Identification of Novel Colored Compounds Containing Pyrrole and Pyrrolinone Structures Formed by Maillard Reactions of Pentoses and Primary Amino Acids

Article abstract of DOI:10.1021/jf980477i

Heating of pentoses with alanine in a ratio of 10:1 in aqueous solution at pH 7.0 generated the yellow 2-[(2-furyl)methylidene]-4-hydroxy-5-methyl-2H-furan-3-one (1), being well in line with data reported in the literature. Decreasing the relative concentrations of the pentose produced further colored nitrogen-containing compounds, among which (S)-4-hydroxy-5-methyl-2-[N-(1′-carboxyethyl)pyrrolyl-2-methylidene]-2H- furan-3-one (2) could be identified by spectroscopic and synthetic experiments. On the other hand, thermal treatment of an aqueous solution of pentose and L-alanine in the presence of furan-2-carboxaldehyde led to the formation of the novel red (2R)-4-oxo-3,5-bis-[(2-furyl)methylidene] tetrahydropyrrolo[1,2-c]-5(S)-(2-furyl)oxazolidine and its 5(R)-(2-furyl)oxazolidine diasteromer (3a/3b), which were characterized by several 1D- and 2D-NMR techniques, LC/MS, and UV-vis spectroscopy as well as by synthesis of the chromophoric substructure. In addition, the red compounds (S)-4-[(E)-1-formyl-2-(2-furyl)ethenyl]-5-(2-furyl)-2-[(E)-(2-furyl)methylidene] -2,3-dihydo-α-amino-3-oxo-1H-pyrrole-1-acetic acid (4b) and the corresponding 2-[(Z)-(2-furyl)-methylidene] isomer (4b) were identified in this Maillard mixture. Quantitative studies on the formation of these colorants clearly demonstrates the key role of 3-deoxypentos-2-ulose as an intermediate in the formation of 4a/4b. Reaction pathways leading to the colorants 2, 3a/3b, and 4a/4b from pentoses and alanine are discussed.

Full text of DOI:10.1021/jf980477i

3902
J. Agric. Food Chem. 1998, 46, 3902−3911
Id en tifica tion of Novel Color ed Com p ou n d s Con ta in in g P yr r ole a n d
P yr r olin on e Str u ctu r es F or m ed by Ma illa r d Rea ction s of P en toses
a n d P r im a r y Am in o Acid s
Thomas Hofmann†
Deutsche Forschungsanstalt f u¨ r Lebensmittelchemie, Lichtenbergstrasse 4, D-85748 Garching, Germany
Heating of pentoses with alanine in a ratio of 10:1 in aqueous solution at pH 7.0 generated the
yellow 2-[(2-furyl)methylidene]-4-hydroxy-5-methyl-2H-furan-3-one (1), being well in line with data
reported in the literature. Decreasing the relative concentrations of the pentose produced further
colored nitrogen-containing compounds, among which (S)-4-hydroxy-5-methyl-2-[N-(1′-carboxyethyl)-
pyrrolyl-2-methylidene]-2H-furan-3-one (2) could be identified by spectroscopic and synthetic
experiments. On the other hand, thermal treatment of an aqueous solution of pentose and L-alanine
in the presence of furan-2-carboxaldehyde led to the formation of the novel red (2R)-4-oxo-3,5-bis-
[(2-furyl)methylidene] tetrahydropyrrolo[1,2-c]-5(S)-(2-furyl)oxazolidine and its 5(R)-(2-furyl)oxazo-
lidine diasteromer (3a /3b), which were characterized by several 1D- and 2D-NMR techniques, LC/
MS, and UV-vis spectroscopy as well as by synthesis of the chromophoric substructure. In addition,
the red compounds (S)-4-[(E)-1-formyl-2-(2-furyl)ethenyl]-5-(2-furyl)-2-[(E)-(2-furyl)methylidene]-
2
,3-dihydo-R-amino-3-oxo-1H-pyrrole-1-acetic acid (4b) and the corresponding 2-[(Z)-(2-furyl)-
methylidene] isomer (4b) were identified in this Maillard mixture. Quantitative studies on the
formation of these colorants clearly demonstrates the key role of 3-deoxypentos-2-ulose as an
intermediate in the formation of 4a /4b. Reaction pathways leading to the colorants 2, 3a /3b, and
4
a /4b from pentoses and alanine are discussed.
Keyw or d s: Maillard reaction; colored compounds; (S)-4-hydroxy-5-methyl-2-[N-(1′-carboxyethyl)-
pyrrolyl-2-methylidene]-2H-furan-3-one; (2R)-4-oxo-3,5-bis[(2-furyl)methylidene]tetrahydropyrrolo-
[
1,2-c]-5(S)-(2-furyl)oxazolidine; (S)-4-[(E)-1-formyl-2-(2-furyl)ethenyl]-5-(2-furyl)-2-[(E)-(2-furyl)-
methylidene]-2,3-dihydo-R-amino-3-oxo-1H-pyrrole-1-acetic acid
INTRODUCTION
beer product was related to the nitrogen content of the
malt used, indicating that amino acids are involved as
catalysts and/or reaction partners in the formation of
browning products during kilning of malt or wort boiling
in beer production.
To gain a more detailed insight into the role of the
amino acid as reactant in the nonenzymatic browning,
we have recently studied Maillard reactions leading to
nitrogen-containing colorants. In an aqueous reaction
mixture of L-proline and furan-2-carboxaldehyde, a
major dehydration product from pentoses, we identified
the previously unknown intense yellow 5-(S)-(2-carboxy-
The Maillard reaction between reducing carbohy-
drates and compounds bearing an amino group is chiefly
responsible for the development of desirable colors and
flavors occurring, for example, during thermal process-
ing of foods, such as roasting of meat or coffee, baking
of bread, or kiln-drying of malt. However, due to the
complexity and multiplicity of the nonvolatile Maillard
reaction products formed, surprisingly few studies have
been aimed at identifying the structures of the com-
pounds responsible for the typical brown color.
Some model studies [e.g., Ledl and Severin (1978,
982)] have been performed to clarify the mechanisms
1
-pyrrolidinyl)-2-hydroxy-(E,E)-2,4-pentadienal-(S)-(2-
1
carboxypyrrolidine)imine as the colored main compound
formed even at low temperatures (Hofmann, 1997a,
of this so-called nonenzymatic browning; however, most
reactions have been carried out with synthetically
related amines instead of food-related amino acids.
In a very recent work, Arnoldi et al. (1997) isolated a
yellow colorant with a three-ring carbocyclic structure
from a xylose/lysine Maillard system. However, al-
though a high amount of lysine was used in the model
experiment, surprisingly, no nitrogen-containing colored
compound was identified by the authors.
However, it is a well-known fact that color develop-
ment in carbohydrate/amino acid mixtures runs in
parallel with the amino acid content. For example,
Narziss and Stippler (1976) found that the color of a
1
998a). In the presence of the primary amino acid
alanine, the novel red compound (S)-4-[(E)-1-formyl-2-
2-furyl)ethenyl]-5-(2-furyl)-2-[(E)-(2-furyl)methylidene]-
,3-dihydo-R-amino-3-oxo-1H-pyrrole-1-acetic acid and
the corresponding 2-[(Z)-(2-furyl)methylidene] isomer
PYRED) were formed from furan-2-carboxaldehyde
(
2
(
upon thermal treatment (Hofmann, 1997b, 1998a). This
was the first time that chromophoric compounds com-
prising four linked rings with an amino acid moiety
incorporated were identified in a Maillard reaction
system. Additional studies with proteins and furan-2-
carboxaldehyde revealed that the ꢀ-amino function of
lysine residues acts as an anchor to form the corre-
sponding lysine derivative of PYRED, linked to a protein
(Hofmann, 1997a, 1998b).
†
Telephone +49-89-289 14181; fax +49-89-289 14183; e-
mail hofmann@dfa.leb.chemie.tu-muenchen.de
1
0.1021/jf980477i CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/05/1998

Novel Colored Pyrroles and Pyrrolinones in the Maillard Reaction
J. Agric. Food Chem., Vol. 46, No. 10, 1998 3903
3
To gain a more detailed insight into the role of the
amino acid in the network of browning reactions, the
present investigation was aimed at characterizing ad-
ditional nitrogen-containing colored compounds in Mail-
lard reactions of pentoses and primary amino acids.
to Figure 2) δ 1.73 [d, 3H, J 7,6 ) 7.08 Hz, H-C(7)], 2.31 [s,
3
3
1
H, H-C(13)], 5.02 [q, 1H, J 6,7 ) 7.08 Hz, H-C(6)], 6.31 [dd,
3
3
H, J 4,5 ) 2.66 Hz, J 4,3 ) 3.98 Hz, H-C(4)], 6.86 (s, 1H, H at
3
4
C-1), 7.08 [dd, 1H, J 3,4 ) 3.98 Hz, J 3,5 ) 1.33 Hz, H-C(3)],
3
4
7
.18 [dd, 1H, J 5,4 ) 2.66 Hz, J 5,3 ) 1.33 Hz, H-C(5)]; UV
4
-1
-1
λ
max ) 405 nm, ꢀ ) 0.7 (10 L mol cm ).
Syn th esis of N-(1′-Car boxyeth yl)-2-for m ylpyr r ole. Eth-
EXPERIMENTAL PROCEDURES
yl 2-(2′-Formylpyrrol-1-yl)propanoate. Thallium(I) ethylate (30
mmol) was added to a solution of 2-formylpyrrole (20 mmol)
in anhydrous acetonitrile (10 mL) under an atmosphere of
argon. Over a period of 60 min, ethyl 2-iodpropionate (20
mmol), freshly prepared from ethyl 2-brompropionate and
potassium iodide in boiling acetone, was added dropwise to
the ice-cooled mixture. After stirring for 12 h at room
temperature, the suspension was applied onto a slurry of silica
gel (25 × 200 mm, 30 g, silica gel 60, Merck) in methanol/
ethyl acetate (20:80, v/v). Elution with the same solvent
mixture, followed by evaporation of the solvent, afforded the
ethyl 2-(2′-formylpyrrol-1-yl)propanoate (14.3 mmol; 72% in
yield) as a colorless oil: GC/MS(EI) 122 (100), 94 (64), 195-
Ch em ica ls. The following compounds were obtained com-
mercially: D-xylose, L-alanine, furan-2-carboxaldehyde, thal-
lium(I) ethylate, 2-formylpyrrole, ethyl 2-bromopropionate,
potassium iodide, 3-hydroxypyrrolidine, 2-tert-butoxycarbony-
loximino-2-phenylacetonitrile, triethylamine, 1,4-dioxane, py-
ridinium chlorochromate, piperidine, and acetic acid (Aldrich,
Steinheim, Germany). Furan-2-carboxaldehyde was distilled
at 30 °C in a high vacuum prior to use. Solvents were of HPLC
grade (Aldrich). DMSO-d
6 3
and CD OD were obtained from
Isocom (Landshut, Germany).
The following compounds were synthesized following closely
the methods recently described in the literature given in
parentheses: 4-hydroxy-5-methyl-2H-furan-3-one (Hofmann
and Schieberle, 1998a), 3-deoxypentos-2-ulose (Hofmann and
Schieberle, 1998a), N-(1-deoxy-D-xylulos-1-yl)-L-alanine (Hof-
mann, 1998c), (S)-4[(E)-1-formyl-2-(2-furyl)ethenyl)]-5-(2-fu-
ryl)-2-[(E)-(2-furyl)methylidene]-2,3-dihydro-R-methyl-3-oxo-
(31), 149(26), 167(22), 93(15), 121(13), 104(10).
N-(1′-Carboxyethyl)-2-formylpyrrole. Ethyl 2-(2′-formylpyr-
rol-1-yl)propanoate (14.0 mmol) was refluxed for 15 min in a
mixture (30 mL; 50:50, v/v) of methanol and aqueous sodium
hydroxide (3 mol/L NaOH). After evaporation of the solvent,
the aqueous phase was extracted with methylene chloride (3
1
H-pyrrole-1-acetic acid (4a ; Hofmann, 1998a) and its 2-[(Z)-
×
10 mL) and then adjusted to pH 3 using hydrochloric acid
5 mol/L). The target compound was then extracted with
(2-furyl)methylene] isomer (4b; Hofmann, 1998a).
(
Isola t ion of 2-[(2-F u r yl)m et h ylid en e]-4-h yd r oxy-5-
methylene chloride (5 × 20 mL). Evaporation of the solvent
and recrystallization from ethyl acetate yielded the N-(1′-
carboxyethyl)-2-formylpyrrole (7.8 mmol; 56% in yield) as
m eth yl-2H-fu r a n -3-on e (1) fr om a Hea ted Xylose/Ala n in e
Solu tion . A solution of xylose (60 mmol) and alanine (6 mol)
in phosphate buffer (40 mL; 1 mmol/L, pH 6.0) was refluxed
for 2.5 h. After cooling, the mixture was extracted with diethyl
+
colorless crystals: LC/MS 168 100, [M + 1] ; IR (KBr) 3109,
-
1
1
2400-3200, 1732, 1614 cm ; UV λ
max1
) 290 nm; H NMR
ether (4 × 30 mL), dried over Na
2
SO
4
, and concentrated to 2
(360 MHz, MeOD-d ; the arbitrary numbering of the carbon
3
3
mL. The raw material was applied onto a column (25 × 350
mm) filled with a slurry of silica gel (100 g, silica gel 60, Merck,
Darmstadt, Germany) in toluene. Chromatography was per-
formed using toluene (300 mL), followed by toluene/ethyl
acetate (8:2; 300 mL) and toluene/ethyl acetate (7:3; 300 mL).
Elution with toluene/ethyl acetate (6:4; 300 mL) gave an
intense yellow fraction, which was further analyzed by RP-
atoms refers to Figure 3) δ 1.73 [d, 3H, J 7,6 ) 7.07 Hz,
3
H-C(7)], 5.72 [quart., 1H, J ) 7.07 Hz, H-C(6)], 6.30 [dd,
6,7
3
3
1H, J
4
,5
) 2.66 Hz, J
4,3
) 3.98 Hz, H-C(4)], 7.06 [dd, 1H,
3
4
3
J 3,4 ) 3.98 Hz, J ) 1.33 Hz, H-C(3)], 7.24 [dd, 1H, J
3,5
5,4
)
4
2.66 Hz, J ) 1.33 Hz, H-C(5)], 9.42 [d, 1H, J ) 0.89 Hz,
5
,3
H-C(1)]; 13C and C/DEPT NMR (360 MHz, MeOD-d ; the
13
3
arbitrary numbering of the carbon atoms refers to Figure 3) δ
1
8-HPLC using a methanol/water gradient as the mobile
17.8 [CH , C(7)], 56.8 [CH, C(6)], 110.9 [CH, C(4)], 126.9 [CH,
3
phase. A peak corresponding to 1 (12 mg; ∼0.1% in yield)
showed UV-vis spectra, LC/MS spectra, and retention time
at RP-18 identical with those of the reference compound
synthesized from 4-hydroxy-5-methyl-2H-furan-3-one and fu-
ran-2-carboxaldehyde (Hofmann, 1998d).
C(3)], 131.1 [CH, C(5)], 132.8 [CH, C(2)], 174.1 [COOH, C(8)],
180.9 [CHO, C(1)].
F or m a tion of 4-Hyd r oxy-5-m eth yl-2-[(E)-N-(1′-ca r box-
yeth yl)p yr r olyl-2-m eth ylid en e]-2H-fu r a n -3-on e fr om a n
Aqu eou s Mixtu r e of 1-(1′-Ca r boxyeth yl)-2-for m ylp yr r ole
a n d 4-Hyd r oxy-5-m eth yl-2H-fu r a n -3-on e. A mixture of
1-(1′-carboxyethyl)-2-formylpyrrole (5 mmol) and 4-hydroxy-
5-methyl-2H-furan-3-one (5 mmol) in phosphate buffer (pH 7.0;
0.2 mol/L; 10 mL) was heated at 90 °C for 60 min, with stirring.
After freeze-drying of the reaction mixture, the residue was
fractionated by flash chromatography on RP-18 material (15.0
g; Lichroprep 25-40 µm, Merck) using methanol/water (60:
40, v/v) as the mobile phase. After application of the crude
material and chromatography with the same eluent, the target
compound was isolated in the effluent >75 mL. Final puri-
fication was performed by RP-HPLC using the following
solvent gradient: starting with a mixture (30:70, v/v) of
acetonitrile and aqueous TFA (0.1% TFA in water), the
acetonitrile content was increased to 100% within 65 min.
Monitoring the effluent at 405 nm gave a peak at 11 min,
which was collected in several runs. The combined eluates
were freeze-dried, yielding the colored compound (0.3 mmol;
6% in yield). The spectroscopic data were very well in line
with those obtained from 2 isolated from the pentose/alanine
mixture.
Isola tion of (S)-4-Hyd r oxy-5-m eth yl-2-[(E)-N-(1′-ca r -
b oxyet h yl)p yr r olyl-2-m et h ylid en e]-2H -fu r a n -3-on e (2)
fr om a Hea ted Aqu eou s Solu tion of Xylose a n d Ala n in e.
A solution of xylose (60 mmol) and alanine (60 mol) in
phosphate buffer (40 mL; 1 mmol/L, pH 6.0) was heated under
reflux for 2.5 h. After cooling, the pH of the mixture was
adjusted to 3.0 and then extracted with ethyl acetate (6 × 30
2 4
mL). The organic layer was dried over Na SO , then concen-
trated to ∼3 mL, and, finally, fractionated by column chro-
matography (35 × 400 mm) on silica gel (150 g, silica gel 60,
Merck). After application of the raw material onto the column
conditioned with ethyl acetate, chromatography was performed
using ethyl acetate (300 mL), followed by ethyl acetate/
methanol (80:20; 300 mL). Elution with ethyl acetate/
methanol (50:50; 500 mL) affords a fraction that was subfrac-
tionated by preparative thin-layer chromatography on silica
gel (20 × 20 cm; 0.5 mm; Merck) using ethyl acetate/methanol
f
(50:50, v/v) as the eluent. The yellow band with R ) 0.7-0.8
was scraped off and suspended in hot methanol. After
filtration and concentration, the colorant was purified by flash
chromatography on RP-18 material (15,0 g; Lichroprep 25-
Isola tion of (2R)-4-Oxo-3,5-bis[(2-fu r yl)m eth ylid en e]-
tetr a h yd r op yr r olo[1,2-c]-5(S)-(2-fu r yl)oxa zolid in e a n d
Its 5(R)-(2-F u r yl)oxa zolid in e Dia ster eom er (3a /b) fr om
a Xylose/Ala n in e Mixt u r e Hea t ed in t h e P r esen ce of
F u r a n -2-ca r boxa ld eh yd e. A solution of xylose (1.34 mol)
and alanine (0.32 mol) dissolved in a mixture of phosphate
buffer (1800 mL; 1 mmol/L, pH 7.0) and methanol (500 mL)
was refluxed for 15 min, furan-2-carboxaldehyde (2 mol) was
4
0 µm, Merk) using methanol/water (60:40, v/v) as the mobile
phase. After application of the raw material, chromatography
with the same eluent afforded the target compound in the
effluent >75 mL. The combined eluates were freeze-dried,
yielding the colored compound as a yellow powder (5 mg,
+
1
∼
0.04% in yield); LC/MS 264, [M + 1] ; H NMR (360 MHz,
MeOD-d ; the arbitrary numbering of the carbon atoms refers
3

3904 J. Agric. Food Chem., Vol. 46, No. 10, 1998
Hofmann
Ta ble 1. Assign m en t of 1H-NMR Sign a ls (350 MHz, CD3OD) of (2R)-4-Oxo-3-[(E)-(2-fu r yl)m eth ylid en e]-5-[(Z)-
2-fu r yl)m eth ylid en e]tetr a h yd r op yr r olo[1,2-c]-5(S)-(2-fu r yl)oxa zolid in e a n d Its 5(R)-(2-F u r yl)oxa zolid in e
(
Dia ster eom er (3a /3b)
δb (ppm)
H at relevant
C atom
a
Ic
Mc
J c (Hz)
COSYd
TOCSYd
3a
3b
H-C(5a)
H-C(5b)
H-C(4)
3.84
4.31
5.25
6.15
6.46
6.51
6.56
6.62
6.66
6.68
7.02
7.36
7.38
7.56
7.81
3.83
4.30
5.25
6.14
6.46
6.51
6.55
6.61
6.66
6.67
7.01
7.36
7.38
7.55
7.80
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
t
t
8.4
8.4
8.4
H-C(4), H-C(5b)
H-C(4), H-C(5a)
H-C(5a), H-C(5b)
H-C(4), H-C(5b), H-C(16)
H-C(4), H-C(5a), H-C(16)
H-C(5a), H-C(5b), H-C(16)
m
s
dd
dd
s
d
dd
d
d
s
d
d
H-C(6)
H-C(14)
H-C(9)
H-C(11)
H-C(13)
H-C(19)
H-C(8)
H-C(18)
H-C(16)
H-C(15)
H-C(10)
H-C(20)
3.5, 1.4
3.5, 1.4
H-C(13), H-C(15)
H-C(8), H-C(10)
H-C(13), H-C(15)
H-C(8), H-C(10)
3.5
3.5, 1.4
3.5
H-C(14)
H-C(14), H-C(15)
H-C(18), H-C(20)
H-C(9), H-C(10)
H-C(19), H-C(20)
H-C(18), H-C(20)
H-C(9)
3.5
H-C(19)
1.4
1.4
1.4
H-C(14)
H-C(9)
H-C(19)
H-C(13), H-C(14)
H-C(8), H-C(9)
H-C(18), H-C(19)
d
a
Numbering of carbon atoms refers to structures 3a and 3b in Figure 5. The 1H chemical shifts are given in relation to CD3OD.
b
c
Determined from 1D spectrum. Observed homonuclear 1H, H connectivities by TOCSY and DQF-COSY.
d
1
Ta ble 2. Assign m en t of 13C-NMR Sign a ls (360 MHz,
CD3OD) of th e Ma jor Dia ster eom er of 4-Oxo-3-[(E)-
added, and heating was continued for another 80 min. After
cooling to room temperature, the solution was extracted with
diethyl ether (10 × 300 mL), and the organic layer was dried
(
2-fu r yl)m eth ylid en e]-5-[(Z)-(2-fu r yl)m eth ylid en e]-
tetr a h yd r op yr r olo[1,2-c]-5-(2-fu r yl)oxa zolid in e (3a )
over Na
2
SO
4
and concentrated to ∼150 mL at 25 °C in vacuo
heteronuclear
(100 mbar). To remove unreacted furan-2-carboxaldehyde, the
1H,13C multiple-quantum
extract was then distilled in high vacuo (0.04 mbar) at 35 °C.
The intensely colored residue was dissolved in a 1:1 mixture
H at
d
coherence
b
relevant
δ
C atoma (ppm) DEPTc via 1J (C,H)
via 2,3J (C,H)
(10 mL) of toluene and ethyl acetate, and aliquots were then
fractionated by column chromatography (35 × 450 mm) on
silica gel (200 g, silica gel 60, Merck). After application of an
aliquot of the crude material (5 mL) onto the column condi-
tioned with toluene, chromatography was performed using
toluene (300 mL) and toluene/ethyl acetate (9:1, v/v; 300 mL).
The fraction, obtained by using toluene/ethyl acetate (8:2, v/v;
C(4)
C(5)
C(6)
62.2 CH
70.1 CH
90.1 CH
99.8 CH
108.4 CH
109.6 CH
111.9 CH
112.7 CH
112.8 CH
119.3 CH
119.7 CH
H-C(4)
H-C(5)
H-C(6)
H-C(11)
H-C(19)
H-C(9)
H-C(14)
H-C(8)
H-C(13)
H-C(18)
H-C(16)
H-C(5), H-C(6), H-C(16)
H-C(4), H-C(6)
2
H-C(4), H-C(5)
C(11)
C(19)
C(9)
C(14)
C(8)
C(13)
C(18)
C(16)
C(3)
H-C(13)
H-C(18), H-C(20)
H-C(8), H-C(10)
H-C(13), H-C(15)
H-C(6), H-C(9), H-C(10)
H-C(11), H-C(14), H-C(15)
H-C(16), H-C(19), H-C(20)
H-C(4), H-C(18)
3
00 mL) as the mobile phase, was further subfractionated by
a further column chromatography (20 × 400 mm) on silica gel
50 g, silica gel 60, Merck). The fraction was concentrated and
(
applied onto water-cooled silica gel, which was conditioned
with n-pentane. After flushing with pentane (200 mL), fol-
lowed by pentane/diethyl ether (9:1, v/v; 200 mL), elution with
pentane/diethyl ether (8:2, v/v; 200 mL) affored a fraction
containing a deep red compound, which was freed from solvent
in vacuo and dissolved in methanol (1 mL). The colored
compound was further purified by flash chromatography using
an RP-18 stationary phase (15.0 g; Lichroprep 25-40 µm,
Merck). The solution was placed onto the column (20 × 1.6
cm), which was conditioned with methanol/water (8:2, v/v).
Flushing with the same solvent mixture (120 mL) afforded a
fraction containing the red colorant. After the solvent had
been removed, the aqueous phase was extracted with ethyl
131.6
141.0
C
C
H-C(4), H-C(5), H-C(16)
H-C(6), H-C(11)
H-C(8), H-C(9)
C(1)
C(10)
C(15)
C(20)
C(12)
C(17)
C(7)
142.5 CH
143.5 CH
147.1 CH
H-C(10)
H-C(15)
H-C(20)
H-C(13), H-C(14)
H-C(18), H-C(19)
H-C(14), H-C(15)
H-C(19), H-C(20)
H-C(9), H-C(10)
150.2
150.6
151.4
189.9
C
C
C
C
C(2)
H-C(4), H-C(11), H-C(16)
a
Numbering of carbon atoms refers to structure 3a in Figure
. b The C chemical shifts are given in relation to CD3OD.
13
5
c
d
1
DEPT-135 spectroscopy. Assignments based on HMQC ( J ) and
HMBC (2 J ) experiments.
,3
acetate (3 × 20 mL) and, after drying over Na
2 4
SO , the red
colorant was isolated in 98% purity by preparative thin-layer
chromatography on silica gel (20 × 20 cm; 0.5 mm; Merck)
using n-pentane/toluene/ethyl acetate (70:15:15, v/v/v) as the
mmol, 79% in yield) was characterized by HRGC/MS: MS-
(EI) 57 (100), 56 (48), 114 (33), 87 (28), 132 (22), 131 (17), 86-
+
(6), 130 (5), 187 (4); MS(CI, isobutane) 188 (100, [M + 1] ).
eluent. A red band at R ) 0.62 was scraped off and dissolved
f
in methanol (20 mL). After filtration, the solvent was evapo-
N-(tert-Butoxycarbonyl)pyrrolidine-3-one. A mixture of
rated to dryness, affording 1 as an intense red solid (1.3 mmol;
N-(tert-butoxycarbonyl)-3-hydroxypyrrolidine (10 mmol) and
pyridinium chlorochromate (15 mmol) in dichloromethane (25
mL) was stirred for 4 h at room temperature. After addition
of diethyl ether (40 mL), the organic phase was separated and
+
∼
0.1% in yield): LC/MS m/z 350 (100, [M + 1] ); UV λmax1
)
4
-1
-1
4
56 nm (ꢀ ) 1.0 × 10 L mol cm ), λmax2 ) 349 nm (ꢀ ) 1.2
4
-1
-1
1
13
×
10 L mol cm ); H and C-NMR data are listed in Tables
and 2.
1
2 4
dried over Na SO and the solvent was distilled off in vacuo.
The residue was purified by flash chromatography as described
recently (Hofmann and Schieberle, 1998b), affording N-(tert-
butoxycarbonyl)pyrrolidine-3-one (3.5 mmol, 35% in yield) as
a colorless oil: MS(EI) 57 (100), 56 (74), 85 (39), 112 (21), 130
Syn th esis of th e 2,4-Bis[(2-fu r yl)m eth ylid en e]p yr r o-
lid in e-3-on e Ch r om op h or e. N-(tert-Butoxycarbonyl)-3-hy-
droxypyrrolidine. 3-Hydroxypyrrolidine (15 mmol), 2-tert-
butoxycarbonyloximino-2-phenylacetonitrile (16.5 mmol), and
triethylamine (22.5 mmol) were stirred for 2 h at room
temperature in 1,4-dioxane/water (20 mL; 1:1, v/v). The
organic solvent was then distilled off in vacuo, and, after
addition of water (50 mL), the oily phase was fractionated by
flash chromatography on a diol phase as described recently
(
19), 105 (13), 103 (11), 129 (6), 185 (4); MS(CI, isobutane) 186
+
(100, [M + 1] ).
N-(tert-Butoxycarbonyl)-2,4-bis[(2-furyl)methylidene]pyrro-
lidine-3-one. A mixture of N-(tert-butoxycarbonyl)pyrrolidine-
3-one (2 mmol), furan-2-carboxaldehyde (5 mmol), piperidine
(100 µL), and acetic acid (100 µL) in ethanol/water (1:1, v/v;
(
Hofmann and Schieberle, 1998b). The target compound (11.9

Novel Colored Pyrroles and Pyrrolinones in the Maillard Reaction
5 mL) was heated for 1 h at 60 °C. The organic solvent was
J. Agric. Food Chem., Vol. 46, No. 10, 1998 3905
2
then evaporated in vacuo (30 mbar) at 35 °C, and the reaction
mixture was adjusted to pH 6.0 with aqueous hydrochloric acid
(
0.1 mol/L). After extraction with ethyl acetate (4 × 10 mL)
and drying over Na SO , the organic layer was concentrated
2
4
in vacuo to ∼1 mL and the target compound was isolated by
semipreparative thin-layer chromatography using silica gel (20
× 20 cm; 0.5 mm; Merck) as the stationary phase and a
mixture of pentane/toluene/ethyl acetate (75:20:5, v/v/v) as the
mobile phase. The band with R ) 0.22 was scraped off and
F igu r e 1. Structure of 2-[(2-furyl)methylidene]-4-hydroxy-5-
methyl-2H-furan-3-one (1).
f
suspended in methanol. Filtration and evaporation of the
solvent gives N-(tert-butoxycarbonyl)-2,4-bis[(2-furyl)methyl-
ene]-3-oxopyrrolidine (0.6 mmol, 30% in yield) as deep red
High -P er for m a n ce Liqu id Ch r om a togr a p h y (HP LC).
The HPLC apparatus (Kontron, Eching, Germany) consisted
of two pumps (type 422), a gradient mixer (M 800), a Rheodyne
injector (100 µL loop), and a diode array detector (DAD, type
440) monitoring the effluent in a wavelength range between
220 and 500 nm. Separations were performed on a stainless
steel column packed with RP-18 (ODS-Hypersil, 5 µm, 10 nm,
Shandon, Frankfurt, Germany) in either an analytical (4.6 ×
250 mm, flow rate ) 0.8 mL/min) or a preparative scale (10 ×
250 mm, flow rate ) 1.8 mL/min).
Liqu id Ch r om a togr a p h y/Ma ss Sp ectr om etr y (LC/MS).
An analytical HPLC column (Nucleosil 100-5C18, Macherey
and Nagel, D u¨ rren, Germany) was coupled to an LCQ-MS
(Finnigan MAT GmbH, Bremen, Germany) using electrospray
ionization (ESI). After injection of the sample (2.0 µL),
analysis was performed using a gradient starting with a 10:
90 (v/v) mixture of acetonitrile and water and increasing the
acetonitrile content to 100% within 15 min.
+
crystals: LC/MS 342 (100, [M + 1] ), 286 (94, [M + 1 -
+
+
+
isobutene] ), 242 (16, [M + 1 - BOC] ), 364 (5, [M + Na] );
1
H NMR (360 MHz; DMSO-d ; DQF-COSY; the arbitrary
numbering of the carbon atoms refers to Figure 6) 1.32 [s, 9H,
6
3
H-C(17-19)], 4.80 [s, 2H, H-C(4)], 6.64 [dd, 1H, J 13,12 ) 3.54
3
Hz, J 13,14 ) 1.77 Hz, H-C(13)], 6.67 [s, 1H, H-C(10)], 6.74
3
3
[
s, 1H, H-C(5)], 6.77 [dd, 1H, J 8,7 ) 3.54 Hz, J 8,9 ) 1.77 Hz,
3
H-C(8)], 7.12 [d, 1H, J 7,8 ) 3.54 Hz, H-C(7)]. 7.33 [d, 1H,
3
3
J
12,13 ) 3.54 Hz, H-C(12)], 7.86 [d, 1H, J 9,8 ) 1.77 Hz,
3 13
H-C(9)], 8.08 [d, 1H, J 14,13 ) 1.77 Hz, H-C(14)]; C NMR
360 MHz; DMSO-d ; DEPT, HMQC, HMBC; the arbitrary
numbering of the carbon atoms refers to Figure 6) δ 27.2 [CH
C(17-19)], 49.7 [CH , C(4)], 80.9 [C, C(16)], 104.5 [CH, C(5)],
12.4 [CH, C(8)], 113.4 [CH, C(13)], 115.9 [CH, C(10)], 118.9
CH, C(12)], 119.2 [CH, C(7)], 126.6 [C, C(3)], 131.3 [C, C(1)],
44.6 [CH, C(9)], 147.6 [CH, C(14)], 150.2 [C, C(6)], 150.7 [C,
C(11)], 151.4 [C, C(15)], 187.7 [C, C(2)]; UV λmax1 ) 461 nm (ꢀ
(
6
3
,
2
1
[
1
UV-Vis Sp ectr ocop y. UV-vis spectra were obtained
using a U-2000 spectrometer (Colora Messtechnik GmbH,
Lorch, Germany).
4
-1
-1
4
)
1.0 × 10 L mol cm ), λmax2 ) 377 nm (ꢀ ) 1.1 × 10 L
-1 -1
mol cm ).
Nu clea r Ma gn etic Reson a n ce Sp ectr oscop y (NMR).
1H, C, DEPT-135, DQF-COSY, TOCSY, HMQC, and HMBC
experiments were performed on a Bruker AC-200 and a Bruker
AM-360 spectrometer (Bruker, Rheinstetten, Germany) using
the acquisition parameters described recently (Hofmann,
1997b). Tetramethylsilane (TMS) was used as the internal
standard.
13
Iden tification an d Qu an tification of (S)-4[(E)-1-For m yl-
2
2
-(2-fu r yl)eth en yl)]-5-(2-fu r yl)-2-[(E)-(2-fu r yl)m eth yliden e]-
,3-d ih yd r o-r-m eth yl-3-oxo-1H-p yr r ole-1-a cetic Acid (4a )
a n d Its 2-[(Z)-(2-F u r yl)m eth ylen e] Isom er (4b) in Hea ted
Aqu eou s Solu tion s of Xylose/Ala n in e, N-(1-Deoxy-D-xy-
lu los-1-yl)-L-a la n in e, a n d 3-Deoxyp en tosu lose/Ala n in e,
Resp ectively, in th e P r esen ce of F u r a n -2-ca r boxa ld e-
h yd e. Solutions of either alanine (30 mmol) and xylose (30
mmol), N-(1-deoxy-D-xylulos-1-yl)-L-alanine (30 mmol) or ala-
nine (30 mmol) and 3-deoxypentos-2-ulose (30 mmol), each in
phosphate buffer (90 mL; 1 mmol/L, pH 7.0), were refluxed
for 15 min. After addition of furan-2-carboxaldehyde (90 mol),
heating was continued for another 80 min. The mixture was
cooled to room temperature and extracted with ethyl acetate
RESULTS AND DISCUSSION
Heating of aqueous solutions of xylose and L-alanine
in ratios of 10:1 or 1:1 led to intense browning of both
reaction mixtures; however, the colorization occurred
much more rapidly in the latter mixture. This is well
in line with the catalytic effect of the amino acid on the
carbohydrate degradation. In the solvent extract of the
(
5 × 30 mL), and the organic layer was dried over Na
2 4
SO and
concentrated at 25 °C under vacuum (100 mbar) to ∼80 mL.
To remove volatile compounds, the extract was then distilled
in high vacuo (0.04 mbar) at 35 °C. The residue was dissolved
in ethyl acetate and fractionated by column chromatography
1
0:1 mixture, 2-[(2-furyl)methylidene]-4-hydroxy-5-
methyl-2H-furan-3-one (1 in Figure 1) could be identi-
fied as one of the main colored reaction products by
comparing the data of the UV-vis and the LC/MS
spectra as well the retention time (RP-18-HPLC) with
those obtained for the synthesized reference compound
(Hofmann, 1998d). This result is well in line with data
reported earlier by Severin and Kr o¨ nig (1972) as well
as Nursten and O’Reilly (1986), who isolated compound
(35 × 450 mm) on silica gel (200 g, silica gel 60, Merck). After
application of the crude material onto the column conditioned
with diethyl ether, chromatography was performed using
diethyl ether (500 mL), followed by ethyl acetate (500 mL).
Elution with ethyl acetate/methanol (8:2, v/v; 300 mL) affords
a colored fraction, which was freed from solvent in vacuo. The
residue was taken up in methanol (2 mL) and was then
analyzed by analytical HPLC. Starting with a mixture of
methanol (20%) and water (80%), the methanol content was
increased to 100% within 45 min. Two peaks eluting after 14.0
and 16.5 min showed UV-vis spectra, MS spectra, and
retention time at RP-18 material identical with those of the
reference compounds of 4a and 4b (Hofmann, 1997b, 1998a).
Using the same HPLC conditions, the colorants 4a /4b were
quantified by comparing the peak areas of 4a and 4b moni-
tored at 414 nm with those of the pure reference compounds
used as external standards.
1
from xylose heated in the presence of isopropyl
ammoniumacetate and glycine, respectively.
Heating of xylose and L-alanine in a 1:1 mixture,
however, gave, besides 1, another colorant, which was
monitored after separation of the solvent extract by RP-
HPLC using either a DAD operating at wavelengths
between 220 and 500 nm or an LC/MS. The intensely
yellow product was characterized by DAD to have an
absorption maximum at 405 nm.
After chromatographic isolation, the determination of
Ga s Ch r om a togr a p h y/Ma ss Sp ectr oscop y (GC/MS).
HRGC was performed with a type 5160 gas chromatograph
1
its chemical structure was performed by H-NMR, LC/
MS, and UV-vis spectroscopy. LC/MS measurements
(Fisons Instruments, Mainz, Germany) using an SE-54 capil-
+
gave an intense [M + 1] ion at m/z 264 (100%), fitting
lary (30 m × 0.32 mm, 0.25 µm; J &W Scientific, Fisons
well with the proposed structure for 2 (Figure 2). The
Instruments) coupled with an MD-800 mass spectrometer
1
(
Fisons Instruments); sample application (0.5 µL) was done
H-NMR spectrum measured in CD3OD showed seven
by on-column injection at 40 °C.
resonance signals. The singlets at 2.31 and 6.86 ppm

3906 J. Agric. Food Chem., Vol. 46, No. 10, 1998
Hofmann
ring system and an alanine moiety in 2. Due to the
chemical shift of the R-hydrogen of the alanine moiety,
the amino acid must be incorporated in the pyrrole ring.
Due to the low amounts that could be isolated from the
Maillard mixture, an unequivocal assignment of the 13C-
NMR data could, however, not be achieved.
In analogy to the formation of 1 from furan-2-
carboxaldehyde and 4-hydroxy-5-methyl-2H-furan-3-one
(
Ledl and Severin, 1978; Hofmann, 1998d), 2 might be
F igu r e 2. Structure of (S)-4-hydroxy-5-methyl-2-[N-(1′-car-
boxyethyl)pyrrolyl-2-methylidene]-2H-furan-3-one (2).
assumed to be formed from 4-hydroxy-5-methyl-2H-
furan-3-one and N-(1′-carboxyethyl)-2-formylpyrrole.
These intermediates were, therefore, used for the syn-
thesis as displayed in Figure 3. 2-Formylpyrrole was
N-alkylated with ethyl 2-iodopropionate, yielding the
ethyl 2-(2′-formylpyrrol-1-yl)propanoate (I), which after
hydrolysis of the carboxylic ester gives rise to N-(1′-
carboxyethyl)-2-formylpyrrole (II). Heating an aqueous
solution of the synthetic aldehyde with 4-hydroxy-5-
methyl-2H-furan-3-one gave a main colored reaction
1
product, which showed identical H-NMR, UV-vis, and
LC/MS spectra as well as the same retention times at
RP-18 as compound 2 isolated from the xylose/alanine
mixture.
In summary, the spectroscopic and synthetic data
obtained are consistent with the proposed structure of
F igu r e 3. Synthetic sequence for the preparation of N-(1′-
carboxyethyl)-2-formylpyrrole.
2
as (S)-4-hydroxy-5-methyl-2-[(E)-N-(1′-carboxyethyl)-
pyrrolyl-2-methylidene]-2H-furan-3-one (Figure 4). The
predominance of the (E) configuration of the meth-
ylidene double bond was evidenced by molecular me-
chanics calculations using an MM3 force field. To our
knowledge, this yellow compound 2, showing an extinc-
were in the range of the chemical shifts of the methyl
group and the methylidene proton, respectively, found
for the colored 2H-furan-3-one 1 (Hofmann, 1998c).
Double-quantum-filtered homonuclear δ,δ correlation
spectroscopy (DQF-COSY) revealed two strongly coupled
4
-1
-1
tion coefficient of 0.7 × 10 L mol cm (in water, pH
1
H spin systems, confirming the presence of a pyrrole
7.0), has previously not been described in the literature.
F igu r e 4. Formation pathway leading to (S)-4-hydroxy-5-methyl-2-[(E)-N-(1′-carboxyethyl)-pyrrolyl-2-methylidene]-2H-furan-
-one (2) from pentoses and L-alanine.
3

Novel Colored Pyrroles and Pyrrolinones in the Maillard Reaction
J. Agric. Food Chem., Vol. 46, No. 10, 1998 3907
A homologuous colorant derived from methylamine as
well as from glycine was reported earlier by Ledl and
Severin (1978), indicating that the formation of this
nitrogen-containing type of chromophore runs indepen-
dently from the amino moiety present.
On the basis of the data obtained, the reaction
pathway displayed in Figure 4 was proposed for the
formation of colorant 2. Dehydration of N-(1-deoxypen-
tulos-1-yl)-L-alanine (I), the Amadori rearrangement
product formed from pentoses and L-alanine, at the
3
-position gives the 3-deoxypentosone (IIa ) after hy-
Figu r e 5. Structure of (2R)-4-oxo-3,5-bis[(2-furyl)methylidene]-
tetrahydropyrrolo[1,2-c]-5(S)-(2-furyl)oxazolidine and its 5(R)-
drolysis. Further dehydration of IIa and cyclization
with one molecule of alanine leads to N-(1′-carboxy-
ethyl)-2-formylpyrrole (IIb). Deamination at the 1-posi-
tion of the Amadori rearrangement product (I) yields
(2-furyl)oxazolidine isomer (3a /3b).
1
-deoxypentosulose (IIIa ), which, upon cyclization and
dehydration, leads to the formation of 4-hydroxy-5-
methyl-2H-furan-3-one (IIIb). As confirmed by syn-
thetic experiments, the condensation reaction between
the methylene-active intermediate IIIb and the alde-
hyde IIb then results in the colored (S)-4-hydroxy-5-
methyl-2-[(E)-N-(1′-carboxyethyl)-pyrrolyl-2-methylidene]-
2
H-furan-3-one (2).
The formation of 2 from N-(1′-carboxyethyl)-2-formyl-
pyrrole and 4-hydroxy-5-methyl-2H-furan-3-one con-
firms the hypothesis of Ledl and Severin (1978, 1982)
that the condensation reaction between methylene-
active compounds and carbonyl compounds is an im-
portant general reaction leading to color development
during the Maillard reaction. The amino acid assisted
conversion of carbohydrates, however, results in a
tremendous variety of reactive carbonyls such as furan-
2
-carboxaldehyde, N-(1′-carboxyethyl)-2-formylpyrrole,
or 1- and 3-deoxyosones, as well as methylene-active
compounds such as 4-hydroxy-5-methyl-2H-furan-3-one.
This large variety of reactants leads, therefore, to the
multiplicity and the low yields of the colored Maillard
reaction products, making the identification of such
browning products a big challenge. To propose pos-
sibilities on how to clarify general reaction types
involved in Maillard-type browning, Hofmann (1998d)
recently reacted aqueous solutions of reducing carbo-
hydrates and amino acids in the presence of a surplus
amount of one carbonyl compound. Because furan-2-
carboxaldehyde is known as one of the main dehydration
products from pentoses (Ledl and Schleicher, 1990;
Hofmann and Schieberle, 1998a), it was chosen for these
experiments. Using this strategy, all methylene-active
color precursors are forced to react with the same
aldehyde, offering the possibility to characterize the key
types of chromophores.
For the identification of further nitrogen-containing
compounds from pentoses and amino acids, the model
mixtures were, therefore, reacted in the presence of
furan-2-carboxaldehyde. When xylose and L-alanine
were reacted in aqueous solution in the presence of
furan-2-carboxaldehyde, the color of the reaction mix-
ture rapidly turned into deep red-brown. An intensely
red reaction product was detected by HPLC analyses,
and diode array detection gave absorption maxima at
F igu r e 6. Synthesis of N-protected 2,4-bis[(2-furyl)meth-
ylidene]pyrrolidine-3-one.
F igu r e 7. UV-vis spectrum of (s) colored Maillard product
3
a /3b and (- - -) synthetic 2,4-bis[(2-furyl)methylidene]pyrro-
lidine-3-one.
LC/MS measurements showed an intense [M + 1]+
ion at m/z 350 fitting well with the structure of 3a /3b.
2
LC/MS revealed a loss of 18 and 96, yielding m/z 332
and 254, respectively, most likely corresponding to the
elimination of one molecule of water and furan-2-
carboxaldehyde. These data are well in line with the
proposed cyclic hemi aminal structure for 3a /3b.
1
The H-NMR spectrum measured in CD OD showed
3
two sets of 15 resonance signals each in a ratio of about
2:1, corroborating the existence of two diastereomeric
forms. Further NMR data, fitting well with the struc-
tures 3a and 3b, are given in Table 1. In the following,
the structure determination of the predominating di-
astereomer 3a is explained in more detail. A total of
three furan rings, each substituted at the 2-position,
were deduced from the characteristic coupling pattern
3
79 and 456 nm. After isolation of this colorant, the
determination of its chemical structure was performed
by several one- and two-dimensional NMR techniques
and, in addition, by LC/MS and UV-vis spectroscopy.
The spectroscopic data were consistent with structure
3
3
, given in Figure 5, existing in the two diastereomers
a and 3b.

3908 J. Agric. Food Chem., Vol. 46, No. 10, 1998
Hofmann
F igu r e 8. Reaction route leading to 4-oxo-3,5-bis[(2-furyl)methylidene]tetrahydropyrrolo[1,2-c]-5-(2-furyl)oxazolidine (3a /3b) from
pentoses and alanine.
of the hydrogens H-C(8)/H-C(9)/H-C(10), H-C(13)/
H-C(14)/H-C(15), and H-C(18)/H-C(19)/H-C(20). This
was further confirmed by DQF-COSY as well as total
correlated spectroscopy (TOCSY), indicating the ex-
pected strongly coupled 1H spin system in the furan
rings. In addition, these homonuclear δ/δ correlation
techniques revealed a coupling of 8.4 Hz between the
triplets resonating at 3.84 and 4.31 ppm and, in addi-
tion, a coupling of 8.4 Hz between these triplets and a
signal resonating at 5.25 ppm. These signals were
assigned as the geminal hydrogen atoms Ha-C(5) and
Hb-C(5) and the vicinal methine proton H-C(4). Using
molecular mechanics calculations in combination with
the Karplus equation revealed dihedral angels of about
formed from benzaldehyde and 5-(hydroxymethyl)pyr-
rolidine-2-one (Thottahil et al., 1986), thus confirming
the proposed structure of 3a .
A comparison of the 13C-NMR spectrum of the pre-
dominant isomer 3a , in which 20 signals appeared, with
the results of the DEPT-135 experiment revealed 12
signals corresponding to 6 quarternary carbon atoms
(Table 2). Unequivocal assignment of these quarternary
carbon atoms could be successfully achieved by means
2
3
of HMBC spectroscopy optimized for J (C,H) and J (C,H)
coupling constants (Table 2). Heteronuclear correla-
tions between the quarternary carbon atom resonating
at 189.9 ppm with both of the methylidene protons
H-C(11) and H-C(16) as well as with the methine
proton H-C(4) led to its unequivocal assignment as the
oxo group C(2).
1
1° and 163° between H-C(4) and Ha-C(5) as well as
between H-C(4) and Hb-C(5), fitting well with the
measured coupling constant of 8.4 Hz and with the (R)
configuration of this chiral center.
Further correlations were observed between H-C(11)
and C(1) as well as between H-C(6) and C(1), indicating
that the (2-furyl)methylidene group C(11-15) is directly
linked at C(1) and not at C(3) of the heterocyclic core.
The fact that the (2-furyl)methylidene group is part of
an exocyclic enamine structure is also well in line with
the high-field shift of H-C(11) in comparison to the
downfield shift of H-C(16). The CH coupling of the
quarternary carbon atom resonating at 131.6 ppm with
the protons H-C(4), H-C(5a), and H-C(5b) and the
olefinic hydrogen H-C(16) enables a more detailed
insight into the structure of 3a , demonstrating that the
second (2-furyl)methylidene group is connected with
C(3) of the heterocyclic core. The oxazolidine ring in
3a was, in addition, confirmed by heteronuclear cor-
relations between the amino acetal proton H-C(6) and
the hydrogens H-C(4) and H-C(5).
The chemical shifts of the signals at 6.56 and 7.36
ppm were in the range expected for olefinic hydrogen
atoms, but no coupling with other hydrogens could be
observed. Heteronuclear multiple-bond coherence ex-
2
3
periments (HMBC) optimized for J (C,H) and J (C,H)
coupling constants (Table 2), however, revealed a cor-
relation between these hydrogens assigned as H-C(11)
and H-C(16) with the furan carbon atoms C(13) and
C(18), respectively. These data demonstrate that two
furan rings are directly linked at the 2-position to a
methylidene group as proposed for structure 3a . Due
3
to the J (C,H) coupling between the singlet at 6.15 ppm
and C(8) of a furan ring, this hydrogen was evidenced
as the proton H-C(6) of an amino acetal of furan-2-
1
13
carboxaldehyde. The H and C chemical shifts of C(6)
are well in line with those found for cyclic amino acetals
In summary, the obtained spectroscopic data are

Novel Colored Pyrroles and Pyrrolinones in the Maillard Reaction
J. Agric. Food Chem., Vol. 46, No. 10, 1998 3909
then yielded the N-BOC-protected 2,4-bis[(2-furyl)-
methylidene]pyrrolidine-3-one (IV).
As outlined in Figure 7, comparison of the UV-vis
spectra of 3a /3b with the synthesized chromophore (IV
in Figure 6) showed nearly identical absorption maxima,
thereby confirming the 2,4-bis[(2-furyl)methylidene]-
pyrrolidine-3-one substructure as the chromophore re-
sponsible for the orange-red color of 3a /3b.
It is obvious from the structure of 3a /3b that, besides
three molecules of furan-2-carboxaldehyde, the C-5
skeleton of the pentose is incorporated. The reaction
pathway outlined in Figure 8 can, therefore, be proposed
for the formation of 3a /b. Dehydration of N-(1-deoxy-
pentulos-1-yl)-L-alanine (I), the Amadori rearrangement
product formed from pentoses and alanine, at the
3-position gives the 3-deoxypentos-2-ulose (II) as a
primary intermediate. After water elimination at the
4-position, this dicarbonyl compound may be reductively
aminated at the aldehyde function by an oxidative
decarboxylation of the amino acid. This reductive
amination is well in line with the finding that 3a /3b is
formed independently from the amino acid moiety; for
example, the colorant is formed also in the presence of
valine or leucine (data not shown). Subsequent cycliza-
tion of the resulting 1-amino-5-hydroxy-3-pentene-2-one
F igu r e 9. Structure of (S)-4-[(E)-1-formyl-2-(2-furyl)ethenyl]-
5
-(2-furyl)-2-[(E)-(2-furyl)methylidene]-2,3-dihydo-R-amino-3-
oxo-1H-pyrrole-1-acetic acid (4b) and the corresponding 2-[(Z)-
2-furyl)methylidene] isomer (4b).
(
consistent with the proposed structure of 3a /3b as (2R)-
-oxo-3,5-bis[(2-furyl)methylidene]tetrahydropyrrolo-
1,2-c]-5(S)-(2-furyl)oxazolidine and its 5(R)-(2-furyl)-
4
[
oxazolidine diastereomer (Figure 5). To the author’s
knowledge, the intense red colored compound 3a /3b,
4
-1
showing an extinction coefficient of 1.0 × 10 L mol
-
1
cm at 456 nm (in water, pH 7.0), has previously not
been described in the literature.
It is obvious from the structure 3a /3b that the 2,4-
bis[(2-furyl)methylidene]pyrrolidine-3-one part is re-
sponsible for the red color of this compound, whereas
the furyloxazolidine moiety should not take part in the
chromophoric system. To confirm the proposed chro-
mophoric substructure in 3a /3b, the 2,4-bis[(2-furyl)-
methylidene]pyrrolidine-3-one substructure was syn-
thesized following the reaction sequence outlined in
Figure 6. N-(tert-Butoxycarbonyl)-3-hydroxypyrrolidine
(IV) by an intramolecular Michael addition then gives
rise to 5-hydroxymethyl-3-oxopyrrolidine (V). Conden-
sation with two molecules of furan-2-carboxaldehyde
forms the colored compound VI, which can be stabilized
by reaction with a further molecule of furan-2-carbox-
aldehyde to give the 4-oxo-3,5-bis[(2-furyl)methylidene]-
tetrahydropyrrolo[1,2-c]-5-(2-furyl)oxazolidine (3a /3b).
(
(
II), prepared by N-protection of 3-hydroxypyrrolidine
I), was oxidized under mild conditions yielding the
N-(tert-butoxycarbonyl)-3-hydroxypyrrolidine (III). Con-
densation with two molecules of furan-2-carboxaldehyde
In an aqueous xylose/L-alanine solution, thermally
treated in the presence of furan-2-carboxaldehyde, we
F igu r e 10. Formation pathway leading to (S)-4-[(E)-1-formyl-2-(2-furyl)ethenyl]-5-(2-furyl)-2-[(E)-(2-furyl)methylidene]-2,3-dihydro-
R-amino-3-oxo-1H-pyrrole-6-acetic acid (4a ) and its 2-[(Z)-(2-furyl)methylidene] isomer (4b) from N-(1-deoxy-D-xylulos-1-yl)-L-
alanine.

3910 J. Agric. Food Chem., Vol. 46, No. 10, 1998
Hofmann
Ta ble 3. F or m a tion of P YRED (4a /4b) fr om Sever a l C-5
P r ecu r sor s
CONCLUSION
a
The identification of novel nitrogen-containing colored
compounds formed from pentoses and alanine clearly
demonstrates that either the complete amino acid
moiety can be incorporated into colored structures or
only the nitrogen atom can be transferred into colorants
via the Strecker reaction with deoxyosones. Such
studies provide useful information to extend the knowl-
edge on chromophores generated by Maillard-type reac-
tions during food processing and will help to construct
a route map of reactions leading to color development
in heated foodstuffs.
amount of PYRED
precursors reacted in the presence
of furan-2-carboxaldehyde
xylose/alanine
N-(1-deoxy-D-xylulos-1-yl)-L-alanine
mg
%
1.2
1.8
6.8
0.01
0.02
0.06
3
-deoxypentos-2-ulose/alanine
a
A solution of the precursors (each 30 mmol) in phosphate
buffer (90 mL; 1 mmol/L, pH 7.0) was refluxed for 20 min. After
addition of furan-2-carboxaldehyde (90 mmol), heating was con-
tinued for another 90 min.
additionally identified the red colorants (S)-4[(E)-1-
formyl-2-(2-furyl)ethenyl)]-5-(2-furyl)-2-[(E)-(2-furyl)m-
ethylidene]-2,3-dihydro-R-methyl-3-oxo-1H-pyrrole-1-
acetic acid and its 2-[(Z)-(2-furyl)methylene] isomer
ACKNOWLEDGMENT
(
PYRED; 4a /4b in Figure 9) by comparison of the UV-
I thank the Institute of Organic Chemistry, Technical
University of Munich, for the NMR measurements and
the Institute of Food Chemistry, Technical University
of Munich, for the LC/MS analyses. I am grateful to Mr.
J . Stein for his excellent technical assistance.
vis and LC/MS spectra as well as the HPLC retention
times with those obtained for the reference compounds
(Hofmann, 1997b, 1998a).
In a recent investigation (Hofmann, 1997b, 1998a),
PYRED was found among the main colored reaction
products formed from the reaction of alanine with furan-
LITERATURE CITED
2
-carboxaldehyde. To study whether PYRED was formed
in the xylose/alanine/furan-2-carboxaldehyde mixture
exclusively from the reaction between furan-2-carbox-
aldehyde and alanine or, also, from other pentose-
derived intermediates, we heated solutions of (i) xylose/
alanine, (ii) the Amadori rearrangement product N-(1-
deoxy-D-xylulos-1-yl)-L-alanine, or (iii) 3-deoxypentos-
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8
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13
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1
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4
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