Synthesis of analogues of ochratoxin A

Article abstract of DOI:10.1080/14786419.2011.613385

Four analogues of ochratoxin A (OTA) differing for the aminoacidic moiety were synthesised using ochratoxin (OTα) as the starting material. The condensation reaction between protected amino acids and OT, carried out in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC?HCl) and N-hydroxybenzotriazole (HOBt) as coupling agents, followed by deprotection and PTLC purification afforded OTA alanine, leucine, serine and tryptophane analogues in satisfactory yields (33-47%, based on OT).

Full text of DOI:10.1080/14786419.2011.613385

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Synthesis of analogues of ochratoxin A
Pierluigi Plastina a , Alessia Fazio a , Mohamed Attya b , Giovanni
Sindona b & Bartolo Gabriele a
a Dipartimento di Scienze Farmaceutiche, Università della
Calabria, Arcavacata di Rende 87036, Cosenza, Italy
b Dipartimento di Chimica, Università della Calabria, Arcavacata
di Rende 87036, Cosenza, Italy
Published online: 11 Oct 2011.
To cite this article: Pierluigi Plastina , Alessia Fazio , Mohamed Attya , Giovanni Sindona & Bartolo
Gabriele (2012) Synthesis of analogues of ochratoxin A, Natural Product Research: Formerly Natural
Product Letters, 26:19, 1799-1805, DOI: 10.1080/14786419.2011.613385
To link to this article: http://dx.doi.org/10.1080/14786419.2011.613385
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Natural Product Research
Vol. 26, No. 19, October 2012, 1799–1805
Synthesis of analogues of ochratoxin A
a
a
b
b
*
Pierluigi Plastina , Alessia Fazio , Mohamed Attya , Giovanni Sindona and
Bartolo Gabrielea*
a
`
Dipartimento di Scienze Farmaceutiche, Universita della Calabria, Arcavacata di Rende 87036,
Cosenza, Italy; Dipartimento di Chimica, Universita della Calabria, Arcavacata di Rende 87036,
b
`
Cosenza, Italy
(Received 17 December 2010; final version received 25 June 2011)
Four analogues of ochratoxin A (OTA) differing for the aminoacidic moiety were
synthesised using ochratoxin ꢀ (OTꢀ) as the starting material. The condensation
reaction between protected amino acids and OTꢀ, carried out in the presence of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC ꢀ HCl) and
N-hydroxybenzotriazole (HOBt) as coupling agents, followed by deprotection
and PTLC purification afforded OTA alanine, leucine, serine and tryptophane
analogues in satisfactory yields (33–47%, based on OTꢀ).
Keywords: ochratoxin A; OTA; ochratoxin analogues; synthesis; diastereomeric
analogues
1. Introduction
The mycotoxin ochratoxin A (OTA; Figure 1, R ¼ Bn) is a secondary metabolite produced
by some fungi of the genera Aspergillus and Penicillium (Van der Merwe, Steyn, Fourie,
Scott, & Theron, 1965). It occurs in raw and improperly stored food products, and in
particular, it has been found in cereal based foods, and also in coffee, cocoa, grape juice,
beer and wine (Anli & Bayram, 2009; Cramer, Koenigs, & Humpf, 2008; Duarte, Pena, &
Lino, 2010; Jalili, Jinap, & Radu, 2010). OTA has been reported to be nephrotoxic,
mutagenic, genotoxic, teratogenic, hepatotoxic, neurotoxic and immunotoxic, in both
animals and humans (Clark & Snedeker, 2006; el Khoury & Atoui, 2010; Mally et al.,
2005; O’Brien & Dietrich, 2005; Pfohl-Leszkowicz & Manderville, 2007; Reddy et al.,
2010), and was classified as a possible carcinogen to humans (group 2b) by the
International Agency for Research on Cancer (IARC; IARC Monographs on the
Evaluation of Carcinogenic Risks to Humans, 1993).
The mechanism by which ochratoxin A exerts its high acute toxicity is still unclear.
OTA was found to competitively inhibit phenylalanyl-tRNA-synthetase-catalysed reac-
tions in both bacterial and eukaryotic systems thus blocking protein synthesis (Bunge,
Dirheimer, & Roschenthaler, 1978; Creppy et al., 1979). However, these data were not
¨
confirmed by successive experiments with recombinant expressed and purified phenyla-
lanyl-tRNA-synthetase (Roth, Eriani, Dirheimer,
& Gangloff, 1993). Moreover,
computationally calculated binding affinities of ochratoxin A to the phenylalanyl-
tRNA-synthetase resulted very low (McMasters & Vedani, 1999). Another possibility that
*Corresponding authors. Email: p.plastina@unical.it; b.gabriele@unical.it
ISSN 1478–6419 print/ISSN 1478–6427 online
ß 2012 Taylor & Francis
http://dx.doi.org/10.1080/14786419.2011.613385
http://www.tandfonline.com

1800
P. Plastina et al.
Figure 1. Structure of ochratoxin A (R ¼ Bn) and its analogues.
could explain OTA toxicity is the enhanced lipid peroxidation that it induces. In fact, OTA
stimulates the production of free radicals in bacteria and in rat microsomes (Hoehler,
Marquardt, McIntosh, & Hatch, 1997).
Several structure-activity relationship studies have been carried out in order to
determine the role of each functional group played in OTA toxicity (Cramer, Harrer,
Nakamura, Uemura, & Humpf, 2010; Mally et al., 2004; Muller et al., 2004; Xiao et al.,
¨
1996; Xiao, Marquardt, Frohlich, & Ling, 1995), and the question of whether the amino
acidic moiety played a role has also been raised. Several analogues of ochratoxin A in
which L-phenylalanine residue was replaced with other amino acidic moieties were
consequently synthesised (Steyn, Vleggaar, Du Preez, Blyth, & Seegers, 1975). All these
compounds caused in vitro toxicity in monkey kidney epithelial cells in varying degrees.
Moreover, these derivatives had inhibitory effects on yeast amino acyl-tRNA synthetase-
catalysed reactions and on growth and protein synthesis of hepatoma culture cells (Creppy
et al., 1983). In particular, among the series of compounds tested by Creppy et al.,
analogues with L-alanine, L-valine, L-tyrosine and L-serine residues resulted as the most
toxic, although to a lesser extent than OTA.
Interestingly, some of these analogues were also reported to occur in nature (Hadidane
et al., 1992). However, to the best of our knowledge, the only method for preparing OTA
analogues differing for amino acidic residue, reported so far in literature, consists of a
semi-synthetic sequence involving acid hydrolysis of OTA, followed by acid azide coupling
method of the resulting 5-chloro-8-hydroxy-3-methyl-1-oxoisochromane-7-carboxylic
acid, known as ochratoxin ꢀ (OTꢀ), with triethylammonium salt of various amino acids
(Steyn et al., 1975). Moreover, spectroscopic characterisation of these compounds was not
complete (Steyn et al., 1975).
In this article we expanded the scope of our previous work on the synthesis of
ochratoxin A (Gabriele et al., 2009) to the preparation of four OTA analogues, differing
for the amino acidic residue. OTA analogues have been fully characterised by IR, MS, 1H
NMR and 13C NMR spectroscopy, and will be useful for further toxicological studies.
2. Results and discussion
Recently, we reported a new total synthesis of ochratoxin A, prepared in 9% overall yield
from commercially available but-2-ynal and dimethyl 3-oxopentanedioate (Gabriele et al.,
2009). The key step of the synthetic strategy consisted of the condensation reaction
between protected L-phenylalanine and ochratoxin ꢀ (OTꢀ), carried out in the presence of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCꢀHCl) and N-hydro-
xybenzotriazole (HOBt) as coupling agents, to give a mixture of protected diastereoi-
somers 4a and 5a in ca. 86% total yield, based on OTꢀ. Deprotection of the crude mixture
of 4a and 5a with TFA in CH2Cl2 at room temperature for 6 h led to a mixture of OTA 1a

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Scheme 1. Synthesis of ochratoxin A and its analogues.
Table 1. Synthesis of ochratoxin A analogues 1b–e and of their corresponding 3-(S) diastereoi-
somers 2b–e.
Entry
R
1
Yield of 1 (%)a
2
Yield of 2 (%)a
1
2
3
Me
i-Bu
CH2OH
1b
1c
1d
47
43
48
2b
2c
2d
41
39
28
H2C
4
1e
33
2e
28
N
H
a
Note: Isolated yield based on starting OTꢀ.
and its 3-(S) diastereoisomer 2a in 81% total yield based on starting OTꢀ. Separation of
the two diastereoisomers by PTLC afforded pure 1a and 2a in 46% and 34% isolated
yields, respectively (Scheme 1, R ¼ Bn).
In order to prepare analogues of ochratoxin A for further studies aimed at establishing
the structure-activity relationship of OTA, we have applied a similar synthetic strategy to
prepare OTA analogues with different amino acidic residues. We started our investigations
with the synthesis of OTA L-alanine analogue 1b. Commercially available L-alanine
tert-butyl ester 3b was reacted with OTꢀ under the same conditions described before
(Gabriele et al., 2009), leading to a mixture of diastereoisomers 4b and 5b, in ca. 97% total
yield, based on OTꢀ. Deprotection of this crude mixture led to a mixture of 1b and its
diastereoisomer 2b in 94% total yield based on starting OTꢀ. Separation of the two
diastereoisomers by PTLC afforded pure 1b and 2b in 47% and 41% isolated yields,
respectively (Scheme 1, R ¼ Me and Table 1, entry 1). For the preparation of OTA
L-leucine analogue 1c, we started by protecting L-leucine with tert-butyl group, affording
L-leucine tert-butyl ester 3c. Following the general synthetic strategy, OTA L-leucine
analogue 1c and its diastereoisomer 2c were obtained in 43% and 39% isolated yields,
respectively (Scheme 1, R ¼ i-Bu and Table 1, entry 2). In the case of amino acids bearing a
functional group in the side chain, such as L-serine (R ¼ CH2OH) and L-tryptophane
(R ¼ 3-indolylmethyl), protection of the side chain functionality with the tert-butyl group
was also needed. OTA serine and tryptophane analogues (1d and 1e, respectively)

1802
P. Plastina et al.
together with their diasteroisomers (2d and 2e) were eventually obtained in good
yields after complete deprotection and PTLC separation (Scheme 1 and Table 1, entries
3 and 4).
3. Experimental
3.1. General
Melting points were determined on a Reichert Thermovar apparatus and are uncorrected.
1H NMR and 13C NMR spectra were recorded on a Bruker DPX Avance 300
spectrometer at 25ꢁC in CDCl3 or DMSO-d6 solutions at 300 MHz and 75 MHz,
respectively, with Me4Si as internal standard. Chemical shifts (ꢁ) and coupling constants
(J) are given in ppm and in Hz, respectively. IR spectra were taken with a Perkin–Elmer
Paragon 1000 PC FT-IR spectrometer. Mass spectra were obtained using a Shimadzu
GCMS-QP2010 apparatus at 70 eV ionisation voltage. The electrospray ionisation mass
spectra were acquired on an ABSciex API 2000 mass spectrometer equipped with a turbo
ion spray ionisation source. The spectra in the positive ion mode were obtained under the
following conditions: ionspray voltage (IS) 4500 V; curtain gas 10 psi; temperature 400ꢁC;
ion source gas (1) 50 psi; ion source gas (2) 70 psi; declustering and focusing potentials 20
V and 200 V, respectively. Microanalyses were carried out with a Carlo Erba Elemental
Analyzer Mod. 1106. All reactions were analysed by TLC on silica gel 60 F254 and by GLC
using a Shimadzu GC-2010 gas chromatograph and capillary columns with poly-
methylsilicone þ 5% phenylsilicone as the stationary phase. Column chromatography was
performed on silica gel 60 (Merck, 70–230 mesh). PTLC separations were carried out using
SiO2 plates (Merck, 20 ꢂ 20 cm, 0.5 mm thickness). MnO2, 2-butynol, 3-oxopentanedioic
acid dimethyl ester, NaH (95% purity), LDA (2 M in THF/heptane/ethylbenzene),
acetaldehyde, SO2Cl2, LiOH, L-alanine tert-butyl ester, L-leucine, L-serine, L-tryptophane,
acetic acid tert-butyl ester, EDCꢀHCl, HOBt and TFA were purchased from Sigma-
Aldrich (Milan, Italy). HClO4 (70%) was purchased from Merck (Milan, Italy).
3.2. Procedure for the preparation of compounds 3c–e
3.2.1. Preparation of L-Leucine tert-Butyl Ester (3c)
Concd HClO4 (70%; 1.5 mL, 2.5 g, 17.4 mmol) was slowly added to a suspension of
L-leucine (1.43 g, 10.9 mmol) in acetic acid tert-butyl ester (27 mL, 23.3 g, 200 mmol) under
nitrogen at 0ꢁC. After stirring at room temperature for 12 h, water (55 mL) followed by 1
M HCl (30 mL) were added. The mixture was basified to pH 9 by the addition of 10% aq
K2CO3, and then extracted with CH2Cl2 (3 ꢂ 25 mL). The combined organic layers were
dried over Na2SO4. After filtration, the solvent was eliminated by rotary evaporation, and
the crude product was purified by column chromatography (silica gel, hexane-AcOEt,
8 : 2), to give pure 3c as a colourless oil (1.51 g, 74%).
3.2.2. Preparation of O-tert-Butyl L-Serine tert-Butyl Ester (3d)
Compound 3d was isolated as a colourless oil in 83% yield after column chromatography
purification, following the same procedure described for the preparation of 3c, starting
from L-serine (1.15 g, 10.9 mmol).
3.2.3. Preparation of N-tert-Butyl L-Tryptophane tert-Butyl Ester (3e)
Compound 3e was isolated as a colourless oil in 91% yield after column chromatography
purification, following the same procedure described for the preparation of 3c, starting
from L-tryptophane (2.22 g, 10.9 mmol).

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3.3. Procedure for the preparation of Ochratoxin A analogues 1b–e and 2b–e
3.3.1. Preparation of Ochratoxin A alanine analogue (1b) and its 3-(S)
diastereoisomer (2b)
EDCꢃHCl (208 mg, 1.08 mmol) was added to a stirred soln of 3b (145 mg, 1.0 mmol), OTꢀ
(269 mg, 1.05 mmol) and BtOH (142 mg, 1.05 mmol) in anhyd CHCl3 (5 mL) under
nitrogen at 0ꢁC. After additional stirring of the mixture at 0ꢁC for 15 min and at 25ꢁC for
20 h, 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
evaporation; this gave a crude mixture of diastereomers 4b and 5b which was added to a
soln of TFA (12.3 g, 108 mmol) in anhyd CH2Cl2 (20 mL) at 25ꢁC under nitrogen. The
reaction mixture was stirred at 25ꢁC and monitored by TLC. When the reaction was
completed (ca. 6 h), the solvent and excess TFA were removed by rotary evaporation. The
resulting residual oil was dissolved in CH2Cl2, washed with H2O (3 ꢂ 10 mL) and dried
(Na2SO4). After filtration and removal of the solvent by rotary evaporation, a colourless
solid was obtained, which was crystallised from benzene to give a mixture of 1b and 2b
(yield: 323.5 mg, 94% from OTꢀ). A mixture of the two diastereomers 1b and 2b (100 mg)
dissolved in CHCl3 was then subjected to preparative TLC on plates coated with silica gel
(20 plates, 20 ꢂ 20 cm, 0.25 mm thickness, benzene–acetone–HCO2H, 79 : 20 : 1); this gave
pure 1b (colourless solid, m.p. 91–93ꢁC, yield: 47 mg, Rf ¼ 0.44) and 2b (colourless solid,
m.p. 121–124ꢁC, yield: 41 mg, Rf ¼ 0.34).
3.3.2. Preparation of Ochratoxin A leucine analogue (1c) and its 3-(S)
diastereoisomer (2c)
The same procedure described before was used, starting from 3c (187 mg, 1.0 mmol) and
affording 1c (colourless solid, m.p. 94–97ꢁC, yield: 43 mg, Rf ¼ 0.44) and 2c (colourless
solid, m.p. 125–128ꢁC, yield: 39 mg, Rf ¼ 0.34).
3.3.3. Preparation of Ochratoxin A serine analogue (1d) and its 3-(S)
diastereoisomer (2d)
The same procedure described before was used, starting from 3d (217 mg, 1.0 mmol) and
affording 1d (colourless solid, m.p. 105–108ꢁC yield: 48 mg, Rf ¼ 0.26) and 2d (colourless
solid, m.p. 134–137ꢁC, yield: 28%, Rf ¼ 0.17).
3.3.4. Preparation of Ochratoxin A tryptophane analogue (1e) and its 3-(S)
diastereoisomer (2e)
The same procedure described before was used, starting from 3e (316 mg, 1.0 mmol) and
affording 1e (colourless solid, m.p. 142–147ꢁC, yield: 33 mg, Rf ¼ 0.36) and 2e (colourless
solid, m.p. 187–191ꢁC, yield: 28 mg, Rf ¼ 0.29).
4. Conclusions
In conclusion, we have shown that our synthetic method, based on the EDC ꢀ HCl/HOBt
promoted coupling of ochratoxin ꢀ with a suitably protected amino acid, is an efficient
and general method for preparing several different analogues of ochratoxin A in
satisfactory yields (33–47 %, based on OTꢀ). The compounds 1b–e were characterised by
IR, MS, 1H NMR and 13C NMR spectroscopy, and will be used for further studies aimed
at establishing the structure–activity relationships and the molecular mechanism of action
of OTA. Moreover, our method represents a useful entry to the diastereomeric OTA

1804
P. Plastina et al.
analogues 2b–e, which have not been reported so far and whose toxicological and
eco-toxicological evaluation can provide additional insights into the mechanism of OTA
toxicity.
Acknowledgement
Mohamed Attya gratefully acknowledges Calabria QUASIORA laboratory for his post-doctoral
grant.
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