSO4•5 H2O (112 mg, 0.44 mmol) and
activated Cu17 (613 mg, 9.6 mmol) were refluxed for 15 h under Ar
in 4 N KOH (5.8 mL). After cooling to r.t. the mixture was filtered
and the filtrate acidified with 2 N HCl (ice-cooling). The aqueous so-
lution was then continuously extracted with Et2O overnight. The sol-
vent was removed and the residue chromatographed on Sephadex
LH-20 with MeOH to yield 9 (73 mg, 87%) as a brown powder; mp
200–202°C (dec.).
1H NMR (400 MHz, CD3OD): δ = 4.94 (br s, 1 H, OH), 6.87 (dd, 2
H, J = 8.7, 7.6 Hz, 3-H, 5-H), 7.92 (d, 2 H, J = 8.7 Hz, 2-H, 6-H).
13C NMR (100.6 MHz, CD3OD): δ = 116.32 (d, J = 1.5 Hz, C-3, C-
5), 123.01 (C-1), 133.28 (d, J = 58.8 Hz, C-2, C-6), 163.63 (d, J = 8.4
Hz, C-4), 170.37 (d, J = 74.3 Hz, C=O).
HRMS (EI): m/z Calcd for 12C613CH6O3: 139.0351; Found: 139.0351.
Bromination of Ethyl 4-Hydroxy[1-13C]benzoate (4):
1H NMR (300 MHz, acetone-d6): δ = 6.90 (dd, 1 H, J = 8.2, 8.2 Hz,
H-5), 7.46 (dd, 1 H, J = 8.2, 1.8 Hz, H-6), 7.52 (dd, 1 H, J = 1.8, 1.8
Hz, H-2).
To a cooled (0°C) solution of 4 (1.34 g, 8.02 mmol) in CHCl3 (20 mL)
was added dropwise over a 6 h period a solution of Br2 (0.41 mL,
8.02 mmol) in CHCl3 (4 mL). The mixture was stirred at 0°C for 10
h, then for an additional 15 h at r.t. Finally, the reaction mixture was
treated with 1 N NaHSO3 solution (3 × 10 mL) and extracted with
CHCl3 (2 × 30 mL). The organic layer was washed with H2O (2 ×
10 mL) and brine (10 mL), dried (MgSO4) and concentrated. Flash
chromatography (silica gel, CHCl3/acetone, 25:1) afforded the mono-
brominated ester 6 (1.76 g, 89%); mp 101–102°C, and the dibromi-
nated ester 7 (78 mg, 3%); mp 89–91°C, as colorless solids.
13C NMR (150 MHz, acetone-d6): δ = 115.66 (C-5), 117.37 (d, J =
61 Hz, C-2), 122.91 (C-1), 123.90 (d, J = 69.2 Hz, C-6), 145.47 (C-
3), 150.72 (d, J = 7.0 Hz, C-4), 167.93 (d, J = 74 Hz, C=O).
MS: m/z (%) = 155 (100) [M+], 149 (19), 139 (13), 138 (97), 110 (19),
97 (12), 83 (13), 81 (14), 71 (14), 69 (24), 57 (19), 55 (24).
HRMS (EI): m/z Calcd for 12C613CH6O4: 155.0292; Found: 155.0296.
Ethyl 4-Methoxy[1-13C]benzoate (10):
Ethyl 3-Bromo-4-hydroxy[1-13C]benzoate (6):
A mixture of 4 (920 mg, 5.5 mmol), dried K2CO3 (3.8 g, 27.5 mmol)
and MeI (0.45 mL, 7.15 mmol) in anhyd acetone (35 mL) was heated
at reflux under Ar for 20 h. After the mixture was cooled to r.t., H2O
was added and the solution concentrated under vacuum. The aqueous
residue was extracted with Et2O (3 × 50 mL) and the combined or-
ganic layers were dried (MgSO4) and evaporated. The residue was pu-
rified by flash chromatography (silica gel, petroleum ether/EtOAc,
4:1) to afford 10 (970 mg, 97%) as a white solid; mp 185 °C.
1H NMR (300 MHz, CDCl3): δ = 1.36 (t, 3 H, J = 7.1 Hz, CH3), 3.85
(s, 3 H, OCH3), 4.34 (q, 2 H, J = 7.1 Hz, OCH2), 6.91 (dd, 2 H, J =
11.6, 11.6 Hz, H-3, H-5), 8.00 (dd, 2 H, J = 11.6, 2.7 Hz, H-2, H-6).
13C NMR (75.5 MHz, CDCl3): δ = 14.40 (CH3), 55.39 (OCH3), 60.62
(OCH2), 113.57 (C-3, C-5), 122.99 (C-1), 131.55 (d, J = 58.9 Hz, C-
2, C-6), 163.30 (d, J = 8.2 Hz, C-4), 166.38 (d, J = 76.4 Hz, C=O).
MS: m/z (%) = 182 (3) [M++1], 181 (27) [M+], 180 (1), 167 (2), 166
(2), 153 (19), 149 (13), 137 (9), 136 (100), 108 (6), 93 (6), 78 (8), 65
(3), 64 (2).
1H NMR (300 MHz, CDCl3): δ = 1.38 (t, 3 H, J = 7.1 Hz, CH3), 4.35
(q, 2 H, J = 7.1 Hz, OCH2), 6.10 (br s, 1 H, OH), 7.05 (dd, 1 H, J =
8.6, 8.6 Hz, H-5), 7.92 (ddd, 1 H, J = 8.6, 2.1, 0.6 Hz, H-6), 8.19 (dd,
1 H, J = 2.1, 2.1 Hz, H-2).
13C NMR (75.5 MHz, CDCl3): δ = 14.33 (CH3), 61.16 (OCH2),
110.04 (C-3), 115.75 (d, J = 1.6 Hz, C-5), 124.36 (C-1), 130.99 (d, J
= 59.0 Hz, C-6), 133.92 (d, J = 60 Hz, C-2), 156.17 (d, J = 7.4 Hz, C-
4), 165.20 (d, J = 77 Hz, C=O).
MS: m/z (%) = 247 (37) [M+, 12C813CH981BrO3], 245 (27) [M+,
12C813CH979BrO3], 219 (24), 217 (29), 202 (90), 200 (100), 124 (11),
97 (19), 93 (14), 64 (12).
HRMS (EI): m/z Calcd for 12C813CH981BrO3: 246.9745; Found:
246.9725; m/z Calcd for 12C813CH979BrO3: 244.9766; Found: 244.9789.
Ethyl 3,5-Dibromo-4-hydroxy[1-13C]benzoate (7):
1H NMR (300 MHz, CDCl3): δ = 1.39 (t, 3 H, J = 7.1 Hz, CH3), 4.36
(q, 2 H, J = 7.1 Hz, OCH2), 6.37 (s, 1 H, OH), 8.14 (d, 2 H, J = 1.7 Hz,
2-H, 6-H).
HRMS (EI): m/z Calcd for 12C913CH12O3: 181.0820; Found:
181.0816.
13C NMR (75.5 MHz, CDCl3): δ = 14.30 (CH3), 61.55 (OCH2),
109.67 (C-3, C-5), 125.07 (C-1), 133.59 (d, J = 60 Hz, C-2, C-6),
153.12 (d, J = 6.3 Hz, C-4), 164.00 (d, J = 77 Hz, C=O).
MS: m/z (%) = 327 (17) [M+, 12C813CH881Br2O3], 325 (35) [M+,
12C813CH881Br79BrO3], 323 (17) [M+, 12C813CH879Br2O3], 299 (28),
297 (57), 295 (28), 282 (48), 281 (12), 280 (100), 278 (49), 246 (13),
244 (13), 173 (12), 171 (12), 82 (12), 80 (12), 64 (11), 63 (15).
4-Methoxy[1-13C]benzyl Alcohol (11):
To a solution of 10 (660 mg, 3.6 mmol) in anhyd THF (10 mL) at 0°C
was added dropwise a solution of 1 M LiAlH4 (3.6 mL, 3.6 mmol) un-
der Ar. After stirring overnight, H2O was added (ice-cooling) and the
mixture was acidified with 2 N HCl (10 mL) and extracted with
EtOAc (4 × 30 mL). The combined organic layers were washed with
brine, dried (MgSO4) and evaporated to give 11 (520 mg, 100%) as a
colorless oil.
3-Bromo-4-hydroxy[1-13C]benzoic Acid (8):
1H NMR (300 MHz, CDCl3): δ = 3.81 (s, 3 H, OCH3), 4.61 (d, 2 H, J
= 3.7 Hz, CH2O), 6.89 (dd, 2 H, J = 11.4, 3.1 Hz, H-3, H-5), 7.29 (d,
2 H, J = 11.4 Hz, H-2, H-6).
Ester 6 (458 mg, 1.86 mmol) was stirred overnight at r.t. with 2 N
NaOH (2.5 mL). After cooling to 0°C and acidification with 2 N HCl
the aqueous phase was extracted with t-BuOMe (3 x 10 mL). The
combined organic layers were washed with brine (5 mL) and dried
(Na2SO4). Evaporation of the solvent yielded 8 (457 mg, 100%) as a
white solid; mp 165–166°C.
13C NMR (75.5 MHz, CDCl3): δ = 55.71 (OCH3), 65.45 (d, J = 47.8
Hz, CH2O), 114.38 (d, J = 2.3 Hz, C-3, C-5), 129.05 (d, J = 57.7 Hz,
C-2, C-6), 133.54 (C-1), 159.63 (d, J = 8.2 Hz, C-4).

SYNTHESIS
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1050
MS: m/z (%) = 139 (100) [M+], 138 (50), 136 (30), 124 (5), 123 (28),
122 (90), 110 (49), 108 (21), 107 (4), 106 (8), 95 (15), 93 (9), 92 (7),
80 (10), 79 (14), 78 (36), 67 (5), 66 (9), 65 (7), 52 (7).
HRMS (EI): m/z Calcd for 12C713CH10O2: 139.0714; Found:
139.0670.
chromatography (silica gel; petroleum ether/EtOAc, 3:1) to yield 15
(820 mg, 88%) as a light-brown powder; mp 58–60°C.
1H NMR (300 MHz, CDCl3): δ = 3.90 (s, 3 H), 3.94 (s, 6H), 6.87 (dd,
1 H, J = 8.5, 8.5 Hz, H-6), 7.54 (br s, 1 H, H-2), 7.68 (d, 1 H, J = 8.5
Hz, H-5).
13C NMR (150 MHz, CDCl3): δ = 51.97 (CH3), 56.00 (OCH3), 110.28
(C-2), 112.02 (d, J = 60.7 Hz, C-5), 122.72 (C-1), 123.49 (d, J = 29.1
Hz, C-6), 148.54 (C-3), 152.89 (d, J = 7.0 Hz, C-4), 166.78 (d, J =
75.0 Hz, C=O).
4-Methoxy[1-13C]benzyl Bromide (12):
To a cold (0°C) solution of 11 (0.71 g, 5.1 mmol) and pyridine
(0.4 mL, 5.1 mmol) in anhyd Et2O (10 mL) was added dropwise a so-
lution of PBr3 (0.19 mL, 2.0 mmol) in Et2O (10 mL). The mixture was
stirred for 2 h at r.t. and then quenched with ice-water (10 mL). The
organic layer was separated, washed with ice-cold 1 N HCl (5 mL)
and dried (MgSO4). Evaporation yielded the crude bromide 12
(820 mg, 80%) which was directly used for the next step.
MS: m/z (%) = 198 (7) [M++1], 197 (100) [M+], 182 (9), 167 (9), 166
(85), 150 (5), 138 (11), 126 (6), 123 (9), 122 (10), 120 (6), 108 (6), 95
(8), 94 (4), 93 (4), 80 (12), 78 (9), 77 (4), 59 (7), 52 (7).
HRMS (EI): m/z Calcd for 12C913CH12O4: 197.0781; Found:
197.0775.
Diethyl 4-Methoxy[1-13C]benzylphthalimidomalonate (13):
A solution of 12 (0.43 g, 2.13 mmol) and sodium diethyl phthalimi-
domalonate (0.7 g, 2.13 mmol) in anhyd DMF (5 mL) was heated to
100°C for 15 h. The mixture was then cooled to 20°C, H2O (10 mL)
and Et2O (20 mL) were added and the phases separated. The aqueous
phase was extracted with Et2O (2 x 20 mL) and the combined organic
phases were washed with H2O and dried (Na2SO4). The oily residue
was purified by column chromatography (silica gel, petroleum ether/
EtOAc, 3:2) to yield 13 (890 mg, 98%) as a yellow oil.
3,4-Dimethoxy[1-13C]benzyl Alcohol (16):
To a cooled (0°C) solution of 15 (410 mg, 2.16 mmol) in anhyd THF
(10 mL) was added dropwise under Ar a 1 M solution of LiAlH4 in
THF (2.7 mL). After stirring for 5 h at r.t., H2O was slowly added
(ice-cooling) and the mixture acidified with 2 N HCl (10 mL). The
aqueous layer was extracted with EtOAc (3 × 50 mL) and the com-
bined organic phases were washed with brine, dried (MgSO4) and
evaporated in vacuo. Alcohol 16 (350 mg, 99%) was obtained as a
light yellow oil.
1H NMR (300 MHz, CDCl3): δ = 1.30 (t, 6H, J = 7.2 Hz, CH3), 3.66
(s, 3 H, OCH3), 3.72 (d, 2 H, J = 5.7 Hz, CH2), 4.29 (q, 4H, J = 7.2 Hz,
OCH2), 6.58 (dd, 2 H, J = 8.7, 7.7 Hz), 7.15 (d, 2 H, J = 8.7 Hz), 7.70
(m, 4 H).
1H NMR (300 MHz, CDCl3): δ = 3.87 (s, 3 H, OCH3), 3.88 (s, 3 H,
OCH3), 4.61 (d, 2 H, J = 3.5 Hz, CH2O), 6.81-6.92 (m, 3 H, H-2, H-
5, H-6).
13C NMR (75.5 MHz, CDCl3): δ = 56.17 (OCH3), 56.29 (OCH3),
65.38 (d, J = 47.8 Hz, CH2O), 110.88 (d, J = 58.3 Hz, C-2), 111.46
(C-5), 119.73 (d, J = 58.9 Hz, C-6), 134.08 (C-1), 148.83 (d, J = 6.4
Hz, C-4), 149.40 (C-3).
13C NMR (75.5 MHz, CDCl3): δ = 14.30, 37.30 (d, J = 75.5 Hz, CH2),
55.44, 63.19, 68.97, 113.67, 123.71, 127.20, 131.80, 132.18 (d, J =
58.9 Hz), 134.50, 158.90, 166.37, 167.15.
MS: m/z (%) = 426 (12), 280 (16), 279 (100), 233 (4), 232 (4), 207
(3), 205 (4), 187 (7), 161 (5), 160 (6), 133 (4), 123 (8), 122 (85), 104
MS: m/z (%) = 170 (8) [M++1], 169 (100) [M+], 168 (9), 152 (23), 140
(26), 138 (16), 122 (6), 110 (9), 108 (6), 98 (7), 94 (6), 78 (5), 66 (6),
52 (3).
(8), 76 (4).
HRMS (EI): m/z Calcd for
12
C
13CH23NO7: 426.1508; Found:
HRMS (EI): m/z Calcd for 12C813CH12O3: 169.0826; Found:
169.0823.
22
426.1547.
D,L-[1′-13C]Tyrosine (14):
3,4-Dimethoxy[1-13C]benzaldehyde (17):
A mixture of 13 (1.5 g, 3.5 mmol) and aq HBr (48%, 20 mL) was
heated at reflux for 5 h. The solvent was then evaporated under re-
duced pressure, the residue suspended in H2O (15 mL) and the phthal-
ic acid formed removed by filtration. The filtrate was extracted with
Et2O, filtered through activated carbon and adjusted to pH 6 with 2 N
NH4OH. The amino acid was filtered off, washed with H2O and dried.
An additional amount of tyrosine was obtained by concentration of
the mother liquor; total yield: 350 mg (55%).
A solution of alcohol 16 (330 mg, 1.95 mmol) in anhyd CH2Cl2
(10 mL) was stirred with pyridinium chlorochromate (840 mg, 3.9
mmol) and molecular sieves 4 Å (1 g) at 20°C for 1 h. After Et2O
(20 mL) was added, the mixture was filtered through a short column
with silica gel and Celite. The solvent was removed in vacuo and the
residue purified by flash column chromatography (silica gel, petro-
leum ether/EtOAc, 3:1); yield: 290 mg (89%) of 17 as a light-pink
solid; mp 41–44°C.
1H NMR (300 MHz, CD3OD/CF3CO2H): δ = 2.94–3.03 (m, 2 H),
4.06 (br s., 1 H), 6.70 (dd, 2 J = 7.8 Hz, H-3′, H-5′), 7.02 (d, 2 H, J =
7.8 Hz, H-2′, H-6′).
1H NMR (300 MHz, CDCl3): δ = 3.95 (s, 3 H, OCH3), 3.98 (s, 3 H,
OCH3), 6.99 (dd, 1 H, J = 8.1, 7.9 Hz, H-5), 7.42 (dd, 1 H, J = 1.8,
1.7 Hz, H-2), 7.47 (dd, 1 H, J = 8.1, 1.8 Hz, H-6), 9.86 (d, 1 H, J =
24.2 Hz, CHO).
13C NMR (75.5 MHz, CD3OD/CF3CO2H) δ = 36.82 (d, J = 46.6 Hz,
C-3), 55.74 (C-2), 117.26 (C-1′), 131.94 (d, J = 57.5 Hz, C-2′, C-6′),
158.51 (C-4), 171.81 (C=O).
13C NMR (75.5 MHz, CDCl3): δ = 56.00 (OCH3), 108.98 (d, J = 59.5
Hz, C-2), 110.43 (C-5), 126.84 (d, J = 59.3 Hz, C-6), 130.16 (C-1),
149.64 (C-3), 154.50 (d, J = 7.1 Hz, C-4), 190.87 (d, J = 55.4 Hz,
CHO).
MS: m/z (%) = 183 (3) [M++1], 182 (7) [M+], 180 (4), 137 (5), 109
(15), 108 (100), 107 (2), 92 (5), 80 (2), 79 (2), 78 (8), 74 (3), 68 (1),
53 (1), 52 (1).
MS: m/z (%) = 168 (9) [M++1], 167 (100) [M+], 166 (47), 152 (29),
149 (4), 138 (5), 136 (9), 135 (68), 123 (4), 122 (3), 120 (3), 108 (4),
107 (7), 106 (4), 105 (2), 96 (21), 92 (5), 80 (7), 78 (13), 77 (10), 68
(3), 66 (5), 64 (6), 52 (6).
HRMS (EI): m/z Calcd for 12C813CH11NO3: 182.0772; Found:
182.0772.
Methyl 3,4-Dimethoxy[1-13C]benzoate (15):
HRMS (EI): m/z Calcd for 12C813CH10O3: 167.0678; Found:
167.0671.
To a solution of 9 (730 mg, 4.7 mmol) in acetone (100 mL) under Ar
were added anhyd K2CO3 (9.7 g, 70.5 mmol) and MeI (2.5 mL,
40.2 mmol) and the mixture was heated at reflux for 25 h. After the
mixture was cooled to r.t., H2O (50 mL) was added and the organic
solvent evaporated under reduced pressure. The aqueous phase was
then extracted with Et2O (3 × 50 mL) and the combined organic phas-
es were washed with brine, dried (MgSO4) and concentrated under re-
duced pressure. The oily residue was purified by column
3,4-Dimethoxy[1-13C]phenol (18):
A solution of 17 (280 mg, 1.68 mmol) in MeOH (5 mL) was stirred
under an Ar atmosphere with 30% H2O2 (0.32 mL) and concd H2SO4
(1 drop) for 6 h at r.t. After evaporation of the organic solvent the res-
idue was suspended in H2O and extracted with Et2O (3 × 20 mL). The
organic phases were washed with brine, dried (MgSO4) and the sol-

SYNTHESIS
July1998
1051
vent was evaporated under reduced pressure. The red oily residue was
purified by column chromatography (silica gel, petroleum ether/
EtOAc, 3:2). 18 (185 mg, 71%) was obtained as a light-brown solid;
mp 66°C.
(1) Gibson, F. Biochem. Soc. Trans. 1973, 1, 317.
Olson, R.E.; Rudney, H. Vitam. Horm. 1983, 40, 1.
Clarke, C.F.; Williams, W.; Teruya, J.H. J. Biol. Chem. 1991,
266, 16636.
1H NMR (300 MHz, CDCl3): δ = 3.73 (s, 3 H, OCH3), 3.79 (s, 3 H,
OCH3), 6.37 (ddd, 1 H, J = 8.6, 8.6, 2.8 Hz, H-6), 6.47 (dd, 1 H, J =
7.7, 2.8 Hz, H-2), 6.71 (dd, 1 H, J = 10.8, 8.6 Hz, H-5).
13C NMR (75.5 MHz, CDCl3): δ = 56.10 (OCH3), 57.06 (OCH3),
101.20 (d, J = 69.1 Hz, C-2), 106.57 (d, J = 67.7 Hz, C-6), 113.22 (C-
5), 143.18 (C-4), 150.17 (C-3), 150.92 (C-1).
Marbois, B.N.; Clarke, C.F. J. Biol. Chem. 1996, 271, 2995.
(2) Review: Inouye, H.; Leistner, E. In The Chemistry of Quinonoid
Compounds, Patai, S.;
Rappoport, Z., Eds.; Wiley: New York, 1988; Vol. 2, p 1294.
(3) Zenk, M.H. Z. Naturforsch. (B) 1964, 19, 856.
(4) Schmid, H.V.; Zenk, M.H. Tetrahedron Lett. 1971, 4151.
Inouye, H.; Ueda, S.; Inoue, K.; Matsumura, H. Phytochemistry
1979, 18, 1301.
(5) Heide, L.; Tabata, M. Phytochemistry 1987, 26, 1651.
Boehm, R.; Li, S.; Melzer, M.; Heide, L. Phytochemistry 1997,
44, 419.
(6) Hanefeld, U.; Floss, H.G.; Laatsch, H. J. Org. Chem. 1994, 59,
3604.
(7) Gill, M.; Steglich; W. Prog. Chem. Org. Nat. Prod. 1987, 51,
98.
(8) Baldwin, J.E.; Bansal, H.S.; Chondrogianni, J.; Gallagher, P.T.;
Taha, A.A.; Taylor, A.; Thaller; V. J. Chem. Res. (S) 1984, 176;
J. Chem. Res. (M) 1984, 1815.
(9) Viswanatha, V.; Hruby, V.J. J. Org. Chem. 1979, 44, 2892.
(10) Musso, H.; Döpp, D. Chem. Ber. 1964, 97, 1147.
Musso, H.; Döpp, D.; Kuhles, J. Chem. Ber. 1965, 98, 3937.
(11) Müller, R.; Wagener; A.; Schmidt, K.; Leistner; E. Appl. Micro-
biol. Biotechnol. 1995, 43, 985.
(12) Mueller, M.E.; Leete, E. J. Org. Chem. 1981, 46, 3151.
(13) Woodward, R.B., private communication to Bergmann, E.D.,
quoted in Org. React. 1959, 10, 219.
MS: m/z (%) = 156 (6) [M++1], 155 (100) [M+], 141 (5), 140 (60), 112
(22), 110 (3), 97 (4), 94 (9), 82 (3), 70 (6), 69 (2), 56 (3), 53 (1).
HRMS (EI): m/z Calcd for 12C713CH10O3: 155.0704; Found:
155.0684.
1,2,4-Trihydroxy-[4-13C]benzene (19):
To a solution of BBr3 (7.6 mL, 7.6 mmol) in CH2Cl2 (10 mL) was
added dropwise a solution of 18 (390 mg, 2.5 mmol) in CH2Cl2 at
–78°C. After stirring for 1.5 h at –78°C and 1.5 h at r.t., H2O (5 mL)
was added and the mixture acidified with 2 N HCl (20 mL). The phas-
es were separated and the aqueous layer was extracted with EtOAc (4
× 30 mL). The combined organic phases were washed with brine,
dried (MgSO4) and the solvent was removed under reduced pressure.
The resulting oil was purified by column chromatography (silica gel,
petroleum ether/EtOAc, 1:1) and yielded 19 (240 mg, 76%) as a grey-
ish solid; mp 139°C (sublimation).
1H NMR (300 MHz, CD3COCD3): δ = 6.16 (m, 1 H, H-3), 6.37 (m, 1
H, H-6), 6.50 (dd, 1 H, J = 8.5, 8.5 Hz, H-5), 7.18 (br s, 1 H, OH),
7.49 (br s, 2 H, OH).
13C NMR (75.5 MHz, CD3COCD3): δ = 104.04 (d, J = 68.4 Hz, C-3),
106.54 (d, J = 66.5 Hz, C-5), 116.39 (C-6), 138.70 (C-1), 146.55 (C-
2), 151.78 (C-4).
(14) Lowenthal, J.; Pepper, J.M. J. Am. Chem. Soc. 1950, 72, 3292.
(15) Prof. M.H. Zenk kindly informed us about an efficient synthesis
of optically pure L-[1'-13C]tyrosine from [4-13C]phenol accor-
ding to Reference 18. The [4-13C]phenol was obtained by decar-
boxylation of 5.
We are grateful for the financial support of this work by the Deutsche
Forschungsgemeinschaft (SFB 369) and the Fonds der Chemischen
Industrie.
(16) Matsumoto, M.; Kobayashi, H.; Hotta, Y. J. Org. Chem. 1984,
49, 4740.
(17) Brewster, R.Q.; Groening, T. Org. Synth. Coll. Vol. II, 1943,
445.
(18) Winkel, C.; Aarts, M.W.MM.; van der Heide, F.R.; Buitenhuis,
E. G.; Lugtenburg, J. Recl. Trav. Chim. Pays-Bas 1989, 108,
139.