Hydrolysis of Ochratoxins by Carboxypeptidase A
Chem. Res. Toxicol., Vol. 14, No. 3, 2001 303
extracted with ethyl acetate. The combined ethyl acetate
extracts were dried (MgSO ), filtered, and evaporated under
4
Ta ble 1. Hyd r olyses of Och r a toxin s a n d An a logu es by
Ca r boxyp ep tid a se A
reduced pressure, which yielded salicyl azide (0.862 g, 5.29
mmol, 73%).
Km
kcat
kcat/Km
(µM) (min-1) (min
-1
µM-1
toxin
)
The salicyl azide (0.862 g) in ethyl acetate (10 mL) was
treated with a solution of Phe (1.5 g, 9.09 mmol) and triethyl
amine (0.918 g, 9.09 mmol) in water (10 mL). The reaction was
monitored by TLC (5:1 benzene/acetic acid mixture), and the
mixture was stirred for 2 days at ambient temperature, where-
ochratoxin A
ochratoxin B
bromoochratoxin B
N-(2-hydroxybenzoyl)-
phenylalanine
N-(5-fluoro-2-hydroxybenzoyl)-
phenylalanine
N-(5-chloro-2-hydroxybenzoyl)-
phenylalanine
N-(5-bromo-2-hydroxybenzoyl)-
phenylalanine
ochratoxin A methyl ester
ochratoxin B methyl ester
5.6
36.8
6.6
266
2717
283
47
74
43
3163
267
521
289
0.8
2372
after the organic layer was separated and 10 mL of 1 N Na
CO was added to the aqueous phase, which was extracted with
ethyl acetate.
2
-
16.0
5.5
4276
2869
3724
3
The alkaline aqueous phase was acidified (1 N HCl to pH 2)
and extracted with chloroform. The combined organic layer was
12.9
dried (MgSO
4
), filtered, and evaporated under reduced pressure.
NDa
-
-
-
-
The resulting oil was purified by column chromatography (silica
gel, 100 g, using a 5:1 benzene/acetic acid mixture) to yield N-(2-
hydroxybenzoyl)phenylalanine (0.929 g, 45%): λmax (MeOH) 207
NDa
a
Not detected.
(
(
ꢀ ) 35 600 M cm ), 237 (sh) (ꢀ ) 10 050 M cm-1), 300 nm
-
1
-1
-1
(d, J C-F ) 26.2 Hz), 111.27 (d, J C-F ) 23.8 Hz), 52.97 (s), 37.29
(s).
-
1
-1
-1
ꢀ ) 4500 M cm ); IR NH (3428, 1534 cm ), CO (1722, 1645
-
1
1
cm ); H NMR (300 MHz, CDCl
.63, 5.54 Hz), 5.06 (m, 1H), 6.72 (d, 1H, J H-H ) 7.39 Hz), 6.80
m, 1H), 7.18 (q, 2H, J H-H ) 2.09, 7.56 Hz), 7.29 (m, 4H), 7.38
3
) δ 3.28 (oc, 2H, J H-H ) 14.01,
Meth od . Kinetic data of peptide bond hydrolysis of the
ochratoxins were obtained by measuring the amount of Phe that
formed. Reactions were performed using an end point assay,
and reaction mixtures which contained 10 mM Tris-HCl (pH
8.0), 50 mM NaCl, 1 µg of carboxypeptidase A, and ochratoxin
(ranging between 1 and 1000 µM). The reactions were termi-
nated after 5 min using concentrated formic acid (5 µL), after
which the mixture was deproteinated, dried under vacuum, and
dissolved in a 1:1 acetonitrile/water mixture (containing 1%
formic acid). All reactions were compared to blanks, in which
the enzyme was omitted, to compensate for trace amounts of
Phe in the incubation mixtures. Electrospray ionization mass
spectrometry/mass spectrometry (ESI-MS/MS) was used to
detect the formed Phe, and an external standard of Phe (ring-
D5) was used. This was added after termination of the reactions
to quantify the Phe that formed. ESI-MS/MS was carried out
using a VG Quattro II triple-quadrupole instrument (Micro-
mass). The electrospray capillary was set at 3.5 kV and the cone
voltage at 30 V. The source temperature was 60 °C. Nitrogen
was used as the drying and nebulizing gas. The flow rates for
drying and nebulizing were set at 350 and 20 L/h, respectively.
Data were acquired in the parent ion scan mode of operation,
scanning for parent ions m/z 166 (Phe) and m/z 171 (Phe, ring-
D5) in the first mass spectrometer and keeping the second mass
spectrometer static, monitoring the collision-induced dissociation
5
(
(
1
1
1
3
m, 1H), 11.88 (br, 1H); C NMR (75 MHz, CDCl
3
) δ 175.69,
69.77, 161.62, 135.32, 134.77, 129.41, 128.93, 127.60, 125.71,
19.02, 118.70, 113.83, 52.95, 37.32.
3) N-(5-Ch lor o-2-h ydr oxyben zoyl)ph en ylalan in e. 5-Chlo-
(
rosalicylic acid (1.1 g, 6.5 mmol) was coupled to Phe (1.3 g, 7.9
mmol) in a manner similar to that of the salicylic acid reaction
described above. However, the crude product (1.4 g) was not
cleaned by column chromatography, but was recrystallized from
chloroform to yield N-(5-chloro-2-hydroxybenzoyl)phenylalanine
(
983 mg, 4.1 mmol, 47%): mp 172.6 °C [lit. 172-173 °C (15)];
-
1
-1
-1
λ
max (MeOH) 208 (ꢀ ) 38 020 M cm ), 312 nm (ꢀ ) 3940 M
-
1
-1
-1
cm ); IR NH (3368, 1534 cm ), CO (1722, 1632 cm ); ES-MS
+
1
m/z 320/322 [C16
MHz, CDCl ) δ 3.29 (oc, 2H, J H-H ) 12.73, 5.73, 5.63 Hz), 5.06
m, 1H), 6.57 (d, 1H, J H-H ) 7.31 Hz), 6.92 (d, 1H, J H-H ) 8.9
4
H14NO Cl requires (M + 1) 320); H NMR (300
3
(
1
3
Hz), 7.18 (m, 3H), 7.30 (m, 4H), 11.78 (s, 1H); C NMR (75 MHz,
CDCl ) δ 174.81, 168.71, 160.32, 135.07, 134.73, 129.38, 129.05,
27.80, 125.22, 123.71, 120.32, 114.70, 52.94, 37.34.
4) N-(5-Br om o-2-h yd r oxyb en zoyl)p h en yla la n in e. The
coupling of 5-bromosalicylic acid (1 g, 4.6 mmol) and Phe (1 g,
.1 mmol) was accomplished in a manner similar to that for
3
1
(
6
salicylic acid (see above). The only difference is that the reaction
between the 5-bromosalicyl azide and the Phe [catalyzed with
triethylamine (1.22 g, 12.1 mmol)] was completed after 2 h as
evidenced by TLC (1:5 acetic acid/benzene mixture). The crude
coupled product (900 mg, 2.47 mmol) was recrystallized with
chloroform to obtain a yield of 43% (735 mg, 2 mmol): mp 178
(
CID) fragment ions at m/z 120 and 125, respectively. This is
achieved with argon as the collision gas at a pressure of 1.4 ×
mbar in the collision cell using a collision energy setting
of 12 eV.
Kinetic parameters were determined using linear regression
-
3
1
0
-
1
-1
1
analysis of double-reciprocal primary rate plots (16). The
averages of three primary rate plots (Lineweaver-Burk, Eadie-
Hofstee, and Hanes plots) were calculated.
°
[
C; IR NH (3360, 1526 cm ), CO (1747, 1636 cm ); H NMR
300 MHz, CO(CD ] δ 3.18 (oc, 2H, J H-H ) 14.05, 4.97, 9.42
Hz), 4.97 (m, 1H), 6.86 (d, 1H, J H-H ) 8.87 Hz), 7.28 (m, 5H),
.52 (q, 1H, J H-H ) 2.46, 8.87 Hz), 7.97 (d, 1H, J H-H ) 2.46
3 2
)
7
Resu lts
1
3
Hz), 8.42 (m, 1H); C NMR [75 MHz, CO(CD
3
)
2
] δ 172.24,
69.13, 160.86, 137.93, 137.23, 130.20, 129.71, 128.92, 127.24,
20.46, 116.88, 110.36, 54.28, 37.25.
5) N-(5-F lu or o-2-h yd r oxyben zoyl)p h en yla la n in e. 5-Flu-
Hydrolyses of the ochratoxins and analogues by car-
boxypeptidase A using ESI-MS/MS are summarized in
Table 1. The enzymatic hydrolysis of the toxins could
clearly be demonstrated by the formation of Phe in all
cases, except in the case of the methylated ochratoxins.
Hydrolysis of the toxins progressed linearly over a period
of at least 10 min. As little as 10 nM Phe could accurately
be detected in the reaction mixtures using this method,
which allowed for very accurate assessments of the
hydrolysis of the toxins. This sensitivity could not be
achieved using either spectrophotometric (17) or HPLC-
based methods (18, and references therein).
1
1
(
orosalicylic acid (500 mg, 3.185 mmol) was coupled to Phe (661
mg, 4.01 mmol) in a manner similar to that for the salicylic acid
reaction mentioned above. The only difference is that the N-(5-
fluoro-2-hydroxybenzoyl)phenylalanine (25.7% yield, 248 mg,
0
.818 mmol) was not cleaned with column chromatography, but
recrystallized from chloroform: mp 162.9 °C; λmax (MeOH) 204.1
-
1
-1
-1
-1
(
(
ꢀ ) 17 000 M cm ), 309 nm (ꢀ ) 2300 M cm ); IR NH
-
1
-1
1
3368, 1534 cm ), CO (1747, 1619 cm ); H NMR (300 MHz,
) δ 3.29 (oc, 2H, J H-H ) 14.20, 5.63, 5.54 Hz), 5.06 (m,
H), 6.55 (d, 1H, J H-H ) 7.34 Hz), 6.92 (m, 2H), 7.14 (m, 2H),
CDCl
1
7
3
1
3
3
.30 (m, 3H), 11.58 (s, 1H); C NMR (75 MHz, CDCl ) δ 175.59
Discu ssion
(s), 168.84 (d, J C-F ) 2.6 Hz), 157.83 (d, J C-F ) 1.6 Hz), 155.16
(d, J C-F ) 239.4 Hz), 135.07 (s), 129.36 (s), 129.05 (s), 127.78
(s), 122.16 (d, J C-F ) 23.2 Hz), 120.01 (d, J C-F ) 7.4 Hz), 113.60
The relative affinity and turnover number of carboxy-
peptidase A for the various toxins have been determined.