Acetylformoin - A Chemical Switch in the Formation of Colored Maillard Reaction Products from Hexoses and Primary and Secondary Amino Acids

Article abstract of DOI:10.1021/jf980512l

Thermal treatment of a methanolic solution of N-(1-deoxy-D-fructos-1-yl)-L-proline and furan-2-carboxaldehyde led to the rapid formation of colored compounds, among which 4-hydroxy-2-methoxy-2-methyl-5-[(E)-(2-furyl)methylidene]methyl-2H-furan-3-one (1) could be identified as one of the main colored compounds by NMR, LC/MS, and UV-vis spectroscopy. Studies on its formation revealed that condensation of the hexose dehydration product acetylformoin (2a/2b) with furan-2-carboxaldehyde formed the labile 2,4-hydroxy-2-methyl-5-[(E)-(2-furyl)methylidene]methyl-2H-furan-3-one (3a/3b), which then reacts with methanol to the more stable colorant 1. Reacting furan-2-carboxaldehyde with N-(1-deoxy-D-fructos-1-yl)-L-alanine, however, completely stalled the formation of acetylformoin and, consequently, also of colorant 1. Further model studies revealed acetylformoin as a chemical switch in the Maillard reaction determining different reaction pathways in the presence of primary and secondary amino acids; for example, the reaction with glycine methyl ester and glycine revealed 2,4-dihydroxy-2,5-dimethyl-1-methoxycarbonylmethyl- (4) and 2,4-dihydroxy-2,5-dimethyl-1-carboxymethyl -3-oxo-2H-pyrrole (5), respectively, whereas the reaction with L-proline resulted in the formation of pyrrolidino- (6) and bispyrrolidino-hexose-reductone (7). Dry heating of these amino reductones in the presence of furan-2-carboxaldehyde produced the colorants 2,4-dihydroxy-2-methyl-5-[(E)-(2-furyl)methylidene]methyl-1- methoxycarbonylmethyl-3-oxo-2H-pyrrole (8) from 4 and 3-hydroxy-4-(E)-(2-furyl)methylidene] methyl-3-cyclopentene-1,2-dione (9) from 6 and 7. Quantitative studies revealed 3-hydroxy-4-methyl-3-cyclopentene-1,2-dione (10) and methylene-reductinic acid (11) as the key intermediates in the formation of colorant 9.

Full text of DOI:10.1021/jf980512l

3918
J. Agric. Food Chem. 1998, 46, 3918−3928
Acetylfor m oin sA Ch em ica l Sw itch in th e F or m a tion of Color ed
Ma illa r d Rea ction P r od u cts fr om Hexoses a n d P r im a r y a n d
Secon d a r y Am in o Acid s
Thomas Hofmann†
Deutsche Forschungsanstalt fu¨r Lebensmittelchemie, Lichtenbergstrasse 4, D-85748 Garching, Germany
Thermal treatment of a methanolic solution of N-(1-deoxy-D-fructos-1-yl)-L-proline and furan-2-
carboxaldehyde led to the rapid formation of colored compounds, among which 4-hydroxy-2-methoxy-
2-methyl-5-[(E)-(2-furyl)methylidene]methyl-2H-furan-3-one (1) could be identified as one of the main
colored compounds by NMR, LC/MS, and UV-vis spectroscopy. Studies on its formation revealed
that condensation of the hexose dehydration product acetylformoin (2a /2b) with furan-2-carboxal-
dehyde formed the labile 2,4-hydroxy-2-methyl-5-[(E)-(2-furyl)methylidene]methyl-2H-furan-3-one
(3a /3b), which then reacts with methanol to the more stable colorant 1. Reacting furan-2-
carboxaldehyde with N-(1-deoxy-D-fructos-1-yl)-L-alanine, however, completely stalled the formation
of acetylformoin and, consequently, also of colorant 1. Further model studies revealed acetylformoin
as a chemical switch in the Maillard reaction determining different reaction pathways in the presence
of primary and secondary amino acids; for example, the reaction with glycine methyl ester and
glycine revealed 2,4-dihydroxy-2,5-dimethyl-1-methoxycarbonylmethyl- (4) and 2,4-dihydroxy-2,5-
dimethyl-1-carboxymethyl -3-oxo-2H-pyrrole (5), respectively, whereas the reaction with L-proline
resulted in the formation of pyrrolidino- (6) and bispyrrolidino-hexose-reductone (7). Dry heating
of these amino reductones in the presence of furan-2-carboxaldehyde produced the colorants 2,4-
dihydroxy-2-methyl-5-[(E)-(2-furyl)methylidene]methyl-1-methoxycarbonylmethyl-3-oxo-2H-pyr-
role (8) from 4 and 3-hydroxy-4-[(E)-(2-furyl)methylidene] methyl-3-cyclopentene-1,2-dione (9) from
6 and 7. Quantitative studies revealed 3-hydroxy-4-methyl-3-cyclopentene-1,2-dione (10) and
methylene-reductinic acid (11) as the key intermediates in the formation of colorant 9.
Keyw or d s: Acetylformoin; amino-hexose-reductones; pyrrolinone-reductone; colored compounds;
Maillard reaction; nonenzymatic browning
INTRODUCTION
presence of a surplus amount of one carbohydrate-
derived carbonyl compound, for example, furan-2-car-
boxaldehyde, forcing all methylene-active compounds to
react with the same aldehyde and reducing the product
multiplicity to the key chromophores (Hofmann, 1998a,b).
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 during the thermal processing of foods
such as roasting of meat, baking of bread, kiln drying
of malt, or roasting of coffee.
Despite extensive studies, due to the complexity and
multiplicity of the nonvolatile Maillard reaction prod-
ucts formed, surprisingly little is known about the
structures of the compounds responsible for the typical
brown color. It is well-known from the literature that
the amino acid-catalyzed conversion of reducing carbo-
hydrates results in a tremendous variety of reactive
aldehydes as well as methylene-active intermediates
(Ledl and Schleicher, 1992). Results of model studies
(Severin and Kro¨nig, 1972; Hofmann, 1998a-c; Hof-
mann and Heuberger, 1998) revealed that indiscrimi-
nate condensation reactions between these reactive
intermediates lead to the multiplicity and the low yields
of colored reaction products.
In comparison to pentoses, less information is as yet
available regarding the color formation from hexoses.
On the basis of the finding that aqueous conditions
destabilize certain colorants in Maillard reactions, Ledl
and Severin (1982) used alcohol instead of water to gain
insights into the structures of possible labile colorants.
The authors identified colored compounds in a heated
ethanolic solution of N-(1-deoxy-D-fructos-1-yl)piperidine
and furan-2-carboxaldehyde and proposed acetylformoin
as a key intermediate in colorant formation. Because
this model reaction has been carried out with the
synthetically related piperidine instead of more food-
related amino acids, it is not clear whether the results
obtained can be transferred to real food systems.
Recent studies (Hofmann, 1998c), therefore, aimed at
the characterization of colored compounds formed from
hexoses and primary amino acids, among which 2H,7H,-
8aH-pyrano[2,3-b]pyran-3-ones and 2H-pyran-3-ones
were identified as the main colored compounds. Studies
on their formation revealed the 3-deoxy-2-hexosulose as
the key intermediate of these colorants (Hofmann,
1998d; Hofmann and Heuberger, 1998). However,
colored compounds formed via the 1-deoxyosone path-
To obtain more detailed information on the chemical
species of the chromophoric compounds involved in color
formation of pentoses, we, in recent investigations,
reacted solutions of xylose and amino acids in the
† Telephone +49-89-289 14181; fax +49-89-289 14183;
e-mail hofmann@dfa.leb.chemie.tu-muenchen.de.
10.1021/jf980512l CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/26/1998

Colored Maillard Reaction Products from Acetylformoin
J. Agric. Food Chem., Vol. 46, No. 10, 1998 3919
Ta ble 1. Assign m en t of 1H-NMR Sign a ls (360 MHz) of
Cyclic (3a ; in MeOD-d 3) a n d Op en -Ch a in F or m s (3b; in
CDCl3) of 2,4-Dih yd r oxy-2-m eth yl-5-
way have as yet not been identified in model systems
of glucose and food-related amino acids.
The objectives of the present investigation were,
therefore, to characterize colored Maillard reaction
products formed from methanolic solutions of N-(1-
deoxy-D-fructos-1-yl)-L-proline and N-(1-deoxy-D-fructos-
1-yl)-L-alanine, respectively, heated in the presence of
an additional Maillard-derived carbonyl compound and
to study certain acetylformoin-derived reaction inter-
mediates as precursors in colorant formation. Because
furan-2-carboxaldehyde was shown to be formed from
hexoses (Hofmann and Schieberle, 1998) and also dur-
ing thermal processing of foods such as wheat and rye
bread crusts (Schieberle and Grosch, 1987), as well as
roasted coffee, barley, and chicory (Kanjahn et al., 1996),
it was chosen as a suitable carbonyl compound for these
experiments.
[(E)-(2-fu r yl)m eth ylid en e]m eth yl-2H-fu r a n -3-on e
H at
δb
relevant
C atoma
3a
3b
Ic Mc J c (Hz)
COSYd
H-C (1)
H-C(10)
H-C(9)
1.49 2.47
6.54 6.55
6.71 6.84
6.94 7.64
7.19 7.45
7.62 7.58
3 s
1 dd 3.5, 1.4 H-C(9), H-C(11)
1 d
1 d 15.92
1 d 15.92
1 d
3.5
H-C(10), H-C(11)
H-C(6)
H-C(7)
H-C(6)
H-C(11)
HO-C(3,4)
H-C(7)
1.4
H-C(9), H-C(10)
11.05/11.63 1 bs
a
Numbering of carbon atoms refers to structures 3a and 3b in
Figure 3. The 1H chemical shifts are given in relation to the
b
solvent signal. c Determined from 1D spectrum. Observed homo-
d
nuclear 1H,1H connectivities by TOCSY and DQF-COSY.
Ta ble 2. Assign m en t of 13C-NMR Sign a ls (360 MHz) of
Cyclic (3a ; in MeOD-d 3) a n d Op en -Ch a in F or m s (3b; in
CDCl3) of 2,4-Dih yd r oxy-2-m eth yl-5-
EXPERIMENTAL PROCEDURES
Ch em ica ls. The following compounds were obtained com-
mercially: D-glucose, L-proline, L-alanine, glycine, furan-2-
carboxaldehyde, piperidine, pyrrolidine, 2-oxopropanal (40%
aqueous solution), potassium cyanide, glycine methyl ester
hydrochloride (Aldrich, Steinheim, Germany). Furan-2-car-
boxaldehyde was freshly distilled at 30 °C in high vacuum
prior to use. Solvents were of HPLC grade (Aldrich). Deu-
terated solvents were obtained from Isocom (Landshut, Ger-
many).
The following Maillard reaction products were prepared as
described in the literature given in parentheses: N-(1-deoxy-
D-fructos-1-yl)-piperidine (Hofmann and Heuberger, 1998);
N-(1-deoxy-D-fructos-1-yl)-L-alanine (Hofmann et al., unpub-
lished results); N-(1-deoxy-D-fructos-1-yl)-proline (Hofmann
and Schieberle, unpublished results).
Syn th eses. Acetylformoin [3,4-Dihydroxy-3-hexene-2,5-di-
one (2a ) and 2,4-Dihydroxy-2,5-dimethyl-3(2H)-furanone (2b)].
Following a procedure of Goto et al. (1963) with major
modifications, freshly distilled 2-oxopropanal (60 g) was diluted
with water (180 mL) and cooled to 0 °C in an ice bath upon
bubbling of argon. After dropwise addition of an ice-cooled
solution of potassium cyanide (1.74 g) in water (60 mL), the
pH was then adjusted to pH 7.0 with a satured aqueous
NaHCO3 solution. The mixture was then stirred at 2 °C while
the pH was held between 7.1 and 7.3 by adding aqueous
phosphoric acid (5 mol/L). After 60 min, the reaction was
quenched by adjusting the pH to 4.5 with concentrated
phosphoric acid. The water was evaporated in vacuo at 40
°C, the syrupy residue was mixed with ethanol (300 mL), and,
after filtration, the solvent was removed in vacuo. The residue
was taken up in diethyl ether (300 mL) and, after filtration,
the solvent was again evaporated in vacuo. The yellowish
syrup was then fractionated by high-vacuum sublimation (0.1
mbar; 90-100 °C) affording the target compound as intense
yellow crystalls (3.9 mg; 16% in yield): MS/CI 145 (59), 131
(11), 127 (100), 103 (14); MS/EI 144 (33), 101 (72), 73 (28), 55
(100). NMR data of 3,4-dihydroxy-3-hexene-2,5-dione (2a ):1H
NMR (360 MHz, CDCl3; arbitrary numbering of the carbon
atoms refers to structure 2a in Figure 3) δ 2.45 [s, 2 × 3H,
H-C(1) and H-C(6)], 10.83 [bs, 2 × 1H, HO-C(3) and HO-
C(4)];13C NMR (360 MHz, CDCl3, HMQC, HMBC, DEPT;
arbitrary numbering of the carbon atoms refers to structure
2a in Figure 3) δ 25.7 [CH3, C(1) and C(6)], 139.4 [C, C(3) and
C(4)], 205.3 [C, C(2) and C(5)]. NMR data of 2,4-dihydroxy-
2,5-dimethyl-3(2H)-furanone (2b): 1H NMR (360 MHz, MeOD-
d3; arbitrary numbering of the carbon atoms refers to structure
2b in Figure 3) δ 1.42 [s, 3H, H-C(1)], 2.18 [s, 3H, H-C(6)];
13C NMR (360 MHz, MeOD-d3, HMQC, HMBC, DEPT; arbi-
trary numbering of the carbon atoms refers to structure 3b in
Figure 3) δ 13.4 [CH3, C(1)], 22.5 [CH3, C(6)], 102.6 [C, C(2)],
132.5 [C, C(5)], 174.8 [C, C(4)], 197.4 [C, C(3)].
[(E)-(2-fu r yl)m eth ylid en e]m eth yl-2H-fu r a n -3-on e
heteronuclear 1H,13
multiple-quantum coherenced
C
H at
δb
relevant
via
via
C atoma
3a
3b
DEPTc 1J (C,H)
2,3,4J (C,H)
C(1)
C(2)
C(6)
C(10)
C(9)
C(7)
C(5)
C(11)
C(8)
C(4)
C(3)
22.6
25.8
CH3
C
H-C(1)
H-C(6)
102.1 205.2
112.0 113.1
113.4 117.0
115.1 118.3
123.7 131.9
133.0 192.6
145.9 146.2
153.2 151.5
165.5 141.1
196.4 140.4
H-C(1)
H-C(7)
CH
CH
CH
CH
C
CH
C
C
H-C(10) H-C(9), H-C(11)
H-C(9)
H-C(7)
H-C(10), H-C(11)
H-C(6)
H-C(6)
H-C(11) H-C(9), H-C(10)
H-C(7), H-C(10),
H-C(11)
C
a
Numbering of carbon atoms refers to structures 3a and 3b in
Figure 3. The 13C chemical shifts are given in relation to the
b
solvent signal. c DEPT-135 spectroscopy. Assignments based on
d
HMQC (1J ) and HMBC (2,3J ) experiments.
Severin (1982) with some modifications, a mixture of acetyl-
formoin (5 mmol), furan-2-carboxaldehyde (15 mmol), piperi-
dine (100 µL), and acetic acid (100 µL) in chloroform (20 mL)
was heated for 90 min at 80 °C in a closed vial under an
atmosphere of argon. After cooling, the solvent was removed
in vacuo and the target compound was isolated by preparative
thin-layer chromatography on silica gel (20 × 20 cm; 0.5 mm;
Merck, Darmstadt, Germany) using toluene/ethyl acetate (80:
20, v/v) as the mobile phase. An intense colored band at Rf )
0.25-0.32 was scraped off, suspended in ethyl acetate (50 mL),
and filtered, affording a deep yellow residue of pure 2,4-
dihydroxy-2-methyl-5-[(E)-(2-furyl)methylidene]methyl-2H-fu-
ran-3-one after evaporation of the solvent (3.6 mmol; ∼72%
in yield): LC/MS(APCI-) 221 (100, [M + 1 - H2]-); UV-vis
1
λmax ) 390 nm (methanol); H and 13C NMR data as well as
signal assignments are given in Tables 1 and 2.
4-Hydroxy-2-methoxy-2-methyl-5-[(E)-(2-furyl)methylidene]-
methyl-2H-furan-3-one (1). A solution of 2,4-dihydroxy-2-
methyl-5-[(E)-(2-furyl)methylidene]methyl-2H-furan-3-one (2
mmol) in methanol (10 mL) was heated in the presence of
hydrochloric acid (20 µL) for 30 min at 60 °C. The mixture
was then diluted with water (150 mL) and extracted with
diethyl ether (3 × 50 mL). After drying over Na2SO4, the
organic layer was concentrated to ∼2 mL, and the target
compound was isolated by thin-layer chromatography on silica
gel (20 × 20 cm; 0.5 mm; Merck) using pentane/toluene/ethyl
acetate (50:40:10, v/v/v) as the mobile phase. A yellow band
at Rf ) 0.15 was scraped off and suspended in ethyl acetate
(20 mL). After filtration, the solvent was removed, affording
an intense orange residue consisting of pure colorant 1 (1.8
mmol, ∼90% in yield): 1H NMR (360 MHz, DMSO-d6; arbitrary
2,4-Dihydroxy-2-methyl-5-[(E)-(2-furyl)methylidene]methyl-
2H-furan-3-one (3a / 3b). Following a method of Ledl and

3920 J. Agric. Food Chem., Vol. 46, No. 10, 1998
Hofmann
numbering of the carbon atoms refers to structure 1 in Figure
3) δ 1.35 [s, 3H, H-C(1], 3.08 [s, 3H, H-C(12)], 6.59 [dd, 1H,
extracted with ethyl acetate (10 × 50 mL). After drying over
Na2SO4, the organic layer was concentrated to ∼10 mL, and
the crude material was fractionated by chromatography on
silica gel (2 × 60 cm, silica gel 60, Merck), which was
conditioned with toluene/ethyl acetate (10:90, v/v). After
application of the crude material (in aliquots of 5 mL) onto
the column, chromatography was performed using toluene/
ethyl acetate (10:90, v/v; 500 mL; fraction A), followed by ethyl
acetate (500 mL; fraction B), ethyl acetate/methanol (90:10,
v/v; 500 mL; fraction C) and ethyl acetate/methanol (80:20,
v/v; 500 mL; fraction D). Concentration of fractions D and C
afforded compound 6 in fraction D (20 mmol, 4% in yield) and
compound 7 in fraction C (35 mmol; 7% in yield) as white
crystals after recrystallization from methanol. Spectroscopical
data of compound 6: LC/MS (ESI) 198 (100, [M + 1]+), 220
(27, [M + Na]+), 180 (18, [M + 1 - H2O]+);1H NMR (360 MHz;
DMSO-d6, DQF-COSY; arbitrary numbering of the carbon
atoms refers to structure 6 in Figure 5) δ 1.17 [s, 3H, H-C(1)],
3J 10,9 ) 3.5 Hz, 3J 10,11 ) 1.4 Hz, H-C(10)], 6.78 [d, 1H, 3J 9,10
)
3
3.5 Hz, H-C(9)], 6.99 [d, 1H, J 7,6 ) 15.9 Hz, H-C(7)], 7.04
[d, 1H, 3J 6,7 ) 15.9 Hz, H-C(6)], 7.76 [dd, 1H, 3J 11,10 ) 1.4 Hz,
H-C(11)]; 13C NMR (360 MHz, DMSO-d6, HMQC, HMBC,
DEPT; arbitrary numbering of the carbon atoms refers to
structure 1 in Figure 3) 21.9 [(CH3, C(1)], 50.9 [CH3, C(12)],
102.3 [C, C(2)], 111.6 [CH, C(6)], 112.7 [CH, C(10)], 113.1 [CH,
C(9], 118.6 [C, C(7)], 133.3 [C, C(5)], 144.7 [CH, C(11)], 152.1
[C, C(8)], 162.9 [C, C(4)], 196.3 [C, C(3)]; LC/MS (APCI-) 235
(100, [M + 1 - H2]-), 220 (19, [M + 1 - H2 - CH3]+); UV-vis
(in water) λmax ) 441 nm, ꢀ ) 1.1 × 104 L mol-1 cm-1
.
2,4-Dihydroxy-2,5-dimethyl-1-methoxycarbonylmethyl-3-oxo-
2H-pyrrole (4). Following a procedure of Ledl and Fritsch
(1984) with major modifications, acetylformoin (30 mmol) and
glycine methyl ester hydrochloride (30 mmol) were refluxed
in phosphate buffer (150 mL; 0.1 mmol/L; pH 5.5) for 25 min.
After cooling, the mixture was extracted with methylene
chloride (6 × 50 mL) and the aqueous phase was mixed with
silica gel (20 g) and then freeze-dried. The residue was applied
onto the top of a column filled with a slurry of silica gel (200
g) in ethyl acetate. Chromatography was performed with ethyl
acetate (400 mL), followed by ethyl acetate/methanol (90:10,
v/v; 400 mL), affording an orange fraction. Thin-layer chro-
matography on RP-18 material using water/methanol (70:30)
as the eluent revealed a yellow, fluorescent compound at Rf )
0.61. The solvent was removed in vacuo, and the residue was
taken up in methanol (3 mL). Upon dropwise addition of ethyl
acetate, the target compound was obtained as an orange
powder. Recrystalliztation from methanol/ethyl acetate (80:
20, v/v) affords compound 4 as yellow-orange crystals (4.5
mmol; 15% in yield): MS(EI) 43 (100), 56 (89), 215 (78, M+),
172 (86, M+ - CH3CO), 84 (55), 149 (53), 115 (52), 86 (47),
156 (35), 140 (32), 144 (25), 199 (20, M+ - CH3): 1H NMR
(360 MHz, MeOD-d3; arbitrary numbering of the carbon atoms
2
1.80-1.84 [m, 4H, H-C(8,9)], 2.44 [d, 1H, J 6a,6b ) 15.92 Hz,
2
Ha-C(6)], 2.56 [m, 1H, J 6b,6a ) 15.92 Hz, Hb-C(6)], 3.55-3.72
[m, 4H, H-C(7,10)], 4.91 [bs, 1H, HO-C(2)], 7.32 [bs, 1H, HO-
C(4)]; 13C NMR (360 MHz; DMSO-d6, DEPT, HMQC, HMBC;
arbitrary numbering of the carbon atoms refers to formula 6
in Figure 5): δ 24.6 [CH3, C(1)], 25.7 [CH2, C(8,9)], 48.2 [CH2,
C(7,10)], 70.2 [C, C(2)], 125.8 [C, C(5)], 152.2 [C, C(4)], 193.1
[C, C(3)]. Spectroscopical data of compound 7: LC/MS (ESI)
251 (100, [M + 1]+), 273 (11, [M + Na]+), 233 (19, [M + 1 -
H2O]+); 1H NMR (360 MHz; DMSO-d6, DQF-COSY; arbitrary
numbering of the carbon atoms refers to structure 7 in Figure
5) δ 1.13 [s, 3H, H-C(1)], 1.76-1.78 [m, 4H, H-C(8′,9′)], 1.82-
2
1.84 [m, 4H, H-C(8,9)], 2.44 [d, 1H, J 6a,6b ) 15.92 Hz, Ha-
2
C(6)], 2.57 [m, 1H, J 6b,6a ) 15.92 Hz, Hb-C(6)], 2.89-3.00 [m,
4H, H-C(7′,10′)], 3.50-3.61 [m, 4H, H-C(7,10)], 4.82 [bs, 1H,
HO-C(2)]; 13C NMR (360 MHz; DMSO-d6, DEPT, HMQC,
HMBC; arbitrary numbering of the carbon atoms refers to
structure 7 in Figure 5) δ 24.6 [CH3, C(1)], 25.7 [CH2, C(8,9)],
48.2 [CH2, C(7,10)], 70.2 [C, C(2)], 125.8 [C, C(5)), 152.2 [C,
C(4)], 193.1 [C, C(3)].
refers to structure 4 in Figure 4) δ 1.26 [s, 3H, H-C(1)], 2.16
2
[s, 3H, H-C(6)], 3.73 [s, 3H, H-C(9)], 4.15 [d, 1H, J 7a,7b
)
2
18.13 Hz, Ha-C(7)], 4.23 [d, 1H, J 7a,7b ) 18.13 Hz, Ha-C(7)];
13C NMR (360 MHz, MeOD-d3, HMQC, HMBC, DEPT; arbi-
trary numbering of the carbon atoms refers to structure 4 in
Figure 4) δ 11.5 [CH3, C(1)], 22.0 [CH3, C(6)], 43.3 [CH3, C(9)],
53.4 [CH2, C(7)], 88.2 [C, C(2)], 128.2 [C, C(5)], 169.1 [C, C(4)],
172.7 [C, C(8)], 193.1 [C, C(3)]; UV-vis (water) λmax ) 363 nm,
2,4-Dihydroxy-2-methyl-5-[(E)-(2-furyl)methylidene]methyl-
1-methoxycarbonylmethyl-3-oxo-2H-pyrrole (8). A mixture of
2,4-dihydroxy-2,5-dimethyl-1-methoxycarbonylmethyl-3-oxo-
2H-pyrrole (2 mmol) and furan-2-carboxaldehyde (5 mmol) in
phosphate buffer (5 mL; pH 7.0; 0.5 mol/L) was heated for 10
min under reflux. After cooling, the mixture was extracted
with ethyl acetate (4 × 10 mL) and the combined organic layers
were dried over Na2SO4 and concentrated to ∼1 mL. The
target compound was then isolated by thin-layer chromatog-
raphy on silica gel (20 × 20 cm; 0.5 mm; Merck) with ethyl
acetate/methanol (90:10, v/v) as the mobile phase. The band
at Rf ) 0.2 was scraped off, suspended in methanol, and
filtered. Removing the solvent revealed the target compound
as a yellow residue (0.25 mmol; ∼5% in yield):1H NMR (360
MHz, MeOD-d3; arbitrary numbering of the carbon atoms
refers to formula 8 in Figure 6) δ 1.15 [s, 3H, H-C(1)], 3.70
[s, 3H, H-C(9)], 4.34 [d, 1H, 2J 7a,7b ) 18 Hz, Ha-C(7)], 4.53 (d,
ꢀ ) 0.6 × 104 L mol-1 cm-1
.
2,4-Dihydroxy-2,5-dimethyl-1-carboxymethyl-3-oxo-2H-pyr-
role (5). Compound 4 (1.0 mmol) was dissolved in phosphate
buffer (10 mL, 0.2 mol/L, pH 7.5) and, after addition of porcine
liver esterase (2000 units; Sigma, Deistenhofen, Germany),
was stored for 12 h at 37 °C. The mixture was freeze-dried
and the residue suspended in methanol/ethyl acetate (10 mL,
50:50, v/v). After filtration and concentration to 1 mL, the
target compound was purified by flash chromatography on RP-
18 material (15.0 g; Lichroprep 25-40 µm, Merck) using a
mixture (20:80, v/v) of methanol and trifluoroacetic acid (0.2%
TFA in water) as the mobile phase. After application of the
crude material, chromatography with the same eluent afforded
compound 5 in the effluent >70 mL. The combined eluates
were freeze-dried, yielding the colored compound as an orange
powder (0.44 mmol, ∼44% in yield): LC/MS(APCI-) 200 (100,
[M + 1 - H2]-);1H NMR (360 MHz, MeOD-d3; arbitrary
numbering of the carbon atoms refers to structure 5 in Figure
4) δ 1.28 [s, 3H, H-C(1)], 2.17 [s, 3H, H-C(6)], 4.17 [d, 1H,
2
3
1H, J 7b,7a ) 18 Hz, Hb-C(7)], 6.54 [dd, 1H, J 13,12 ) 3.5 Hz,
3J 13,14 ) 1.7 Hz, H-C(13)], 6.69 [d, 1H, J 12,13) 3.5 Hz,
3
H-C(12)], 6.81 [d, 1H, 3J 10.6 ) 15.9 Hz, H-C(10)], 6.84 [d, 1H,
2J 6,10 ) 15.9 Hz, H-C(6)], 7.50 [d, 1H, J 14,13 ) 1.7 Hz,
3
H-C(14)]; UV-vis λmax ) 402 nm, ꢀ ) 1.4 × 104 L mol-1 cm-1
.
N-(1-Methyl-1,2,3-trihydroxy-2-cyclopentene-4-ylidene)piperi-
dinium Betaine (Piperidino-hexose-reductone). A mixture of
N-(1-deoxy-D-fructos-1-yl)piperidine (200 mmol) and acetic acid
(200 mmol) in ethanol (150 mL) was refluxed for 20 h. After
cooling, the mixture was concentrated to ∼50 mL in vacuo and
then cooled at -20 °C. The crystals formed were filtered off
and recrystallized from methanol, affording the piperidino-
hexose-reductone as a white solid (39 mmol, ∼20% in yield):
LC/MS (ESI) 212 (100, [M + 1]+), 234 (38, [M + Na]+), 194
2
2J 7a,7b ) 18.2 Hz, Ha-C(7)], 4.25 [d, 1H, J 7a,7b ) 18.2 Hz, Ha-
C(7)].
N-(1-Methyl-1,2,3-trihydroxy-2-cyclopentene-4-ylidene)pyrro-
lidinium Betaine (Pyrrolidino-hexose-reductone; 6) and N-[1-
Methyl-1,2-dihydroxy-3-(1-pyrrolidino)-2-cyclopenten-4-ylidene]-
pyrrolidinium Betaine (Bispyrrolidino-hexose-reductone; 7).
Following a procedure of Papst et al. (1984) with some
modifications, a mixture of glucose (500 mmol) and pyrrolidine
(500 mmol) in ethanol (150 mL) was refluxed for 90 min, acetic
acid (500 mmol) was added, and heating was continued for
additional 20 h. After cooling, the solvent was removed, and
the syrupy residue was redissolved in water (300 mL) and then
(12, [M + 1 - H2O]+); H NMR (360 MHz; DMSO-d6, DQF-
1
COSY) δ 1.17 (s, 3H, -CH3), 1.51-1.61 (m, 6H, 3 × -CH2-),
2.40 [d, 1H, 2J 6a,6b ) 15.92 Hz, -CHaHb-C(OH)], 2.52 [m, 1H,
2J 6b,6a ) 15.92 Hz, -CHaHb-C(OH)], 3.55-3.63 (m, 4H, 2 ×
-CH2-N), 4.94 [bs, 1H, HO-C(CH3)-], 7.64 (bs, 1H, HO-C);
13C NMR (360 MHz; DMSO-d6, DEPT, HMQC, HMBC) δ 23.9

Colored Maillard Reaction Products from Acetylformoin
J. Agric. Food Chem., Vol. 46, No. 10, 1998 3921
Ta ble 3. Assign m en t of 1H-NMR Sign a ls (360 MHz,
DMSO-d 6) of 3-Hyd r oxy-4-[(E)-(2-fu r yl)m eth ylen e]m eth yl-
3-cyclop en ten e-1,2-d ion e (9)
Ta ble 4. Assign m en t of 13C-NMR Sign a ls (360 MHz,
DMSO-d 6) of 3-Hyd r oxy-4-[(E)-(2-fu r yl)m eth ylen e]m eth yl-
3-cyclop en ten e-1,2-d ion e (9)
H at
heteronuclear 1H,13
multiple-quantum coherenced
C
relevant
connectivityd
with
C atoma
δb
Ic
Mc
J c (Hz)
relevant
C atoma
via
via
H-C(6)
H-C(10)
H-C(9)
H-C(7)
H-C(1)
H-C(11)
HO-C(3)
3.12
6.65
6.85
7.00
7.30
7.86
10.85
2
1
1
1
1
1
1
s
dd
d
d
d
δb
DEPTc 1J (C,H)
2,3J (C,H)
3.5, 1.8
3.5
15.9
15.9
1.8
H-C(9), H-C(11)
H-C(10)
C(6)
C(10)
C(9)
C(1)
C(7)
C(2)
C(11)
C(8)
C(3)
C(4)
C(5)
33.5
113.0
114.3
117.8
123.6
138.3
145.6
152.3
155.1
181.9
196.9
CH2 H-C(6) H-C(1)
CH
CH
CH
CH
C
CH
C
C
H-C(10) H-C(9), H-C(11)
H-C(9) H-C(7), H-C(10), H-C(11)
H-C(1) H-C(6), H-C(7)
H-C(7) H-C(1)
H-C(1)
H-C(7)
d
bs
H-C(10)
H-C(2) H-C(1), H-C(6), H-C(7)
H-C(11) H-C(9), H-C(11)
H-C(8) H-C(9), H-C(10), H-C(11)
H-C(3) H-C(1), H-C(6)
H-C(4)
a
Numbering of carbon atoms refers to structure 9 in Figure 7.
The 1H chemical shifts are given in relation to DMSO-d6.
b
c Determined from 1D spectrum. Observed homonuclear 1H,1H
d
C
C
connectivities by DQF-COSY.
H-C(5) H-C(6)
a
(-CH2-), 25.7 (-CH3), 26.0 (2 × -CH2-), 41.2 [-CH2-
C(OH)], 48.1 (2 × -CH2-N), 69.5 [--(OH)-CH3], 125.8
[N-C(CH2)-], 150.8 [-C(OH)-], 194.1 (-CO-).
Numbering of carbon atoms refers to structure 9 in Figure 7.
The 13C chemical shifts are given in relation to DMSO-d6.
b
c DEPT-135 spectroscopy. Assignments based on HMQC (1J ) and
d
HMBC (2,3J ) experiments.
3-Hydroxy-4-methyl-3-cyclopentene-1,2-dione (10) and 2,3-
Dihydroxy-4-methylene-2-cyclopenten-1-one (Methylene-reduc-
tinic Acid, 11). Following a procedure of Ledl et al. (1982), a
mixture of piperidino-hexose-reductone (60 mmol) and con-
centrated hydrochloric acid (8 mL) in water (140 mL) was
refluxed for 5 min. After cooling, the solution was extracted
with diethyl ether (6 × 30 mL), the organic layer was dried
over Na2SO4, and the solvent was removed to ∼4 mL. Cooling
to -30 °C afforded the methylene-reductinic acid (11) as yellow
crystals (16.2 mmol; ∼27% in yield). The mother liquor was
fractionated by column chromatography on silica gel (50 g),
which was conditioned with ethyl acetate. After elution with
ethyl acetate (400 mL), the solvent was removed under vacuo
and the residue was fractionated by high-vacuum sublimation
(0.1 Torr) affording yellow crystalls of 3-hydroxy-4-methyl-3-
cyclopentene-1,2-dione (10) between 60 and 80 °C (1.2 mmol;
2% in yield). The spectroscopical data of 3-hydroxy-4-methyl-
3-cyclopentene-1,2-dione (10) are well in line with those
reported by Ledl et al. (1982): 1H NMR (360 MHz; CDCl3,
TOCSY; arbitrary numbering of the carbon atoms refers to
structure 10 in Figure 8) δ 2.30 [t, 3H, 0.9 Hz, H-C(1)], 3.05
[q, 2H, 0.9 Hz; H-C(6)]; MS(EI) 126 (100, M+), 98 (71), 70 (76),
69 (64), 55 (81), 43 (51), 42(38), 41(57), 39(55). Spectroscopical
data of methylene-reductinic acid (11): MS(EI) 126 (100, M+),
Rea ction of N-(1-Deoxy-D-fr u ctos-1-yl)-L-a la n in e or
N-(1-Deoxy-D-fr u ctos-1-yl)-L-p r olin e, Resp ectively, w ith
F u r a n -2-ca r boxa ld eh yd e. A mixture of N-(1-deoxy-D-fruc-
tos-1-yl)-L-alanine (10 mmol) or N-(1-deoxy-D-fructos-1-yl)-L-
proline (10 mmol), respectively, in methanol (30 mL) was
refluxed for 5 h in the presence of furan-2-carboxaldehyde (40
mmol). After cooling, the solvent was removed in vacuo, and
the residue was dissolved in water (200 mL) and extracted
with ethyl acetate (8 × 50 mL). The combined organic layers
were dried over Na2SO4, concentrated to ∼100 mL, and then
distilled under high vacuum at 35 °C to remove the volatile
fraction. The residue was dissolved in ethyl acetate (5 mL)
and then fractionated by column chromatography (35 × 450
mm) on silica gel (150 g, silica gel 60, Merck), which was
conditioned with toluene/ethyl acetate (90:10, v/v). Chroma-
tography was performed using toluene/ethyl acetate (90:10;
300 mL; fraction A), toluene/ethyl acetate (70:30; 300 mL;
fraction B), toluene/ethyl acetate (60:40; 300 mL; fraction C),
and toluene/ethyl acetate (50:50; 300 mL; fraction D). The
latter two fractions were collected and freed from solvent under
vacuum at 25 °C; the colored residues were taken up in ethyl
acetate (1 mL) and were then further fractionated by thin-
layer chromatography on silica gel (20 × 20 cm; 0.5 mm;
Merck) using toluene/ethyl acetate (60:40, v/v) as the mobile
phase. In the reaction mixture containing N-(1-deoxy-D-
fructos-1-yl)-L-proline, a yellow band at Rf ) 0.44 was scraped
off and suspended in ethyl acetate (20 mL). After filtration,
the solvent was removed, affording an intense yellow residue
consisting of 4-hydroxy-2-methoxy-2-methyl-5-[(E)-(2-furyl)-
methylidene]methyl-2H-furan-3-one (1; 0.05 mmol, ∼0.5% in
yield) exclusively in the mixture containing N-(1-deoxy-D-
fructos-1-yl)-L-alanine.
1
69 (50), 55 (29), 52 (38), 43 (50), 42 (25), 41 (31), 39 (37); H
NMR (360 MHz; DMSO-d6, TOCSY; arbitrary numbering of
the carbon atoms refers to structure 11 in Figure 8) δ 2.83 [s,
2H, H-C(1)], 4.93 [s, 1H, Ha-C(1)], 5.34 [s, 1H, Hb-C(1)], 9.29
[bs, 1H, HO-C(4)], 10.95 [bs, 1H, HO-C(3)]; 13C NMR (360
MHz; DMSO-d6, DEPT, HMQC, HMBC; arbitrary numbering
of the carbon atoms refers to structure 11 in Figure 8) δ 35.8
[CH2, C(6)], 104.5 [CH2, C(1)], 128.8 [C, C(4)], 136.2 [C, C(3)],
157.2 [C, C(2)], 194.3 [C, C(5)].
3-Hydroxy-4-[(E)-(2-furyl)methylidene]methyl-3-cyclopentene-
1,2-dione (9). A mixture of methylene-reductinic acid (5 mmol),
furan-2-carboxaldehyde (10 mmol), and concentrated hydro-
chloric acid (0.1 mL) in ethanol/water (2:1; 20 mL) was heated
for 1 h at 80 °C. The organic solvent was removed in vacuo,
the pH of the aqueous layer was adjusted to 3.0 with aqueous
hydrochloric acid (1 mol/L), and the aqueous phase was then
extracted with ethyl acetate (5 × 20 mL). The organic layer
was dried over Na2SO4, concentrated to ∼2 mL, and then
fractionated by column chromatography on silica gel (2 × 60
cm, silica gel 60, Merck), which was conditioned with toluene.
After application of the raw material onto the column, chro-
matography was performed using toluene/ethyl acetate (80:
20, v/v; 400 mL). Chromatography with ethyl acetate (800 mL)
affords compound 9 as deep red crystals upon concentration
(1.9 mmol, ∼38% in yield): LC/MS 187 (100, [M + 1 - H2O]+),
177 (87), 205 (43, [M + 1]+), 149 (37), 159 (27); UV-vis (water,
pH 6.0) λmax ) 441 nm, ꢀ ) 1.8 × 104 L mol-1 cm-1; 1H and 13C
NMR data as well as signal assignments are given in Tables
3 and 4.
Detection of Acetylfor m oin (2a /2b) in Dr y-Hea ted
Mixt u r es of Am a d or i R ea r r a n gem en t P r od u ct s. N-(1-
Deoxy-D-fructos-1-yl)-L-alanine (10 mmol) or N-(1-deoxy-D-
fructos-1-yl)-L-proline (10 mmol) was dry-heated for 15 min
at 160 °C. After cooling, the reaction mixtures were suspended
in ethyl acetate/methanol (95:5, v/v; 30 mL), filtrated, concen-
trated in vacuo to ∼300 µL, and analyzed by GC/MS. A peak,
showing the same retention time and the identical MS
spectrum as synthetic acetylformoin, was detected in the
heated N-(1-deoxy-D-fructos-1-yl)-L-proline mixture in a yield
of ∼2 µg/mmol. In the N-(1-deoxy-D-fructos-1-yl)-L-alanine
mixture, acetylformoine was, however, not detected. GC/MS-
(EI): 101 (100), 43 (96), 144 (67), 73 (65), 55 (41). GC/MS(CI,
isobutane): 145 (100, [M + 1]+).
F or m a tion of 2,4-Dih yd r oxy-2,5-d im eth yl-1-m eth oxy-
ca r bon ylm eth yl- (4) a n d 2,4-Dih yd r oxy-2,5-d im eth yl-1-
ca r boxym eth yl-3-oxo-2H-p yr r ole (5) u p on Dr y Hea tin g
of Acetylfor m oin (2a /2b) w ith Glycin e Meth yl Ester a n d
Glycin e, Resp ectively. Acetylformoin (1 mmol) was inti-

3922 J. Agric. Food Chem., Vol. 46, No. 10, 1998
Hofmann
mately mixed with glycine methyl ester (1 mmol) or glycine
(1 mmol), respectively, and then dry-heated for 4 min at 150
°C. For identification of compound 4, the reaction mixture
taken up in methanol (2 mL) and separated by preparative
thin-layer chromatography on silica gel (20 × 20 cm; 0.5 mm;
Merck) using ethyl acetate/methanol (90:10, v/v) as the mobile
phase. A band with Rf ) 0.46-0.57 was scraped off, suspended
in methanol, filtered, and concentrated to ∼1 mL prior to
HPLC analysis. For identification of compound 5, the reaction
mixture was dissolved in water (5 mL) and extracted with
ethyl acetate (3 × 5 mL), and the aqueous phase was freeze-
dried and taken up in methanol (2 mL). After membrane
filtration, both reaction mixtures were analyzed by RP-HPLC
starting with a mixture (10:90, v/v) of acetonitrile and aqueous
ammonium formiate buffer (20 mmol/L; pH 3.5) and increasing
the acetonitrile content within 50 min to 30%. When the
effluent was monitored at λ ) 360 nm, a peak of a fluorescent
compound was detected at 9.2 and 4.8 min, respectively,
showing UV-vis and LC/MS data as well as retention times
identical with those of synthetic 4 and 5 (∼6% in yield).
F or m a tion of P yr r olid in o-h exose-r ed u cton e (6) a n d
Bisp yr r olid in o-h exose-r ed u ct on e (7) fr om Acet ylfor -
m oin (2a /2b) a n d L-P r olin e. Acetylformoin (1 mmol) and
L-proline (1 mmol) were intimately mixed and then dry-heated
for 15 min at 180 °C. After cooling, the mixture was dissolved
in methanol (2 mL), filtered, and separated by preparative
thin-layer chromatography on silica gel (20 × 20 cm; 0.5 mm;
Merck) using ethyl acetate/methanol (95:5, v/v) as the mobile
phase. Two bands at Rf ) 0.30 and 0.65 were scraped off and
dissolved in methanol. After filtration and concentration,
analysis of both fractions by HPLC revealed the pyrrolidino-
hexose-reductone (band with Rf ) 0.30; ∼1.2% in yield) and
the bispyrrolidino-hexose-reductone (band at Rf ) 0.65; ∼0.5%
in yield) by comparison of the retention times as well as the
UV-vis and the LC/MS data with those obtained for the
synthetic reference compounds.
acetonitrile content within 50 min to 30%. When the effluent
was monitored at λ ) 360 nm, a peak of a fluorescent
compound was detected at 4.8 min showing UV-vis and LC/
MS data and retention time identical with those of the
synthetic 2,4-dihydroxy-2,5-dimethyl-1-carboxymethyl-3-oxo-
2H-pyrrole. Quantification, performed by using the synthetic
pyrrolinone 5 as the external standard, revealed a yield of
∼0.3%.
F or m a tion of 2,4-Dih yd r oxy-2-m eth yl-5-[(E)-(2-fu r yl)-
m et h ylid en e]m et h yl-1-m et h oxyca r b on ylm et h yl-3-oxo-
2H-p yr r ole (8) in a Mixtu r e of Acetylfor m oin /Glycin e
Meth yl Ester Hea ted in th e P r esen ce of F u r a n -2-ca r -
boxa ld eh yd e. Acetylformoin (2 mmol) and glycine methyl
ester (2 mmol) were intimately mixed and then dry-heated for
4 min at 150 °C. After addition of furan-2-carboxaldehyde (4
mmol), heating was continued for another 2 min. The reaction
mixture was cooled, suspended in methanol (5 mL), and
filtered. After concentration, the mixture was separated by
preparative thin-layer chromatography on silica gel (20 × 20
cm; 0.5 mm; Merck) using ethyl acetate/methanol (90:10, v/v)
as the mobile phase. A yellow band at Rf ) 0.4-0.5 was
scraped off, dissolved in methanol, and, after filtration and
concentration, analyzed by HPLC connected to a DAD as well
as an LC/MS. Spectroscopical data as well as retention time
were identical with those obtained for the synthetic reference
compound 8.
Id en tifica tion a n d Qu a n tifica tion of 3-Hyd r oxy-4-[(E)-
(2-fu r yl)m eth yliden e]m eth yl-3-cyclopen ten e-1,2-dion e (9)
in Sever a l P r ecu r sor System s. After the reaction mixtures
had cooled, detailed in Figure 5, the mixtures were suspended
in water (20 mL) and filtered, the pH was adjusted to 5.0, and
the mixtures were then extracted with ethyl acetate (3 × 20
mL). After drying of the organic layer with Na2SO4, the
solvent was evaporated in vacuo and the residue was taken
up in methanol (1 mL). After membrane filtration, the
mixtures were analyzed by RP-HPLC starting with a mixture
(90:10, v/v) of acetonitrile and aqueous ammonium formiate
buffer (20 mmol/L; pH 3.5) and increasing the acetonitrile con-
tent within 50 min to 80%. When the effluent was monitored
at λ ) 440 nm, a peak of a colored compound was detected at
25.4 min, showing UV-vis and LC/MS data as well retention
time identical with those of synthetic 3-hydroxy-4-[(E)-(2-
furyl)methylidene]methyl-3-cyclopentene-1,2-dione. Quanti-
tation was performed by using the synthetic colorant as the
external standard.
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
(Fisons Instruments, Mainz, Germany) using an SE-54 capil-
lary (30 m × 0.32 mm, 0.25 µm; J &W Scientific, Fisons
Instruments) coupled with an MD-800 mass spectrometer
(Fisons Instruments); sample application (0.5 µL) was done
by on-column injection at 40 °C.
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).
Id en tifica tion of P yr r olid in o-h exose-r ed u cton e (6)
a n d Bisp yr r olid in o-h exose-r ed u ct on e (7) in
a Dr y-
Hea ted Mixtu r e of L-P r olin e a n d Glu cose. L-Proline (20
mmol) and glucose (10 mmol) were powdered in a mortar and
then dry-heated for 15 min at 180 °C. The dry-heated mixture
was dissolved in methanol (30 mL), filtered, concentrated to
∼10 mL, and then ultracentrifugated with a cutoff of 1000 Da
(Amicon, Witten, Germany). The filtrate was further fraction-
ated by column chromatography on silica gel (2 × 60 cm, silica
gel 60, Merck), which was conditioned with toluene/ethyl
acetate (50:50, v/v). Chromatography was performed with
toluene/ethyl acetate (50:50; 300 mL; fraction A) and ethyl
acetate (300 mL; fraction B), followed by ethyl acetate/
methanol (90:10, v/v; 300 mL; fraction C) and ethyl acetate/
methanol (80:20, v/v; 300 mL; fraction D). Fractions C and D
were further fractionated by thin-layer chromatography on
silica gel (20 × 20 cm; 0.5 mm; Merck) using ethyl acetate/
methanol (95:5, v/v) as the mobile phase. Two bands at Rf )
0.30 and 0.65 were scraped off and dissolved in methanol. After
filtration and concentration, HPLC analysis coupled to a diode
array detector (DAD) or an LC/MS revealed the pyrrolidino-
hexose-reductone (band with Rf ) 0.30; ∼0.2% in yield) and
the bispyrrolidino-hexose-reductone (band at Rf ) 0.65; ∼0.05%
in yield).
Dr y Hea tin g of N-(1-Deoxy-D-fr u ctos-1-yl)-L-p r olin e
w ith Glycin e. N-(1-Deoxy-D-fructos-1-yl)-L-proline (10 mmol)
and glycine (10 mmol) were intimately mixed and then dry-
heated at 160 °C for 30 min. After cooling, the mixture was
dissolved in methanol (100 mL), filtered, concentrated to ∼4
mL, and fractionated by flash chromatography on RP-18
material (15.0 g; Lichroprep 25-40 µm, Merck) using a
mixture of (10:90, v/v) of methanol and trifluoroacetic acid
(0.2% TFA in water) as the mobile phase. After application
of the crude material, chromatography with the same eluent
afforded compound 5 in the effluent >100 mL. The combined
eluates were freeze-dried, and the residue was taken up in
methanol and then analyzed by RP-HPLC starting with a
mixture (10:90, v/v) of acetonitrile and aqueous ammonium
formiate buffer (20 mmol/L; pH 3.5) and increasing the
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, Du¨rren, Germany) was coupled to an LCQ-MS
(Finnigan MAT GmbH, Bremen, Germany) using electrospray
ionization (ESI) or negative atmospheric chemical pressure
ionization (APCI-). After injection of the sample (2.0 µL),
analysis was performed using a gradient starting with a
mixture (10:90, v/v) of acetonitrile and water and increasing
the acetonitrile content to 100% within 15 min.
UV-Vis Sp ectr oscop y. UV-vis spectra were obtained by
means of a U-2000 spectrometer (Colora Messtechnik Gmbh,
Lorch, Germany).

Colored Maillard Reaction Products from Acetylformoin
J. Agric. Food Chem., Vol. 46, No. 10, 1998 3923
Severin (1982) to be formed via the hexose dehydration
product acetylformoin (2a /2b), which is well-known to
be formed by dehydration of 1-deoxyosone (I) at the
6-position as outlined in Figure 2. For further con-
firmation of the assumed structure, synthetic acetyl-
formoin, which could be shown to consist of a mixture
of 3,4-dihydroxy-3-hexene-2,5-dione (2a , in Figure 3)
and 2,4-dihydroxy-2,5-dimethyl-3(2H)-furanone (2b, in
Figure 3) was, therefore, condensed with furan-2-
carboxaldehyde, affording the labile colorants 3,4-dihy-
droxy-6-[(E)-(2-furyl)methylidene]-3-hexene-2,5-dione (3a,
Figure 3) and 2,4-dihydroxy-2-methyl-5-[(E)-(2-furyl)-
methylidene]methyl-2H-furan-3-one (3b, Figure 3). The
equilibrium between the forms 3a and 3b was unequivo-
cally deduced from the 1H- and the 13C-NMR data.
Although the existence of two forms was supposed
earlier by Ledl and Severin (1982), it was as yet not
confirmed by NMR spectroscopic data, which are sum-
marized in Tables 1 and 2. Reacting 3a /3b with
methanol revealed the corresponding stable methyl
acetal, showing spectroscopic data identical with those
of the colorant isolated from the Maillard mixture,
thereby confirming the proposed structure 1 (Figure 3).
The corresponding 2-ethoxy derivative was reported by
Ledl and Severin (1982) to be formed from N-(1-deoxy-
D-fructos-1-yl)piperidine, which does, however, not occur
in foods. The formation of 1 from glucose in the
presence of the food-related secondary amino acid
L-proline demonstrated that compound 3a /3b might be
involved in color formation during the thermal process-
ing of foods.
Rea ction of Acetylfor m oin w ith P r im a r y a n d
Secon d a r y Am in o Acid s. The data indicated that
compound 1 was exclusively generated from hexoses in
the presence of the secondary amino acid L-proline. To
gain more detailed information on the influence of
primary and secondary amino acids on the formation
of the colorants 1 and 3a /3b, N-(1-deoxy-D-fructos-1-yl)-
L-alanine or N-(1-deoxy-D-fructos-1-yl)-L-proline, respec-
tively, was dry-heated, and the amounts of acetylfor-
moin (2a /2b) formed were determined. In the presence
of the latter, the Amadori rearrangement product
acetylformoin was determined in amounts of ∼2 µg/
mmol. In contrast, acetylformoin could not be detected
in the heated N-(1-deoxy-D-fructos-1-yl)-L-alanine mix-
ture, thereby explaining the lack of colorant 1 in
Maillard mixtures of primary amino acids.
F igu r e 1. Structure of the yellow 4-hydroxy-2-methoxy-2-
methyl-5-[(E)-(2-furyl)methylidene]methyl-2H-furan-3-one (1).
Nu clea r Ma gn etic Reson a n ce Sp ectr oscop y (NMR).
1H, 13C, 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,
1997). Tetramethylsilane (TMS) was used as the internal
standard.
RESULTS AND DISCUSSION
On the basis of the finding that aqueous conditions
destabilize certain colorants in Maillard reactions, Ledl
et al. (1983) used methanol instead of water to clarify
the structures of possible labile colorants. To gain an
insight into the formation of such compounds from
hexoses, methanolic solutions of N-(1-deoxy-D-fructos-
1-yl)-L-alanine and N-(1-deoxy-D-fructos-1-yl)-L-proline,
respectively, were heated in the presence of furan-2-
carboxaldehyde, leading to a rapid colorization of both
mixtures. After separation of the reaction mixtures, the
colorants generated were registered by RP-HPLC using
either a diode array detector (DAD), monitoring in the
wavelength range between 220 and 500 nm, or an LC/
MS.
A yellow compound, exhibiting an absorption maxi-
mum at 441 nm, was detected in the mixture containing
N-(1-deoxy-D-fructos-1-yl)-L-proline; however, it was
lacking in a heated solution of N-(1-deoxy-D-fructos-1-
yl)-L-alanine, indicating that its formation was strongly
dependent on the amino acid. LC/MS measurements
with negative atmospheric pressure chemical ionization
revealed an [M + 1 - H2]- ion at m/z 235 (100%). A
loss of 15 to a signal at m/z 220, most likely correspond-
ing to the cleavage of a methyl group, and a methyl
signal at 3.7 ppm in the 1H NMR spectrum made a
methyl acetal structure very likely. Because, in addi-
tion, a furan ring, two E-configured vicinal olefinic
protons showing a coupling constant of 18 Hz, and an
1
additional methyl group were observed in the H NMR
spectrum, the structure of the colorant was assumed as
4-hydroxy-2-methoxy-2-methyl-5-[(E)-(2-furyl)methyli-
dene]methyl-2H-furan-3-one (1, Figure 1). The corre-
sponding 2-ethoxy derivative was reported by Ledl and
Ledl and Fritsch (1984) reported on the pyrrolinone
formation in aqueous solutions of acetylformoin and
primary amines. To study whether this type of reaction
might be the reason for the absence of acetylformoin in
F igu r e 2. Formation of acetylformoin [3,4-dihydroxy-3-hexene-2,5-dione (2a ) and 2,4-dihydroxy-2,5-dimethyl-3(2H)-furanone
(2b)] via 1-deoxy-2,3-hexodiulose as the key intermediate.

3924 J. Agric. Food Chem., Vol. 46, No. 10, 1998
Hofmann
mary amino acids, thereby explaining the absence of
detectable amounts of acetylformoin in heated N-(1-
deoxy-D-fructos-1-yl)-L-alanine. As proposed in Figure
4, the formation of the pyrrolinone-reductones 4 and 5
might run via Schiff base formation of the amino acid
with the open-chain form of acetylformoin (2a ), followed
by cyclization. However, the amine-induced ring open-
ing of the cyclic hemiacetal form of acetylformoin (2b),
followed by cyclic hemi aminal formation, might also
result in compounds 4 and 5 (Figure 4).
Also in the presence of L-proline, the acetylformoin
was converted to several secondary reaction products.
N-(1-Methyl-1,2,3-trihydroxy-2-cyclopentene-4-ylidene)-
pyrrolidinium betaine (pyrrolidino-hexose-reductone; 6
in Figure 5) and N-[1-methyl-1,2-dihydroxy-3-(1-pyrroli-
dino)-2-cyclopenten-4-ylidene]pyrrolidinium betaine (bis-
pyrrolidino-hexose-reductone; 7 in Figure 5) were iden-
tified in yields of about 1.2 or 0.5%, respectively, by
comparison of the retention times (RP-18) and UV-vis
as well as LC/MS data with those obtained for the
synthetic reference compounds. On the basis of the
results of 14C-labeling experiments applied to the cor-
responding piperidino-hexose-reductone (Simon, 1962),
the formation pathways leading from acetylformoin (2a /
2b) and proline to the amino-hexose-reductones 6 and
7 are displayed in Figure 5. Proline-induced ring
opening of acetylformoin (2b) results in 4-hydroxy-5-
[1-(2-carboxy)pyrrolidino]-4-hexene-2,3-dione (I), which,
upon decarboxylation and enolization, gives rise to the
intermediate II. Ring closure via the nucleophilic
enamine group forms the pyrrolidino-hexose-reductone
(6). Schiff base formation of intermediate II with a
molecule of pyrrolidine, which is formed by decarboxyl-
ation of proline under roasting conditions (data not
shown), followed by enolization and nucleophilic ring
closure, yields the corresponding bispyrrolidino-hexose-
reductone (7). To study whether the amino-hexose-
reductones 6 and 7 were also formed from carbohydrates
and secondary amino acids, we reacted glucose and
proline under dry-heating conditions. After chromato-
graphical purification, we identified the amino-hexose-
reductones 6 and 7 by comparison of the UV-vis and
F igu r e 3. Formation of 3,4-dihydroxy-6-[(E)-(2-furyl)meth-
ylidene]-3-hexene-2,5-dione (3a ), 2,4-dihydroxy-2-methyl-5-
[(E)-(2-furyl)methylidene]methyl-2H-furan-3-one (3b), and 4-hy-
droxy-2-methoxy-2-methyl-5-[(E)-(2-furyl)methylidene]methyl-
2H-furan-3-one (1).
the dry-heated N-(1-deoxy-D-fructos-1-yl)-L-alanine mix-
ture, acetylformoin was heated in the presence of the
primary amine glycine methyl ester, the primary amino
acid glycine, and, in comparison, the secondary amino
acid L-proline. HPLC analyses of these reaction mix-
tures showed that in the presence of glycine methyl
ester and glycine the acetylformoin was rapidly decom-
posed to a variety of products, among which 2,4-
dihydroxy-2,5-dimethyl-1-methoxycarbonylmethyl- (4,
Figure 4) and 2,4-dihydroxy-2,5-dimethyl-1-carboxy-
methyl-3-oxo-2H-pyrrole (5, Figure 4), respectively,
could be identified in yields of ∼6% by comparison of
the UV-vis and the LC/MS data as well as the retention
times (RP-18) with those obtained for the synthetic
reference compounds. These results clearly demon-
strated that pyrrolinone-reductones were rapidly formed
under roasting conditions from acetylformoin and pri-
F igu r e 4. Formation of 2,4-dihydroxy-2,5-dimethyl-1-methoxycarbonylmethyl- (4) or 2,4-dihydroxy-2,5-dimethyl-1-carboxymethyl-
3-oxo-2H-pyrrole (5) from the reaction of acetylformoin (2a /2b) with glycine methyl ester or glycine, respectively.

Colored Maillard Reaction Products from Acetylformoin
J. Agric. Food Chem., Vol. 46, No. 10, 1998 3925
F igu r e 5. Reaction pathways leading to pyrrolidino-hexose-reductone (6) and bispyrrolidino-hexose-reductone (7) from
acetylformoin and L-proline.
the LC/MS data as well as the retention times (RP-
HPLC) with those obtained for the synthetic reference
compounds. To our knowledge, this is the first time that
the pyrrolidino-hexose-reductone (6) was identified in
a carbohydrate/proline mixture, whereas the bispyrro-
lidinyl-hexose-reductone (7) was identified by Papst et
al. (1984) in a heated sucrose/proline mixture. Both
amino-hexose-reductones were, however, found as bit-
ter-tasting Maillard reaction products in a heated
ethanolic solution of glucose and pyrrolidine (Papst et
al., 1984).
The data presented indicate that the formation of
either pyrrolinone-reductones (4, 5) or amino-hexose-
reductones (6, 7) from acetylformoin is determined by
the amino acid. Because in foods primary and second-
ary amino acids occur side by side, we studied whether
the formation of either the pyrrolinone 5 or the amino-
hexose-reductones 6 and 7 from glucose is favored in
the presence of primary and secondary amino acid. A
mixture of N-(1-deoxy-D-fructos-1-yl)-L-proline was, there-
fore, dry-heated in the presence of glycine. HPLC
analysis of the products formed revealed that the

3926 J. Agric. Food Chem., Vol. 46, No. 10, 1998
Hofmann
F igu r e 7. Structure of 3-hydroxy-4-[(E)-(2-furyl)methylidene]-
methyl-3-cyclopentene-1,2-dione (9).
Ta ble 5. F or m a tion of 3-Hyd r oxy-4-[(E)-
(2-fu r yl)m eth ylid en e]m eth yl-3-cyclop en ten e-1,2-d ion e (9)
u p on Dr y Hea tin g of Sever a l P r ecu r sor sa
precursors reacted
amount of 9
in the presence of
furan-2-carboxaldehyde
mg
%
F igu r e 6. Structure of 2,4-dihydroxy-2-methyl-5-[(E)-(2-furyl)-
methylidene]methyl-1-methoxycarbonylmethyl-3-oxo-2H-pyr-
role (8).
bispyrrolidino-hexose-reductone (7)
pyrrolidino-hexose-reductone (6)
3-hydroxy-4-methyl-3-cyclopentene-
1,2-dione (10)
0.9
3.6
23.1
0.9
3.5
22.7
methylene-reductinic acid (11)
28.7
28.1
pyrrolinone 5 was preferentially formed with a yield of
∼0.3%, whereas the formation of the amino-hexose-
reductones 6 and 7 was nearly stalled. Ledl and Fritsch
(1984) obtained similar results in heated aqueous solu-
tions of glycine ethyl ester and the synthetically related
N-(1-deoxy-D-fructos-1-yl)-piperidine. However, this is
the first time that the key role of acetylformoin as a
chemical switch in the Maillard reaction determining
the formation of pyrrolinone reductones (5) and amino-
hexose-reductones (6, 7) could be demonstrated with
food-related amino acids, thereby implying that similar
reactions can be expected to participate in nonenzymatic
browning during the thermal processing of foods.
F or m a tion of Color ed Com p ou n d s fr om Acetyl-
for m oin in th e P r esen ce of P r im a r y a n d Secon d -
a r y Am in o Acid s. To study the influence of primary
and secondary amino acids on the formation of colored
Maillard reaction products, a mixture of acetylformoin
(2a /2b) and glycine methyl ester or L-proline, respec-
tively, was dry-heated in the presence of furan-2-
carboxaldehyde. In the deeply colored mixture contain-
ing glycine methyl ester, a colored compound was
detected, which could be identified as 2,4-dihydroxy-2-
methyl-5-[(E)-(2-furyl)methylidene]methyl-1-methoxy-
carbonyl-methyl-3-oxo-2H-pyrrole (8, Figure 6) by com-
parison of the UV-vis and LC/MS data as well as the
retention time (RP-18) with those obtained for the
synthesized reference compound. A homologous com-
pound formed from 1-ethoxycarbonylmethyl derivative
and 5-(hydroxymethyl)furan-2-carboxaldehyde in aque-
ous solution was earlier reported by Ledl and Fritsch
(1984).
a
The precursor (0.5 mmol) and furan-2-carboxaldehyde (1.5
mmol) were intimately mixed with silica gel (1 g) containing
phosphate buffer (150 µL; pH 5.0, 0.5 mol/L) and then dry-heated
for 10 min at 180 °C.
F igu r e 8. Structures of 3-hydroxy-4-methyl-3-cyclopentene-
1,2-dione (10) and methylene-reductinic acid (11).
pound 9 were as yet available in the literature, the NMR
data are listed in Tables 3 and 4.
The piperidino-hexose-reductone used by Ledl and
Severin (1982) for the synthesis of 9 is not a food-related
compound, and also the reaction conditions chosen are
not suitable to simulate the thermal processing of foods.
It was, therefore, as yet not clear whether the formation
of 9 might also occur under food-relevant conditions. We
therefore studied whether the pyrrolidino- and bispyr-
rolidino-hexose-reductones 6 and 7, formed from the
more food-related glucose and L-proline, might generate
colorant 9 under conditions used in roasting processes.
To elucidate the effectiveness of the amino-hexose-
reductones 6 and 7 in the formation of colorant 9, we
heated the pyrrolidino- as well as the bispyrrolidino-
hexose-reductone in the presence of furan-2-carboxal-
dehyde under dry-heating conditions and determined
the amounts of compound 9 formed (Table 5). Because
studies of Ledl et al. (1982) revealed 3-hydroxy-4-
methyl-3-cyclopentene-1,2-dione and methylene-reduc-
tinic acid as the major hydrolysis products of piperidino-
hexose-reductones, we, in comparison, reacted synthetic
3-hydroxy-4-methyl-3-cyclopentene-1,2-dione (10, Figure
8) and methylene-reductinic acid (11, Figure 8) with
furan-2-carboxaldehyde. The results, given in Table 5,
showed that the methylene-reductinic acid (11) and
3-hydroxy-4-methyl-3-cyclopentene-1,2-dione (10) gener-
ated colorant 9 most effectively with yields of 28.1 and
22.7%, respectively. Also, 6 and 7 were found as
precursors of 9; however, 31- or 8-fold lower amounts
of the colorant were formed compared to the methylene-
reductinic acid. These data indicate that the formation
of colorant 9 from glucose and L-proline via amino-
hexose-reductones as intermediates is possible under
dry-heating conditions, implying that similar reactions
Also, dry-heating of the acetylformoin/L-proline mix-
ture in the presence of furan-2-carboxaldehyde led to
an intense colorization of the reaction mixture.
A
colored compound was registered by HPLC/DAD exhib-
iting an absorption maximum at 441 nm. LC/MS
measurements revealed an [M + 1]+ ion at m/z 205 with
a loss of 18 to the base peak at m/z 187 and a loss of 28
to m/z 177, most likely corresponding to the cleavage of
H2O and CO, respectively. The UV-vis and MS data
were well in line with those of 3-hydroxy-4-[(E)-(2-furyl)-
methylidene]methyl-3-cyclopentene-1,2-dione (9, Figure
7), which was prepared by Ledl and Severin (1982) from
piperidino-hexose-reductone, furan-2-carboxaldehyde,
and concentrated hydrochloric acid. To confirm our
supposition, colorant 9 was synthesized and character-
ized by several NMR measurements. Because neither
the signal assignment nor the 13C NMR data of com-

Colored Maillard Reaction Products from Acetylformoin
J. Agric. Food Chem., Vol. 46, No. 10, 1998 3927
F igu r e 9. Formation pathways leading to 3-hydroxy-4-[(E)-(2-furyl)methylidene]methyl-3-cyclopentene-1,2-dione (9) from
N-(1-deoxy-D-fructos-1-yl)-L-proline.
F igu r e 10. Role of acetylformoin (2b) as a switch determining the formation of the colorants 3a /3b, 8, and 9 in the presence of
primary and secondary amino acids.
might participate in color formation during, for example,
roasting of coffee or baking of bread.
cyclopentene-1,2-dione (10) and methylene-reductinic
acid (11), which were demonstrated by quantitative
measurements to form colorant 9 very effectively in the
presence of furan-2-carboxaldehyde.
Con clu sion s. The data presented indicate that in
the presence of primary and secondary amino acids, the
hexose-derived Maillard intermediate acetylformoin (2a /
2b) predominantly reacts with the primary amino acid,
leading to pyrrolinone-reductones (4, 5) and, in the
presence of a carbonyl compound, to the corresponding
On the basis of these quantitative data, the following
reaction pathway leading to 9 from N-(1-deoxy-D-fructos-
1-yl)-L-proline is displayed in Figure 9. Decomposition
of N-(1-deoxy-D-fructos-1-yl)-L-proline leads to acetyl-
formoin (2a /2b), which, upon reaction with L-proline,
gives rise to pyrrolidino-hexose-reductone (6) and bispyr-
rolidino-hexose-reductone (7) as detailed in Figure 5.
Hydrolysis of 6 and 7 yields 3-hydroxy-4-methyl-3-

3928 J. Agric. Food Chem., Vol. 46, No. 10, 1998
Hofmann
Hofmann, T. Studies on the influence of the solvent on the
contribution of single Maillard reaction products to the total
color of browned pentose/alanine-solutionssa quantitative
correlation by using the color activity concept. J . Agric. Food
Chem. 1998b, in press.
Hofmann, T. Characterization of the most intense coloured
compounds from Maillard reactions of pentoses by applica-
tion of colour dilution analysis (CDA). Carbohydr. Res.
1998c, submitted for publication.
Hofmann, T. Characterisation of precursors and elucidation
of the reaction pathway leading to a novel coloured 2H,7H,-
8aH-Pyrano[2,3-b]pyran-3-one from pentoses by quantita-
tive studies and application of 13C-labeling experiments.
Carbohydr. Res. 1998d , submitted for publication.
Hofmann, T.; Heuberger, S. Determination of the contribution
of coloured Maillard reaction products to the total colour of
browned glucose/alanine solutions and studies on their
formation. Z. Lebensm. Unters. Forsch. 1998, in press.
Kanjahn, D.; J arms, U.; Maier, H. G. Hydroxymethylfur-
fural and furfural in coffee and related beverages. Dtsch.
Lebensm. Rundsch. 1996, 92, 328-331.
Ledl, F.; Fritsch, G. Formation of pyrrolinone-reductones in
heated mixtures of hexoses and amino acids (in German).
Z. Lebensm. Unters. Forsch. 1984, 178, 41-44.
Ledl, F.; Severin, Th. Formation of colored compounds from
hexoses (in German). Z. Lebensm. Unters. Forsch. 1982, 175,
262-265.
Ledl, F.; Fritsch, G.; Severin, Th. Methylene-reductinic acid,
a novel reductone from glucose (in German). Z. Lebensm.
Unters. Forsch. 1982, 175, 208-210.
Papst, H. M. E.; Ledl, F.; Belitz, H.-D. Bitter tasting com-
pounds in heated proline/sucrose mixtures (in German). Z.
Lebensm. Unters. Forsch. 1984, 178, 356-360.
chromophore of type 8 (Figure 10). The condensation
of acetylformoin with a carbonyl compound to the
corresponding colorant of type 3a /3b , the proline-
induced formation of the amino-hexose-reductones 6 and
7 and, consequently, the formation of colored compounds
of type 9, are, therefore, mostly suppressed.
Because in foods primary amino groups of amino acids
as well as lysine residues of proteins were predominant
in comparison to secondary amino groups, the formation
of pyrrolinone-reductones should be favored. Neverthe-
less, the thermal processing of foods with a high content
of L-proline, such as malt, might lead to the formation
of colored compounds with chromophores of type 9.
The results of the present investigation on the reac-
tions of certain Maillard intermediates provide useful
information to extend the knowledge on chromophores
generated by nonenzymatic browning during food pro-
cessing and will help to construct a route map of
reactions leading to color development in heated food-
stuffs.
ACKNOWLEDGMENT
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.
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Hofmann, T. Determination of the chemical structure of novel
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Received for review May 15, 1998. Revised manuscript
received August 10, 1998. Accepted August 13, 1998.
J F980512L