A new and expedient total synthesis of ochratoxin A and d 5-ochratoxin A

Article abstract of DOI:10.1055/s-0028-1088076

A new total synthesis of the mycotoxin ochratoxin A (OTA) is presented, in which it is prepared in 9% overall yield from commercially available substrates. The key step consists of the condensation reaction between protected L-phenylalanine and 5-chloro-8-hydroxy-3-methyl-1-oxoisochromane-7-carboxylic acid (ochratoxin α, OTα). The same strategy could be successfully applied to L-d₅-phenylalanine, leading to the first total synthesis of d₅-OTA, a molecular tracer for the detection and analytical quantification of the natural mycotoxin in food samples by means of stable isotope dilution assay (SIDA). Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-0028-1088076

PAPER
1815
A New and Expedient Total Synthesis of Ochratoxin A and d5-Ochratoxin A
Total
Synthesis
o
a
f
O
chratoxirn A and 5
t
d
-Oc
h
o
ratoxinA lo Gabriele,*a Mohamed Attya,b Alessia Fazio,a Leonardo Di Donna,b Pierluigi Plastina,a Giovanni Sindonab
Dipartimento di Scienze Farmaceutichea and Dipartimento di Chimicab, Università della Calabria,
87036 Arcavacata di Rende (Cosenza), Italy
Fax +39(984)492044; E-mail: b.gabriele@unical.it
Received 5 February 2009; revised 9 February 2009
titative determination of microcomponents in food.6 Thus,
OTA can be detected and quantified in food samples by
use of SIDA if, for example, a deuterated derivative, such
as d5-OTA (2a; Figure 1), is used as an internal standard.5a
Abstract: A new total synthesis of the mycotoxin ochratoxin A
(OTA) is presented, in which it is prepared in 9% overall yield from
commercially available substrates. The key step consists of the con-
densation reaction between protected L-phenylalanine and 5-
chloro-8-hydroxy-3-methyl-1-oxoisochromane-7-carboxylic acid
(ochratoxin a, OTa). The same strategy could be successfully ap-
plied to L-d5-phenylalanine, leading to the first total synthesis of d5-
OTA, a molecular tracer for the detection and analytical quantifica-
tion of the natural mycotoxin in food samples by means of stable
isotope dilution assay (SIDA).
In this paper, we report a new and efficient method for the
total synthesis of OTA (1a) and d5-OTA (2a), together
with their (3S)-diastereomers 1b and 2b, respectively,
starting from but-2-ynal and dimethyl 3-oxopentanedioate
(3) (Schemes 1– 3). The key step of the synthetic strategy
consists of the condensation reaction between protected L-
phenylalanine 7 (Scheme 2) or protected L-d5-phenylala-
nine 9 (Scheme 3) and 5-chloro-8-hydroxy-3-methyl-1-
oxoisochromane-7-carboxylic acid (ochratoxin a, OTa),
which was prepared by a modification of the method orig-
inally proposed by Kraus (Scheme 1).7
Key words: heterocycles, natural products, ochratoxin A, stable
isotope dilution assay, total synthesis
Ochratoxin A (OTA, 1a; Figure 1) is a ubiquitous myco-
toxin produced by some fungi of the Aspergillus and Pen-
icillium species (such as Aspergillus ochraceus and
Penicillium verrucosum), and is found in raw and improp-
erly stored food products.1,2 OTA has been shown to be
nephrotoxic, mutagenic, genotoxic, teratogenic, hepato-
toxic, neurotoxic, and immunotoxic, in both animals and
humans,3 and in 1993 was classified as a possible carcin-
ogen to humans (Group 2B) by the International Agency
for Research on Cancer (IARC).4
OH
O
MeO2C
CO2Me
Me
1) NaH
MeO2C
CO2Me
2) Me
CHO
3
4 (48%)
1) LDA
2) MeCHO
3) H+
OH
O
OH
O
O
Ph
C6D5
O
OH
O
O
OH
O
HO2C
MeO2C
1) SO2Cl2
O
HO2C
N
O
HO2C
N
O
2) LiOH
3) H+
H
H
R2
R1
R2
R1
Me
Me
5 (70%)
Cl
6 (69%)
Cl
Cl
Scheme 1 Synthesis of ochratoxin a (OTa, 6)
1a R1 = H, R2 = Me
1b R1 = Me, R2 = H
2a R1 = H, R2 = Me
2b R1 = Me, R2 = H
Ph
O
Ph
Figure 1 Structures of (–)-ochratoxin A (OTA, 1a), (–)-d5-ochra-
toxin A (d5-OTA, 2a), and their (3S)-diastereomers 1b and 2b, res-
pectively
6
1) t-BuOAc, HClO4
t-BuO
–O2C
NH3
NH2
2) HCl
3) K2CO3
EDC⋅HCl, HOBt
7 (91%)
Ph
O
O
OH
O
OTA contamination occurs in a wide range of foods and
beverages, including cereals, beans, dried vine fruits, cof-
fee, wine, beer, grape juice, pork, poultry, spices, and
chocolate.1,2 The development of sensitive and accurate
methods for the analytical determination of OTA in food
products has therefore become increasingly important.5
Stable isotope dilution assay (SIDA) is currently one of
the most promising methods for the highly sensitive quan-
t-BuO
N
O
TFA
H
1a + 1b (81% from 6)
R2
R1
separation
by PTLC
8a + 8b
Cl
8a R1 = H, R2 = Me; 8b R1 = Me, R2 = H
1a (46%) + 1b (34%)
Scheme 2 Synthesis of (–)-ochratoxin A (OTA, 1a) and its (3S)-dia-
stereomer 1b
SYNTHESIS 2009, No. 11, pp 1815–1820
x
x.
x
x
.
2
0
0
9
Advanced online publication: 27.04.2009
DOI: 10.1055/s-0028-1088076; Art ID: Z01909SS
© Georg Thieme Verlag Stuttgart · New York

1816
B. Gabriele et al.
PAPER
C6D5
t-BuO
ed L-d5-phenylalanine 9, followed by deprotection and
separation of the two resulting diastereomers 2a and 2b by
preparative TLC led to pure 2a and 2b in 47% and 36%
isolated yield, respectively (Scheme 3). To our knowl-
edge, this is the first example of the total synthesis of both
d5-OTA and its (3S)-diastereomer reported in the litera-
ture.13 Both deuterated diastereomers 2a,b were fully
characterized by IR, 1H NMR and 13C NMR spectroscopy,
HRMS, and specific rotation.
C6D5
–O2C
6
1) t-BuOAc, HClO4
NH2
NH3
EDC⋅HCl, HOBt
2) HCl
3) K2CO3
O
9 (90%)
C6D5
t-BuO
O
OH
O
N
O
TFA
H
2a + 2b (82% from 6)
O
R2
R1
separation
by PTLC
10a + 10b
Cl
10a R1 = H, R2 = Me; 10b R1 = Me, R2 = H
In conclusion, we have developed a novel and convenient
total synthesis of ochratoxin A (1a) and its (3S)-diastereo-
mer 1b, with overall yields of ca. 9% and 6%, respective-
ly, over only six steps, starting from commercially
available starting materials. Our strategy has also allowed
the first total synthesis of d5-OTA (2a), which can be used
as an internal standard for the quantitative determination
of the wild-type ochratoxin in foodstuff.
2a (47%) + 2b (36%)
Scheme 3 Synthesis of d5-(–)-ochratoxin A (d5-OTA, 2a) and its
(3S)-diastereomer 2b
Thus, following the method recently disclosed by
Covarrubias-Zúñiga,8 crude but-2-ynal (readily available
by quantitative oxidation of but-2-ynol with MnO2 in
CH2Cl2)9 was reacted with the sodium salt of commercial-
ly available dimethyl 3-oxopentanedioate (3) at –10 °C in
tetrahydrofuran, to give dimethyl 2-hydroxy-4-methyl-
benzene-1,3-dicarboxylate (4) in 48% yield (Scheme 1).
Kraus’s method7 was followed to provide lactone deriva-
Melting points were determined on a Reichert Thermovar apparatus
and are uncorrected. Optical rotations were measured on a Jasco
DIP-1000 12 polarimeter equipped with a sodium lamp (589 nm)
1
and a 10-cm microcell. H NMR (300 MHz) and 13C NMR (75
tive 5: the methyl group of 4 was deprotonated with lithi- MHz) spectra of samples dissolved in CDCl3 or DMSO-d6 were re-
corded on a Bruker DPX Avance 300 spectrometer at 25 °C; TMS
was used as internal standard. IR spectra were recorded on a Perkin-
Elmer Paragon 1000 PC FT-IR spectrometer. GC-MS spectra were
obtained with a Shimadzu QP-2010 GC-MS apparatus (ionization
voltage 70 eV). LC-MS analyses were carried out on a fractionlynx
HPLC system composed of a autosampler/collector, a 600E pump
working in analytical mode, a 486 UV detector (set to 280 nm) and
um diisopropylamide at –78 °C in tetrahydrofuran;
addition of acetaldehyde and acidic workup followed; 5
was obtained in 70% yield. Chlorination of 5 with sulfuryl
chloride in dichloromethane at room temperature, fol-
lowed by hydrolysis of the ester group with lithium hy-
droxide in methanol finally gave OTa (6) in 69% yield.
The overall yield of 6 was therefore 23% over three steps a ZMD mass spectrometer equipped with an ESI source; a 100 × 3.0
mm ONYX-C18 monolithic column was used [flow rate 1.5 mL/
min; run time 20 min; gradient elution with 0.5% HCO2H in H2O
starting from commercially available 3, which is higher
than that previously obtained by Kraus (17% over 4 steps
(solvent A) and MeOH (solvent B); solvent run: linear gradient
starting from acetone and ethyl formate),7 Snieckus and
from 95% A to 5% A in 14 min, linear gradient from 5% A to 95%
coworkers (6% over 5 steps starting from 4-chlorophe-
A in 3 min, isocratic 95% A for 3 min]. The MS conditions were the
following: capillary voltage 3.15 kV, cone voltage 3 V, extractor 2
nol),10 and Donner and Gill [who synthesized (R)-OTa
starting from (R)-propylene oxide over 9 steps with an
V, RF lens 0.2 V, source block and desolvation temperature 120,
250 °C respectively, ion energy 0.5 V, LM resolution 15.0, HM res-
olution 14.5 and multiplier 650 V. The nebulizer gas was set to 650
L/h. ESI-HRMS experiments were performed on a hybrid Q-Star
Pulsar-i QqToF mass spectrometer equipped with an ion-spray ion-
ization source. All samples were acquired at the optimum ion-spray
overall yield of 10%].11
A condensation reaction between OTa (6) and protected
L-phenylalanine 7 was then performed in chloroform as
the solvent at 25 °C for 20 hours in the presence of N-eth-
yl-N¢-(3-dimethylaminopropyl)carbodiimide hydrochlo- voltage of 4.8 kV by direct infusion (5 mL/min) of a soln containing
the appropriate compound dissolved in MeOH–H2O (20 mg/mL).
The N2 gas flow was set at 20 psi and the declustering and the fo-
cusing potentials were kept at 50 and 220 V relative to ground, re-
spectively. Commercially available flavonoids were used as
ride and N-hydroxybenzotriazole (BtOH) as coupling
agents, to give a mixture of protected diastereomers 8a,b
in ca. 86% total yield, based on 6 (Scheme 2). Deprotec-
tion of the crude mixture 8a,b with trifluoroacetic acid in
calibration standard compounds. The accuracy of the measurement
was within 5 ppm. MS2 experiments were performed in the collision
cell q on the isotopically pure (12C) peak of the selected precursor
ions by keeping the first quadrupole analyzer at unit resolution, and
scanning the time-of-flight (ToF) analyzer. The collision energy
was set to 20 eV, for each compound, while the gas pressure of the
collision chamber was regulated at the instrumental parameters
CAD 5, which corresponds to a pressure of the chamber of 6.86 ×
10–3 Torr and a gas thickness of 9.55 × 1015 molecules/cm2. All the
acquisitions were averaged over 30 scans at a TOF resolving power
of 7000. The molecular formula was evaluated by means of Analyst
QS software. All reactions were analyzed by TLC on silica gel 60
F254 and by GLC on a Shimadzu GC-2010 gas chromatograph and
capillary columns with polymethylsilicone with 5% phenylsilicone
as the stationary phase. Column chromatography was performed on
silica gel 60 (Merck, 70–230 mesh). Preparatory TLC separations
dichloromethane at room temperature for six hours led to
a mixture of OTA (1a) and its (3S)-diastereomer 1b in
81% total yield based on starting OTa (6).12 Separation of
the two diastereomers by preparative TLC (PTLC) afford-
ed pure 1a and 1b in 46% and 34% isolated yield, respec-
tively. Both diastereomers were fully characterized by IR,
1H NMR and 13C NMR spectroscopy, HRMS, and specif-
ic rotation.
Our strategy could be successfully applied to the first total
synthesis of d5-OTA (2a) and its (3S)-diastereomer 2b
(Figure 1), by use of L-d5-phenylalanine as deuterated
starting material, with essentially the same overall yields
(Scheme 3). Thus, condensation of OTa (6) with protect-
Synthesis 2009, No. 11, 1815–1820 © Thieme Stuttgart · New York

PAPER
Total Synthesis of Ochratoxin A and d5-Ochratoxin A
1817
were carried out on Merck silica gel plates (20 × 20 cm, 0.25 mm
thickness). MnO2, but-2-ynol, dimethyl 3-oxopentanedioate (3),
NaH (95% purity), 2 M LDA in THF–heptane–ethylbenzene, acet-
aldehyde, SO2Cl2, LiOH, L-phenylalanine, t-BuOAc, HClO4 (70%),
EDC·HCl, BtOH, TFA, and L-d5-phenylalanine (99% D) were com-
mercially available and were used as received.
5-Chloro-8-hydroxy-3-methyl-1-oxoisochromane-7-carboxylic
Acid (Ochratoxin a, OTa, 6)
SO2Cl2 (6.9 g, 51 mmol) was added to a stirring soln of 5 (2.4 g,
10.2 mmol) in anhyd CH2Cl2 (50 mL) under N2 at 25 °C. After ad-
ditional stirring of the mixture at 25 °C for 24 h, the resulting yellow
soln was evaporated in vacuo to give methyl 5-chloro-8-hydroxy-3-
methyl-1-oxoisochromane-7-carboxylate as a yellow solid, which
was suspended in MeOH (50 mL). LiOH (4.46 g, 186 mmol) was
added, and the resulting mixture was allowed to reflux for 5 h. Dur-
ing this time a semisolid product separated from the mixture. After
removal of the solvent under vacuum, H2O (25 mL) and Et2O (25
mL) were added, and the organic layer was separated. The aqueous
layer was acidified to pH 2 with 1 N aq HCl, and then extracted with
Et2O (3 × 30 mL); then the combined organic layers were dried
(Na2SO4). After filtration, the solvent was removed by rotary evap-
oration, and the crude product was crystallized from acetone; this
gave pure ochratoxin a (6).
Dimethyl 2-Hydroxy-4-methylbenzene-1,3-dicarboxylate (4)
But-2-ynol (5.0 g, 71.3 mmol) was added to a suspension of MnO2
(74.4 g, 860 mmol) in anhyd CH2Cl2 (200 mL) at r.t. under N2. After
stirring at r.t. for 12 h, the mixture was filtered, and the solvent was
removed by distillation at atmospheric pressure. The crude but-2-
ynal thus obtained (still containing ca. 0.5 mL CH2Cl2) was used as
such for the next reaction. Dimethyl 3-oxopentanedioate (3; 11.5 g,
66.0 mmol) was added dropwise to a stirred suspension of NaH
(95% purity; 1.9 g, 75.2 mmol) in anhyd THF (120 mL) at –10 °C
under N2, followed by crude but-2-ynal (obtained as described
above). After additional stirring at –10 °C for 6 h, the mixture was
poured into dilute aq HCl (250 mL). The organic layer was separat-
ed, and the aqueous layer was extracted with EtOAc (3 × 50 mL).
The combined organic phases were washed with brine (250 mL)
and then dried (Na2SO4). After filtration of the mixture and removal
of the solvent by rotary evaporation, the residue was purified by col-
umn chromatography (silica gel, hexane–EtOAc, 7:3); this gave
pure 4.
Yield: 1.8 g (69%); colorless solid; mp 245–246 °C (Lit.7 246 °C).
IR (KBr): 3266 (br m), 1732 (s), 1700 (s), 1680 (s), 1610 (s), 1440
(s), 1220 (m), 1200 (s), 1149 (s), 1100 (m), 821 (m) cm–1.
1H NMR (300 MHz, DMSO-d6): d = 10.03 (br s, 2 H, CO2H, OH),
7.98 (s, 1 H, H-6), 4.87–4.60 (m, 1 H, CHCH3), 3.20 (distorted dd,
J = 17.1, 2.4 Hz, 1 H, CHH), 2.87 (distorted dd, J = 17.1, 11.6 Hz,
1 H, CHH), 1.46 (d, J = 6.1 Hz, 3 H, Me).
Yield: 7.1 g (48%); yellow crystals; mp 43–45 °C (Lit.8 44–46 °C).
13C NMR (75 MHz, DMSO-d6): d = 167.0, 165.5, 160.4, 143.1,
136.0, 120.6, 118.1, 112.3, 74.4, 32.2, 20.1.
ESI-MS: m/z = 279 (100) [M + Na]+, 257 (80) [M + H]+.
IR (KBr): 2955 (m), 1730 (s), 1672 (s), 1330 (s), 1298 (m), 1259
(m), 789 (m), 747 (m) cm–1.
1H NMR (300 MHz, CDCl3): d = 11.16 (s, 1 H, OH), 7.74 (d, J =
8.2 Hz, 1 H, H-6), 6.72 (d, J = 8.2 Hz, 1 H, H-5), 3.94 (s, 3 H,
CO2Me), 3.92 (s, 3 H, CO2Me), 2.33 (s, 3 H, Me at C-4).
13C NMR (75 MHz, CDCl3): d = 170.2, 167.6, 159.0, 144.3, 131.0,
123.3, 121.1, 110.8, 52.3, 52.1, 20.1.
ESI-HRMS: m/z [M + H]+ calcd for C11H10ClO5: 257.0217; found:
257.0199.
MS/MS [M + H]+ (ESI+, 30 eV): m/z = 239.00 (88.2) [M – H2O +
H]+, 220.99 (100.0) [M – 2H2O + H]+, 193.00 (59.7) [M – 2H2O –
CO + H]+, 165.00 (15.3) [M – 2H2O – 2CO + H]+, 137.01 (17.4),
102.04 (10.5).
GC-MS (EI, 70 eV): m/z = 224 [M+] (32), 193 (38), 192 (100), 161
(93), 160 (28), 134 (48), 105 (17), 77 (27).
tert-Butyl L-Phenylalaninate (7)
Concd HClO4 (70%; 1.5 mL, 2.5 g, 17.4 mmol) was slowly added
to a suspension of L-phenylalanine (1.8 g, 10.9 mmol) in t-BuOAc
(27.0 mL, 23.3 g, 200 mmol) under N2 at 0 °C. After stirring of the
mixture at 25 °C for 12 h, H2O (55 mL) followed by 1 N HCl (30
mL) were added. The mixture was basified to pH 9 by the addition
of 10% aq K2CO3 soln, and then extracted with CH2Cl2 (3 × 25 mL).
The combined organic layers were dried (Na2SO4). After filtration,
the solvent was removed by rotary evaporation, and the crude prod-
uct was purified by column chromatography (silica gel, hexane–
EtOAc, 1:1); this gave pure 7.
Methyl 8-Hydroxy-3-methyl-1-oxoisochromane-7-carboxylate
(5)
A soln of 4 (6.5 g, 29.0 mmol) in anhyd THF (30 mL) was added
dropwise (15 min) to a 2 M soln of LDA in THF–heptane–ethylben-
zene (36.3 mL, 72.6 mmol) maintained at –78 °C under N2. After
additional stirring of the mixture at –78 °C for 30 min, acetaldehyde
(5.5 g, 124.9 mmol) was added, and the soln was stirred at –78 °C
for 15 min and then at 0 °C for a further 15 min. After quenching of
the mixture with glacial AcOH (10 mL) at 0 °C, H2O (30 mL) and
Et2O (30 mL) were added, and the organic layer was separated. The
aqueous layer was extracted with Et2O (2 × 30 mL), and the com-
bined organic layer was dried (Na2SO4). After filtration, the solvent
was removed by rotary evaporation, and the crude product was crys-
tallized from acetone; this gave pure 5.
Yield: 2.2 g (91%); colorless oil.
IR (film): 2978 (w), 1729 (s), 1603 (w), 1458 (w), 1368 (m), 1154
(s), 847 (m), 740 (m), 700 (m) cm–1.
1H NMR (300 MHz, CDCl3): d = 7.32–7.17 (m, 5 H, Ph), 3.60 (dis-
torted dd, J = 7.6, 5.7 Hz, 1 H, CHNH2), 3.02 (distorted dd, J = 13.6,
5.7 Hz, 1 H, CHH), 2.83 (distorted dd, J = 13.6, 7.6 Hz, 1 H, CHH),
1.51 (br s, 2 H, NH2), 1.42 (s, 9 H, t-Bu).
13C NMR (75 MHz, CDCl3): d = 174.3, 137.9, 129.5, 128.4, 126.7,
81.0, 56.5, 41.6, 28.1.
GC-MS (EI, 70 eV): m/z = 221 [M+] (absent), 130 (16), 121 (28),
120 (100), 103 (19), 91 (45), 77 (25), 74 (98).
Yield: 4.79 g (70%); yellow solid; mp 108–109 °C (Lit.7 108–
110 °C).
IR (KBr): 3419 (br m), 1724 (s), 1660 (m), 1619 (m), 1431 (w),
1239 (m), 1218 (m), 1143 (w), 808 (w) cm–1.
1H NMR (300 MHz, CDCl3): d = 8.02 (d, J = 7.9 Hz, 1 H, H-6), 6.77
(dt, J = 7.9, 0.7 Hz, 1 H, H-5), 4.80–4.65 (m, 1 H, CHCH3), 3.93 (s,
3 H, CO2Me), 3.03–2.90 (m, 2 H, CH2), 1.53 (d, J = 6.4 Hz, 3 H,
CHCH3). (Note: the OH signal was too broad to be detected.)
13C NMR (75 MHz, CDCl3): d = 168.2, 166.2, 162.5, 145.2, 137.9,
117.6, 117.2, 110.2, 75.5, 52.1, 35.1, 20.6.
tert-Butyl N-{[(3R/3S)-5-Chloro-8-hydroxy-3-methyl-1-oxoiso-
chroman-7-yl]carbonyl}-L-phenylalaninate (8a,b)
GC-MS (EI, 70 eV): m/z = 236 [M+] (59), 205 (52), 204 (18), 192
(28), 160 (100), 104 (24), 91 (6), 77 (30).
EDC·HCl (208 mg, 1.08 mmol) was added to a stirred soln of 7 (222
mg, 1.0 mmol), 6 (269 mg, 1.05 mmol), and BtOH (142 mg, 1.05
mmol) in anhyd CHCl3 (5 mL) under N2 at 0 °C. After additional
stirring of the mixture at 0 °C for 15 min and at 25 °C for 20 h,
Synthesis 2009, No. 11, 1815–1820 © Thieme Stuttgart · New York

1818
B. Gabriele et al.
PAPER
CHCl3 (10 mL) and H2O (10 mL) were added. The organic layer
was separated, washed sequentially with 1 N HCl, H2O, 5%
NaHCO3, and brine (10 mL each), and then dried (Na2SO4). After
filtration of the mixture, the solvent was removed by rotary evapo-
ration; this gave a crude mixture of diastereomers 8a,b, which was
used as such for the next step.
CHHCHCH3), 2.89–2.76 (m, 1 H, CHHCHCH3), 1.58 (d, J = 6.3
Hz, 3 H, Me).
13C NMR (75 MHz, CDCl3): d = 174.8, 169.7, 163.1, 159.2, 141.0,
139.1, 136.0, 129.4, 128.7, 127.3, 123.3, 120.6, 110.2, 75.9, 54.5,
37.6, 32.4, 20.7.
ESI-MS: m/z = 426 (100) [M + Na]+, 404 (87) [M + H]+.
ESI-HRMS: m/z [M + H]+ calcd for C20H19ClNO6: 404.0901;
found: 404.0886.
(–)-N-{[(3R)-5-Chloro-8-hydroxy-3-methyl-1-oxoisochroman-
7-yl]carbonyl}-L-phenylalanine (Ochratoxin A, OTA, 1a) and
(+)-N-{[(3S)-5-Chloro-8-hydroxy-3-methyl-1-oxoisochroman-
7-yl]carbonyl}-L-phenylalanine (1b)
MS/MS [M + H]+ (ESI+, 20 eV): m/z = 404.09 (33.7) [M + H]+,
386.08 (5.9) [M – H2O + H]+, 358.08 (81.4) [M – H2O – CO + H]+,
341.06 (17.9) [M – H2O – CO – NH3 + H]+, 239.01 (100.0) [M –
The crude mixture of diastereomers 8a,b, obtained as described
above, was added to a soln of TFA (12.3 g, 108 mmol) in anhyd
CH2Cl2 (20 mL) at 25 °C under N2. The reaction mixture was stirred
at 25 °C and monitored by TLC. When the reaction was completed
(ca. 6 h), the solvent and excess TFA were removed by rotary evap-
oration. The resulting residual oil was dissolved in CH2Cl2, washed
with H2O (3 × 10 mL), and dried (Na2SO4). After filtration and re-
moval of the solvent by rotary evaporation, a colorless solid was ob-
tained, which was crystallized from benzene to give a mixture of 1a
and 1b.
C9H11NO2]+, 221.00 (8.6) [M
[C8H10N]+.
–
C9H13NO3]+, 120.08 (8.1)
tert-Butyl L-d5-Phenylalaninate (9)
Concd HClO4 (70%) (200 mL, 336 mg, 2.34 mmol) was added slow-
ly to a suspension of L-d5-phenylalanine (99% D; 255 mg, 1.5
mmol) in t-BuOAc (3.6 mL, 3.1 g, 26.7 mmol) under N2 at 0 °C. Af-
ter stirring of the mixture at r.t. for 12 h, H2O (15 mL) followed by
1 N aq HCl (10 mL) were added. The mixture was basified to pH 9
by the addition of 10% K2CO3, and then extracted with CH2Cl2 (3 ×
15 mL). The combined organic layers were dried (Na2SO4). After
filtration, the solvent was removed by rotary evaporation, and the
crude product was purified by column chromatography (silica gel,
hexane–EtOAc, 1:1); this gave pure 9.
Yield: 345 mg (81% from 6); colorless solid; mp 168–171 °C (Lit.9
169–172 °C).
Separation of 1a and 1b
A mixture of the two diastereomers 1a and 1b (100 mg) dissolved
in CHCl3 was then subjected to preparative TLC on plates coated
with silica gel (20 plates, 20 × 20 cm, 0.25 mm thickness, benzene–
acetone–HCO2H, 79:20:1); this gave pure 1a (Rf = 0.47) and pure
1b (Rf = 0.43).
Yield: 307.0 mg (90%); colorless oil.
IR (film): 3381 (br s), 1735 (m), 1621 (s), 1212 (m), 1154 (m) cm–1.
1H NMR (300 MHz, CDCl3): d = 3.61 (distorted dd, J = 7.6, 5.8 Hz,
1 H, CHNH2), 3.04 (distorted dd, J = 13.6, 5.8 Hz, 1 H, CHH), 2.84
(distorted dd, J = 13.6, 7.6 Hz, 1 H, CHH), 1.53 (br s, 2 H, NH2),
1.42 (s, 9 H, t-Bu).
GC-MS (EI, 70 eV): m/z = 227 [M+] (absent), 125 (100), 126 (10),
107 (5), 106 (5), 96 (10), 74 (37).
ESI-HRMS: m/z [M + H]+ calcd for C13H15D5NO2: 227.1803;
found: 227.1795.
1a
Yield: 46 mg (46%); colorless solid; mp 110–112 °C; [a]D25 –31.5
(c 5 mg/mL, CHCl3).
IR (KBr): 3029 (m), 2985 (m), 2928 (m), 1742 (m), 1674 (s), 1614
(m), 1534 (s), 1427 (m), 1391 (w), 1214 (s), 1171 (m), 1139 (m),
809 (w), 758 (w), 702 (w) cm–1.
1H NMR (300 MHz, CDCl3): d = 12.74 (br s, 1 H, OH), 8.50 (br d,
J = 6.8 Hz, 1 H, NH), 8.42 (s, 1 H, H-6), 7.34–7.20 (m, 6 H, Ph,
OH), 5.07–4.98 (m, 1 H, CHNH), 4.81–4.68 (m, 1 H, CHCH3),
3.40–3.15 (m, 3 H, CH2Ph, CHHCHCH3), 2.84 (dd, J = 17.6, 11.7
Hz, 1 H, CHHCHCH3), 1.59 (d, J = 6.3 Hz, 3 H, Me).
13C NMR (75 MHz, CDCl3): d = 174.6, 169.7, 163.3, 159.1, 141.0,
139.0, 135.8, 129.3, 128.7, 127.3, 123.2, 120.2, 110.1, 75.9, 54.4,
37.3, 32.3, 20.7.
ESI-MS: m/z = 426 (68) [M + Na]+, 404 (100) [M + H]+.
ESI-HRMS: m/z [M + H]+ calcd for C20H19ClNO6: 404.0901;
MS/MS [M + H]+ (ESI+, 20 eV): m/z = 171.11 (55.7) [M – t-Bu +
H]+, 125.11 (100.0), 124.10 (16.0).
tert-Butyl N-{[(3R/3S)-5-Chloro-8-hydroxy-3-methyl-1-oxoiso-
chroman-7-yl]carbonyl}-L-d5-phenylalaninate (10a,b)
EDC·HCl (208 mg, 1.08 mmol) was added to a stirred soln of 9 (227
mg, 1.0 mmol), 6 (269 mg, 1.05 mmol), and BtOH (142 mg, 1.05
mmol) in anhyd CHCl3 (5 mL) under N2 at 0 °C. After additional
stirring of the mixture at 0 °C for 15 min and at r.t. for 20 h, CHCl3
(10 mL) and H2O (10 mL) were added. The organic layer was sep-
arated, washed sequentially with 1 N aq HCl, H2O, 5% NaHCO3,
and brine (10 mL each), and then dried (Na2SO4). After filtration of
the mixture, the solvent was removed by rotary evaporation; this
gave a crude mixture of diastereomers 10a,b, which was used as
such for the next step.
found: 404.0891.
MS/MS [M + H]+ (ESI+, 20 eV): m/z = 404.08 (34.8) [M + H]+,
386.07 (6.4) [M – H2O + H]+, 358.08 (79.1) [M – H2O – CO + H]+,
341.05 (17.6) [M – H2O – CO – NH3 + H]+, 239.00 (100.0) [M –
C9H11NO2]+, 220.99 (7.2) [M
[C8H10N]+.
–
C9H13NO3]+, 120.08 (6.2)
(–)-N-{[(3R)-5-Chloro-8-hydroxy-3-methyl-1-oxoisochroman-
7-yl]carbonyl}-L-d5-phenylalanine (d5-Ochratoxin A, d5-OTA,
2a) and (+)-N-{[(3S)-5-Chloro-8-hydroxy-3-methyl-1-oxoiso-
chroman-7-yl]carbonyl}-L-d5-phenylalanine (2b)
The crude mixture of diastereomers 10a,b, obtained as described
above, was added to a soln of TFA (12.3 g, 108 mmol) in anhyd
CH2Cl2 (20 mL) at r.t. under N2. The reaction mixture was stirred at
r.t. and monitored by TLC. When the reaction was completed (ca. 6
h), the solvent and the excess TFA were removed by rotary evapo-
ration. The resulting residual oil was dissolved in CH2Cl2, washed
with H2O (3 × 10 mL), and dried (Na2SO4). After filtration and re-
moval of the solvent by rotary evaporation, a colorless solid was ob-
1b
Yield: 34 mg (34%); colorless solid; mp 182–183 °C; [a]D25 +66.7
(c 3 mg/mL, CHCl3).
IR (KBr): 3022 (w), 2925 (s), 2851 (m), 1741 (s), 1673 (s), 1615 (w)
1541 (m), 1427 (m), 1214 (s), 1380 (m), 1170 (m), 1139 (m), 809
(m), 742 (w), 705 (w) cm–1.
1H NMR (300 MHz, CDCl3): d = 12.71 (br s, 1 H, OH), 8.50 (br d,
J = 7.2 Hz, 1 H, NH), 8.40 (s, 1 H, H-6), 7.34–7.18 (m, 5 H, Ph),
7.08 (br s, 1 H, OH), 5.05 (distorted dd, J = 12.6, 6.8 Hz, 1 H,
CHNH), 4.80–4.66 (m, 1 H, CHCH3), 3.39–3.15 (m, 3 H, CH2Ph,
Synthesis 2009, No. 11, 1815–1820 © Thieme Stuttgart · New York

PAPER
Total Synthesis of Ochratoxin A and d5-Ochratoxin A
1819
tained, which was crystallized from benzene; this gave a mixture of
2a and 2b.
Melillo, 84084 Fisciano (Salerno, Italy)] for specific rotation mea-
surements.
Yield: 350 mg (82% from 6); colorless solid; mp 93–95 °C.
References
Separation of 2a and 2b
A mixture of the two diastereomers 2a and 2b (100 mg) dissolved
in CHCl3 was then subjected to preparative TLC on plates coated
with silica gel (20 plates, 20 × 20 cm, 0.25 mm thickness, benzene–
acetone–HCO2H, 79:20:1); this gave pure 2a (Rf = 0.47) and pure
2b (Rf = 0.43).
(1) (a) Bouras, N.; Mathieu, F.; Coppel, Y.; Strelkov, S. E.;
Lebrihi, A. J. Agric. Food Chem. 2007, 55, 8920.
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120. (f) Sweeny, M. J.; Dobson, A. D. Int. J. Food
Microbiol. 1998, 43, 141. (g) MacDonald, S.; Wilson, P.;
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Addit. Contam. 1998, 15, 550. (m) Ramos, A. J.; Labernia,
N.; Marin, S.; Sanchis, V.; Magan, N. Int. J. Food Microbiol.
1998, 44, 133. (n) Hohler, D. Z. Ernaehrungswiss. 1998, 37,
2. (o) Stegen, G.; Jrissen, U.; Pittet, A.; Saccon, M.; Steiner,
W.; Vincenzi, M.; Winkler, M.; Zapp, J.; Schlatter, C. H. R.
Food Addit. Contam. 1997, 14, 211. (p) Trucksess, M. W.;
Giler, J.; Young, K.; White, K. D.; Page, S. W. J. AOAC Int.
1997, 82, 85. (q) Teren, J.; Varga, J.; Hamari, Z.; Rinyu, E.;
Kevei, F. Mycopathologia 1996, 134, 171. (r) Varga, J.;
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Environ. Microbiol. 1996, 62, 4461. (s) Zimmerli, B.; Dick,
R. Food Addit. Contam. 1996, 13, 655. (t) Pittet, A.;
Tornare, D.; Huggett, A.; Viani, R. J. Agric. Food Chem.
1996, 44, 3564. (u) Majerus, P.; Otteneder, H. Dtsch.
Lebensmitt. Rundsch. 1996, 92, 388. (v) Scott, P. M.;
Kanhere, S. Food Addit. Contam. 1995, 12, 591.
2a
Yield: 47 mg (47%); colorless solid; mp 112–113 °C; [a]D25 –32.7
(c 5 mg/mL, CHCl3).
IR (KBr): 3020 (m), 2985 (br m), 2932 (m), 1728 (s), 1677 (s), 1611
(w), 1531 (s), 1426 (m), 1219 (m), 1138 (w), 810 (w), 770 (w) cm–1.
1H NMR (300 MHz, CDCl3): d = 12.72 (br s, 1 H, OH), 8.50 (br d,
J = 7.2 Hz, 1 H, NH), 8.41 (s, 1 H, H-6), 7.73 (br s, 1 H, OH), 5.10–
5.01 (m, 1 H, CHNH), 4.82–4.67 (m, 1 H, CHCH3), 3.40–3.16 (m,
3 H, CH2Ph, CHHCHCH3), 2.84 (distorted dd, J = 17.6, 11.7 Hz, 1
H, CHHCHCH3), 1.58 (d, J = 6.3 Hz, 3 H, Me).
13C NMR (75 MHz, CDCl3): d = 174.8, 169.7, 163.2, 159.2, 140.9,
139.1, 135.8, 129.0 (t, J = 23.8 Hz), 128.2 (t, J = 23.8 Hz), 127.3–
126.3 (m), 123.3, 120.6, 110.2, 75.9, 54.4, 37.5, 32.4, 20.7.
ESI-MS: m/z = 409 (100) [M + H]+.
ESI-HRMS: m/z [M + H]+ calcd for C20H14D5ClNO6: 409.1210;
found: 409.1199.
MS/MS [M + H]+ (ESI+, 20 eV): m/z = 409.11 (43.03) [M + H]+,
391.10 (7.7) [M – H2O + H]+, 363.11 (86.8) [M – H2O – CO + H]+,
346.08 (17.6) [M – H2O – CO – NH3 + H]+, 239.00 (100.0) [M –
C9H6D5NO2]+, 220.99 (8.7) [M – C9H8D5NO3]+, 125.11 (6.6)
[C8H5D5N]+.
2b
Yield: 36 mg (36%); colorless solid; mp 183–185 °C; [a]D25 +60.2
(c 3 mg/mL, CHCl3).
(w) Studer-Rohr, I.; Dietrich, D. R.; Schlatter, J.; Schlatter,
C. Food Chem. Toxicol. 1995, 33, 341. (x) Abarca, M. L.;
Bragulat, M. R.; Castella, G.; Cabanes, F. J. Appl. Environ.
Microbiol. 1994, 60, 2650. (y) Breitholtz-Emanuelson, A.;
Olsen, M.; Oskarsson, A.; Hult, I. K. J. Assoc. Off. Anal.
Chem. 1993, 76, 842. (z) Kuiper-Goodman, T.; Scott, P. M.
Biomed. Environ. Sci. 1989, 2, 179.
IR (KBr): 3072 (w), 2927 (br m), 2855 (w), 1740 (s), 1672 (s), 1626
(m), 1544 (s), 1427 (m), 1380 (w), 1214 (m), 1139 (w), 810 (w), 769
(w) cm–1.
1H NMR (300 MHz, CDCl3): d = 12.70 (br s, 1 H, OH), 8.49 (br d,
J = 7.2 Hz, 1 H, NH), 8.40 (s, 1 H, H-6), 7.16 (br s, 1 H, OH), 5.05
(distorted dd, J = 12.2, 5.9 Hz, 1 H, CHNH), 4.80–4.67 (m, 1 H,
CHCH3), 3.39–3.16 (m, 3 H, CH2Ph, CHHCHCH3), 2.89–2.76 (m,
1 H, CHHCHCH3), 1.58 (d, J = 6.3 Hz, 3 H, Me).
(2) (a) Pohland, A. E.; Nesheim, S.; Friedman, L. Pure Appl.
Chem. 1992, 64, 1029. (b) Micco, C.; Grossi, M.; Miraglia,
M.; Brera, C. Food Addit. Contam. 1989, 6, 333.
13C NMR (75 MHz, CDCl3): d = 174.8, 169.7, 163.1, 159.2, 140.9,
139.1, 135.8, 129.0 (t, J = 23.8 Hz), 128.2 (t, J = 23.8 Hz), 127.2–
126.3 (m), 123.3, 120.6, 110.2, 75.9, 54.5, 37.5, 32.4, 20.7.
(c) Van der Merwe, K. J.; Steyn, P. S.; Fourie, L. J. Chem.
Soc. 1965, 7083.
(3) For recent reviews, see: (a) Pfol-Leszkowicz, A.;
Manderville, R. A. Mol. Nutr. Food Res. 2007, 51, 61.
(b) Clark, H. A.; Snedeker, S. M. J. Toxicol. Environ. Health
B Crit. Rev. 2006, 9, 265. (c) O’Brien, E.; Dietrich, D. R.
Crit. Rev. Toxicol. 2005, 35, 33. (d) Dai, J.; Park, G.; Perry,
J. L.; Il’ichev, Y. V.; Bow, D. A. J.; Pritchard, J. B.; Faucet,
V.; Pfohl-Leszkowicz, A.; Manderville, R. A.; Simon, J. D.
Acc. Chem. Res. 2004, 37, 874. (e) Peraica, M.; Radić, B.;
Lucić, A.; Pavlović, M. Bull. World Health Organ. 1999, 77,
754.
ESI-MS: m/z = 409 (100) [M + H]+.
ESI-HRMS: m/z [M + H]+ calcd for C20H14D5ClNO6: 409.1210;
found: 409.1192.
MS/MS [M + H]+ (ESI+, 20 eV): m/z = 409.12 (41.20) [M + H]+,
391.11 (7.4) [M – H2O + H]+, 363.11 (84.5) [M – H2O – CO + H]+,
346.08 (18.7) [M – H2O – CO – NH3 + H]+, 239.01 (100.0) [M –
C9H6D5NO2]+, 221.00 (9.2) [M – C9H8D5NO3]+, 125.11 (8.0)
[C8H5D5N]+.
(4) Ochratoxin, A. In Some Naturally Occurring Substances:
Food Items and Constituents, Heterocyclic Aromatic Amines
and Mycotoxins, IARC Monographs on the Evaluation of
Carcinogenic Risks to Humans, Vol. 56; IARC and WHO:
Lyon, 1993, 489-521.
Acknowledgment
Thanks are due to Dr. Anna Lisa Piccinelli [Dipartimento di Scienze
Farmaceutiche, Università degli Studi di Salerno, Via Ponte Don
Synthesis 2009, No. 11, 1815–1820 © Thieme Stuttgart · New York

1820
B. Gabriele et al.
PAPER
(5) For leading examples, see: (a) Lindenmeier, M.; Schieberle,
P.; Rychlik, M. J. Chromatogr., A 2004, 1023, 57.
(b) Dell’Asta, C.; Galaverna, G.; Dossena, A.; Marchelli, R.
J. Chromatogr., A 2004, 1024, 275. (c) Monaci, L.;
Palmisano, F. Anal. Bioanal. Chem. 2004, 378, 96.
(d) Soleas, G. J.; Yan, J.; Goldberg, D. M. J. Agric. Food
Chem. 2001, 49, 2733. (e) Becker, M.; Degelmann, P.;
Herderich, M.; Schreier, P.; Humpf, H.-U. J. Chromatogr.,
A 1998, 818, 260.
(6) For a review, see: Rychlik, M.; Asam, S. Anal. Bioanal.
Chem. 2008, 390, 617.
(7) (a) Kraus, G. A. J. Org. Chem. 1981, 46, 201. (b) Kraus, G.
A. US Patent 4346039, 1982; Chem. Abstr. 1983, 98,
P4438v.
(10) Sibi, M. P.; Chttopadhyay, S.; Dankwardt, J. W.; Snieckus,
V. J. Am. Chem. Soc. 1985, 107, 6312.
(11) Donner, C. D.; Gill, M. Aust. J. Chem. 2002, 213.
(12) It is worth noting that the coupling reaction between 8 and 9
has been carried out previously, in the presence of 2-ethoxy-
1-(ethoxycarbonyl)-1,2-dihydroquinoline (EEDQ), to give,
after deprotection, a mixture of 1a and 1b in a total yield of
35–50%, depending on the crystallization conditions. See:
Roberts, J. C.; Woollven, P. J. Chem. Soc. C 1970, 278.
(13) The only method reported in the literature so far for the
synthesis of d5-OTA (2a) was based on a semi-synthetic
sequence involving: (a) acid hydrolysis of OTA to give (R)-
OTa; (b) conversion of (R)-OTa into the corresponding acyl
chloride by reaction with SOCl2; (c) reaction between the
acyl chloride and L-d5-phenylalanine methyl ester to give
methyl-protected d5-OTA; (d) deprotection of the latter with
NaOH in MeOH to give d5-OTA (2a). The overall yield
based on starting OTA was 5% over 4 steps. See ref. 5a.
(8) Covarrubias-Zúñiga, A.; Ríos-Barrios, E. J. Org. Chem.
1997, 62, 5688.
(9) Betzer, J.-F.; Pancrazi, A. Synthesis 1999, 629.
Synthesis 2009, No. 11, 1815–1820 © Thieme Stuttgart · New York