Patulin Synthesis
J. Agric. Food Chem., Vol. 46, No. 12, 1998 5165
F igu r e 2. Structure of geometric isomers 9/10 formed by
reaction of ketone 4 with unlabeled ethyl bromoacetate.
sulfonyl chloride (0.17 mL) was then dropwise added and the
mixture stirred for 2 h at room temperature. After addition
of chloroform (4 × 10 mL), the mixture was filtered over a
paper filter and the combined filtrates were washed with
hydrochloric acid (2 mol/L, 5 mL). After the organic phase
was washed with brine (7 mL), the solution was dried over
Na
synthetic step.
2
-Hydroxy-4H-[ C ]furo[3,2-c]pyran-2(6H)-one (8). One-
2 4
SO and concentrated to 10 mL for use in the next
1
3
4
tenth of the solution containing 7 was evaporated to dryness
and, after addition of a mixture of trifluoroacetic acid/water
(1.65 mL; 10 + 1, v/v), heated for 1 h at 50-70 °C. The solution
was then concentrated to 0.5 mL and, after addition of ethyl
acetate (5 mL), washed with a saturated aqueous solution of
sodium hydrogencarbonate (2.5 mL). The organic phases were
F igu r e 3. 13C NMR spectrum of labeled (A) and unlabeled
2 4
dried over Na SO and concentrated to 200 µL. The crude
(B) patulin. Signals at 29.9 and 206.3 ppm are caused by the
product was purified by high-performance liquid chromatog-
raphy (pump 110 A; Beckman, Munich, Germany) on silica
using ethyl acetate/hexane (1 + 1, v/v) as the solvent. The
effluent (1 mL/min) was monitored at 275 nm using a UV
detector Uvikon 735 LC (Kontron Instruments, Neufahrn,
Germany). The target compound was pooled from 30 runs in
the elution range of 8.5-10.5 mL and, after evaporation of the
solvent acetone-d
6
.
2 2 3
(d, J ) 13.1, H-5′), 4.21 (quart., J ) 7.2, CO CH CH ), 4.31
(dt, J ) 6.0 and 1.8, H-4), 5.23 (d, J ) 1.8, H-1), 6.03 (d, J )
7.5, H-3), 6.35 (d, J ) 1.8, dCH); Z-isomer 10, δ 1.29 (t, J )
2 2 3 3 3 3
7.1, CO CH CH ), 1.39 (CH ), 1.51 (CH ), 3.47 (OCH ), 3.98
(d, J ) 13.3, H-5), 4.10 (dd, J ) 13.0 and 2.6, H-5′), 4.18 (quart.,
J ) 7.1, CO CH CH ), 4.23 (dd, J ) 4.4 and 1.8, H-4), 4.76
(dd, J ) 5.5 and 2.0, H-3), 6.16 (d, J ) 1.8, dCH), 6.25 (H-1).
1
solvent, characterized by H NMR (total yield of the two last
2
2
3
synthetic steps ) 50%). The following signals were obtained:
1
13
H NMR (CD
3
COCD
3
) δ 2.90 (br s, OH), 4.37 (dd, J ) 17.3
C NMR 9 δ 14.0 (CO
(OCH ), 60.5 (C-5), 62.8 (CO
97.6 (C-1), 110.3 (CMe ), 123.9 (dCH), 147.6 (C-2), 165.3
(CdO); 10 δ 15.1 (CO CH CH ), 26.2 (CH ), 27.7 (CH ), 55.4
(OCH ), 57.9 (C-5), 60.3 (CO CH CH ), 71.5 (C-3), 73.8 (C-4),
), 118.6 (dCH), 150.4 (C-2), 165.0 (Cd
2
CH
2
CH
3
), 25.1 (CH
3 3
), 26.2 (CH ), 55.3
and 4.0, H-6), 4.66 (dd, J ) 17.7 and 2.9, H-6′), 6.05 (m, H-7/
H-4), 6.07 (dd, J ) 185 and 9.3, H-3). C NMR (CD
δ 110.6 (d, J ) 69, C-3), 169.4 (d, J ) 68, CdO).
3
2
CH
2
CH ), 68.4 (C-3), 75.0 (C-4),
3
1
3
3
COCD
3
)
2
2
2
3
3
3
Data obtained by high-resolution mass spectrometry of 8
relative intensities in parentheses): m/z 111.9957 (100%),
3
2
2
3
(
95.2 (C-1), 109.6 (CMe
O).
2
5
6
2
4.9517 (61%), 55.9549 (57%), 128.0393 (49%), 83.9646 (46%),
8.9202 (43%), 53.9440 (32%), 42.9997 (31%), 156.0284 (M ,
8%), 138.0393 (23%).
+
Liqu id Ch r om a togr a p h y (LC)/Mu ltip le-Sta ge Ma ss
n
n
Sp ectr om etr y (MS) . LC(MS) spectra were recorded with
an LCQ (Finnigan MAT) ion trap mass spectrometer operating
in the negative electrospray ionization mode with a spray
needle voltage of -4.5 kV and a spray current of 0.2 µA. The
temperature of the capillary was 220 °C and the capillary
voltage -6 V. The sheath and auxiliary gas nitrogen nebulized
the sample solutions with flows of 2.3 or 3.0 mL/min. The ion
Methyl 2-Deoxy-3,4-O-isopropylidene-2-C-[(E)- and (Z)-
ethoxycarbonyl methylene]-R-L-erythro-pentopyranosid-2-ulose.
The mixture of the two geometric isomers 9 and 10 was
1
3
prepared as reported for C-labeled 5 by using unlabeled ethyl
bromacetate. 9 and 10 were separated by high-performance
liquid chromatography on silica using an isocratic HPLC
system (pump 110 A; Beckman, Munich, Germany) and ethyl
acetate/hexane (2 + 8, v/v) as the mobile phase with a flow of
-
3
trap was run with a helium pressure of 10 Torr.
Samples were diluted with methanol/water (98 + 2, v/v) or
ethyl acetate to reach concentrations of 50 µg/mL and were
introduced by means of a syringe pump operating at a flow of
18 µL/min. For HPLC/MS analysis, 3 µL of a solution was
injected onto a spectra series high-performance liquid chro-
matograph (Thermo Separation Products, San J ose, CA)
equipped with a Nucleosil RP18 column (250 × 2.0 mm i.d., 5
µm) and using an isocratic flow of 0.4 mL acetonitrile/water
2
mL/min. The effluent was monitored at 254 nm with a
Uvikon 735 LC (Kontron Instruments, Neufahrn, Germany).
and 10 were present in a ratio of 1.4 to 1 and were singly
9
pooled from 20 runs in the elution ranges of 7.0-7.4 and 7.9-
8
.8 mL, respectively.
The two compounds gave the following NMR signals: 1H
NMR, E-isomer 9, δ 1.30 (t, J ) 7.1, CO
2 2 3 3
CH CH ), 1.40 (CH ),
.53 (CH ), 3.48 (OCH ), 3.64 (dd, J ) 13.1 and 1.5, H-5), 3.67
n
1
3
3
(1 + 9, v/v). LC/(MS) experiments were performed by colli-