Carbohydrate Research

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ACID-CATALYZED DEGRADATION OF D-F~W~-CX%

PHILIP E. SHAW, JAMESH. TATUM,ANDROBERTE. BERRY

U. S. Fruit and Vegetable Products Laboratory *. Winter Haven, FIorida 33880 (U. S. A.)
(Received April 21st, 1967; in revised forrn, June 16tb, 1967)

An attempt to study the nonenzymic browning reactions occurring in stored,

dehydrated orange juice suggested that preliminary studies on a simple model system
might lead to the development of more efficient methods. Such a study would also pro-
vide background information for interpretation of data obtained with the more
complex system present in powdered, dehydrated citrus juice.
A number of different approaches have been used to study nonenzymic browning,
which is usually initiated in foods by reactions of reducing sugars with amines or
acids, especially basic amino acids’. One approach has been the examination of changes
that carbohydrates undergo in presence of the amines or acids2P3. Another has been
the study of the products formed when acidic solutions of sugars are heated. Sugisawa4
used column-chromatographic separation to identify several acids and esters formed
when acidic solutions of D-glucose are heated. Livingston5 reported that malic acid
causes more-rapid browning of D-fructose than does hydrochloric acid at the same PH.
In another approach, involving studies on syrups and on fruit-juice concentrates,
Braverman observed darkening on heating, and a relationship between pH and rate
of reaction; a pH of 3-4 was found optimal for this browning reaction, and a corre-
lation between darkening and appearance of 5-(hydroxymethyI)-2-furaldehyde was
demonstrated. Berry and Taturn’ isolated I(hydroxymethyl)-2-furaldehyde from
dehydrated orange juice that had been stored at elevated temperatures. Further
investigations of these findings indicated that one of the principal precursors of
substances formed in dehydrated orange powders during storage is D-fructose.
To find out if D-fructose is a principal precursor of off-flavors that may develop
in citrus juices on storage, the products of ‘thermal degradation of D-fructose in acid
solution were studied at three pH values: pH 3.5 (in the range commonly encountered
in citrus juice), pH 2.15, and pH 1.0. The highest acidity permitted more rapid accu-
mulation of materials in quantities sufficient for complete~dentilication, and provided
information concerning the effect of pH on the course of acid-catalyzed, nonenzymic
browning;

*One of the laboratories of the Southern Utilization Research and DeveIopment Division, AgrIcuI-
tural Research Service, U. S. Department of AgricuIture. References to products do not constitute
endorsement.

Carbohyd. Res., 5 (1967) 266-273

268 P.E. SHAW, J. H. TATUM, R. E. BERRY

identification was not made until we studied the reaction at pH 1.0, in which much
greater proportions of these acids were formed.
At pH 2.15, all compounds found at pH 3.5 were present, together with 2-(2-
hydroxyacetyl)furan for-mate (10). This compound (previously unreported) was
identified by comparison with a sample synthesized from 9 and formic acid.
Degradation of D-fructose at pH 1.0 with hydrochloric acid afforded all of the
compounds in Table I except 11.5-Methyl-2-ftualdehyde (6) was the only compound
present at this pH that was not found at either of the higher pH levels. Identity in
degradation products when either citric or hydrochloric acid was used would appear
to exclude citric acid as a contributing source for such products as formic or acetic acid.
Identification of isomaltol (7) is of special interest, because it had previously
been found as a degradation product of maltose and of lactose l”*ll. It has not pre-
viously been identified as a degradation product of D-fructose.
The structural assignment for the previously unreported compound 11 was
based on its spectra. The ultraviolet maximum at 298 nm (a 6,300) is similar to that of
2,5-dimethyl+hydroxy-3(2N)-furanone (14) [&,,
EtoH 289 nm (a 6,700)]‘2 and also to
that of 4-hydroxy-2,3,Shexanetrione, which exists in the cyclic form 5a in ethanol
[lzzH 303 nm (E 7,500)] l3 . The infrared spectrum showed a strong, broad hydroxyl
band at 3320 cm-‘; a strong band at 1660 cm-’ and a very strong band at 1610 cm-’
indicated a conjugated carbonyl group. Similar bands were noted”*13 for related
compounds 14 and §a, although the overall spectra of the latter substance and com-
pound 11 (which have the same molecular weight) were quite different. The mass
spectrum indicated a molecular weight of 144 for 11,and the cracking pattern was
similar to, but not identical with, that of 4-hydroxy-2,3,5-hexanetrione (form 5a).
The n.m.r. spectrum of compound 11 could be interpreted in the methyl region
only, because of the small quantity of material available. A methyl singlet at r 7.88
was observed with the use of a time-averaging computer, indicating structure 11
rather than its tautomer lla. This value is in good agreement with the methyl singlets
of 14,15, and 16, found at z 7.80,7.87, and 7.88, respectively’2*‘4.
Consideration of mechanisms of formation indicates that most of these products
can be formed from D-fructose by a simple combination of enolization and dehy-
dration steps. Anet 2*g~15 has shown (Chart I) that acid-catalyzed degradation of
D-fructose occurs primarily through 1,Zenolization (Parh A) with loss of the 3-hydr-
oxyl group by dehydration and then further dehydration to give 5-(hydroxymethyl)-2-
furaldehyde (13). When the less-favorable 2,3-enolization occurs, elimination of the
4-hydroxyl group (Path B) leads to 2-(hydroxyacetyl)furan2.” (9)_ Elimination of the
l-hydroxyl group and dehydration of a furanose form of the 2,Zdiulose thus formed
(Path C) can lead to isomaltol”. However, dehydration of an alternative furanose form
of the 2,3&ulose (Path D) can produce compound 11. On several occasions, the spon-
taneous transformation of 11 into isomaltol(7) has been observed in chloroform. This
could be explained by assuming that a trace of acid and water could reverse Path D to
form the 2,3-diulose, which could give isomaltol by Path C. 4-Hydroxy-2,3,5-hexane-
trione (5) can be formed from the 2,3diulose by further enolization, followed by

Carbohyd.Res., 5 (1967) 266-273

 ACID-CATALYZED DEGRADATION OF D-Fitucl-o= 269

CHzOH HCOH HC=O

I II
C=O COH c=o
I I I
HOCH HOCH -YO
‘CH2
I - I -
HCOH HCOH HCOH
I I
HCOH H$OH 13
HCOH

C%OH C&Oh

CI-Fructose 1.2-.enediol

CHzOH CHzOH
I I
$-OH +O COC&OH
-H;?O -2Hz0
&-OH L=o - Path B
I - I
CHOH CH2
I I
CHOH CHOH 9
I
C&OH CHzOH

2,3 - enediol

- Hz0
1
1 OH

Path c
CH3
c-
C==O
I
q=o 7

AioH
I
CHOH _
I
CH?OH

lla
2.3 -cliulose

CH3 CH3
I 7
c=o c=o Lo
C 0 OH CHJ
I I - H2Q
I Path E
C-OH CHOH CHOH
II - I -1 = I
C-OH C-OH c=o J=L 0
HO
I
CHOH !-OH +o
I I
C&O’+ C&OH CH3

5 5a

14. R=H
15: R=CH3
16. R=COCHs

Carbohyd- Res., 5 (1967) 266-273

270 P. E. !SHAW, J. H. TATUM, R. E. BERRY

dehydrationwithloss of the 6-hydroxyl group (Path E)10*16. The fact that the quantities
of products 5, 7, and 11 isolated were small, relative to 2-(2-hydroxyacetyl)furan (9),
indicates that elimination of a C-4 hydroxyl group is favored over a C-l hydroxyl group,
following 2,3-enolizationz under these conditions.
The three carboxylic acids and two lactones that were identified were probably
produced by further degradation of some of the compounds already mentioned. Thus,
acetic acid and formic acid can be formed by acid-catalyzed degradation of isomaltol”.
Levulinic acid is a known degradation product of 5-(hydroxymethyl)-2-furaldehyde”.
The a- and /3-angelica lactones are probably formed from levulinic acid during the
distillation step’*.
Currently, stored dehydrated citrus powders are being examined for the presence
of the above mentioned and related compounds, which might have been formed by
nonenzymic browning. Methods of analysis that have been developed in the present
study are being applied to stored citrus-juice powders, and a report on these results will
be forthcominglg.

EXPERIhENTAL

Melting points are uncorrected. U.V. spectra were recorded in 95% ethanol with
a Cary-14 spectrophotometer, i.r. spectra were obtained with a Perkin-Elmer 137
Infracord spectrophotometer, and mass spectra were determined with a Bendix Model
12-100 Time-of-Flight mass spectrometer. Mass-spectral data give m/e values, and
peak intensities expressed, in parentheses, as percent of the most intense peak. G.1.c.
analyses were performed with an F &M Model 810 instrument on both a silicone
column (G-E. SE-30,23x on Gas-Chrom Z; 9.5ft x 0.25 in. stainless-steel column;
83 ml of helium per min; injection port at 190”, on-column injection; column oven
initially at 65” and programmed at 15” per min to 150”; 5:l splitter at oven temper-
ature; and flame-ionization detector at 210°) and on a poly(oxyethylene) column
(Carbowax 20M, 20% on 60-80 mesh Gas-Chrom P; 4 ft x 0.25 in. stainless-steel
column; 130 ml of helium per min; injection port at 190”, on-column injection;
column oven at 50” for 4 min, and then programmed at 60” per min to 130°, isothermal
until 20 min into run, and then programmed at 60” per min to 170”, isothermal until
48 min into run, and then programmed at 60” per min to 200”, and then isothermal;
5:l splitter at oven temperature; and flame-ionization detector at 210”). G.1.c. samples
were collected in capiliary tubes cooled with liquid nitrogen.
Thin-layer chromatograms’ were developed in 200:47:15:1 benzene-ethanol-
water-cone . ammonia” (upper phase).
Footnotes to each compound indicate the method of preparation for comparison
samples.
Acid-catalyzed degradation of mfructoseg. - A. At pW 3.5. A solution of 240 g
of D-fructose (Fisher Scientific Co., Fair Lawn, N. J.) in 440 ml of water, adjusted to
pH 3.5 by the addition of 0.044 g of citric acid, was heated for 5 h at 100”. Sodium
chloride (50 g) was added, and the mixture was cooled, and extracted with one 300~ml
Carbohyd. Res., 5 (1967) 266-273
ACID-CATALYZED DEGRADATION OF D-J%TJCl-OSE 271

portion and three 200-ml portions of ether. The extracts were combined, and evapor-
ated to dryness under diminished pressure at room temperature. At this point,
5-(hydroxymethyl)-2-furaldehyde (13) could be crystallized by dissolving the residue
in ether and cooling the solution in a freezer. As the distillation described below
separated the minor components from 13,this crystallization step was usually omitted.
Compound 13 was recrystallized from ether, and identzed by its i-r. spectrum, its
mass spectrum [m/e 126 (59), 109 (S), 97 (68), 69 (30), 53 (20), 52 (1 1), 51 (IQ, 50
(12), 41 (loo), and 39 (60’/,11, and by t.1.c. comparison, with an authentic sample
(Fluka AG, Buchs S G, Switzerland).
The total residue from the combined ether extracts was distilled for 1 h at 60”,
with a trap cooled in liquid nitrogen as the receiver. The yellow solid that collected in
the receiver was dissolved in ether, and the ether solution was decanted from a small
amount of water that was usually present. The ether solution was dried, concentrated
to a small volume, and cooled, and the precipitate that formed was collected to give
2-(2-hydroxyacetyl)furan (9), m.p. SO-Sl”, m/e 126 (22), 95 (loo), 39 (18 “/,), identical
(by i.r., mass-spectral, and t.1.c. comparison) with an authentic sampIe’. The mother
liquor was analyzed by g.l.c., and the following compounds were collected in sufficient
quantities to permit positive identification by i-r., mass-spectral, and t.1.c. comparison
with authentic samples : furfural(4, Fisher Scientific Co.), m/e 99 (6), 96 (loo), 95 (79),
62 (2), 51(3), 50 (4), 42 (S), 40 (12), 39 (81%); a-angelica lactone (3, K&K Laboratories
Inc., Plainview, N. J.), m/e 98 (87), 55 (lOO), 43 (85%); /3-angelica lactone (8, K&K
Laboratories, Inc.), m/e 98 (83), 83 (57), 69 (19), 55 (loo), 43 (81%); isomaltol* (7),
m/e 126 (63), 111 (loo), 84 (7), 55 (21), 43 (32%); 4-hydroxy-2,3,5-hexanetrione” (S),
m-p. 78-79”, m[e 144 (33), 101 (59), 73 (33), 55 (36), 43 (100%); Ievufinic acid
(12, Eastman Organic Chemicals, Rochester, N. Y.), m/e 116 (IO), 56 (23), 55 (15), 45
(13), 43 (loo), 31 (46%); 4-hydroxy-2-(hydroxymethyl)-5-methyl-3(2H)-furanone (ll),
RUXIX 298 nm (E 6,300), vzLfhrn 3320 s, 2870 w, 1660 s, 1610 vs, 1460 w, 1420 m,
1380 W, 1270 W, 1205 s, 1180 m, 1080 m, 1060 m, 1020 m, 965 m, 910 m, 880 w,
810 w, 780 w, cm-l; m/e 144 (37), 101 (31), 73 (14), 72 (20), 55 (25), 45 (25), 44 (75),
43 (10073. Compounds 9 and 13 were also isolated from the g.1.c. column.
B. At pN 2.15. The amount of citric acid added was 6.10 g. One additional
compound was isolated, which was found to be 2-(2-hydroxyacetyl)furan formate (IO),
m-p. 55.0-55-S”, I.,, 271 nm (e 18,000), 223 nm (E 4,600), ~2;~‘~ 3090 w, 2900 w,
1725 s, 1680 s, 1570 m, 1465s, 1390 m, 1265 m, 1255w, 1220 w, 1175 s, 1155 s, 108Om,
1045 m, 1035 m, 1015 m, 945 m, 910 m, 895 w, 883 m, 770 s, cm-l;m/e 154 (12), 95
(lOO), 40 (62x), identical (by i-r., mass-spectral, and t.1.c. comparison) with the sample
prepared from 9 and formic acid.
Authentic 10 was prepared as follows: a solution of 9 (0.04 g) in 0.4 ml of 90%
formic acid was kept for 22 h at room temperature. The formic acid was removed by
warming under a nitrogen stream, and the residue was dissolved in 0.5 ml of chloro-
form and spotted onto an 8 x &inch plate coated with a I-e-layer of silica gel
.TM

*J. E. Hodge kindly provided us withan authenticsample.

Carbohyd. Res., 5 (1967) 266-273
272 P. E. SHAW, J. H. TATUM, R. E. BERRY

IS-254 (BrinkmannInstruments, Inc., Westbury, L.I., N.Y.). The less-polar, absorb-

ing band was scraped off, and eluted with acetone. Concentration of the eluate, and
crystallization of the residue from ether-pentane gave 10,m-p. 55-55.5”. Three re-
crystallizations from ether-pentane afforded an analytical sample, m-p. 55.5-56.0”.
And. Calc. for C,HsO,: C, 54.55; H, 3.92. Found: C, 54.36; H, 4.04.
An indication of the relative proportions of products formed from 240 g of
D-fructose at pH 2.15 was obtained hy g.1.c. analysis of the total crude extract, and of
the distillate, on a silicone column by the cut-and-weigh method. From 1.16 g of
crude extract (87% of 13,9% of 9, and 4% of lower-boiling material) there was
ob*&ined 0.30 g of distilllate (72% of 9, 17% of a mixture of 7 and 5, 4% of 11,and
7% of lower-boiling material).
At pH 1.0.Hydrochloric acid was added to pH 1.0. Analysis of the B-fructose
degradation mixture showed the presence of all compounds in Table I except 11.
In addition, 5-methyl-2-furaldehyde (6) was produced; m/e 110 (100) 109 (85), 81(13),
53 (69), 51 (22), 50 (16), 43 (15), 39 (18%); identical (by i.r., mass-spectral and t.1.c.
comparison) with an authentic sample (K&K Laboratories, Inc.).
Formic acid and acetic acid came off the silicone column together, as a single,
broad peak in quantities larger than were found at the other two levels of acidity.
The leading half of the peak was collected, and shown by infrared analysis to be formic
acid. The infrared spectrum of the material collected from the trailing half of the peak
revealed it to be mostly acetic acid together with some formic acid. Acetic acid, but not
formic acid, could be isolated on the Carbowax column.

SUMMARY

Interest in nonenzymic browning of dehydrated food-products led to this study

of the acid-catalyzed degradation of D-fructose. The two major furan derivatives
formed in this degradation are 5-(hydroxymethyl)-2-furaldehyde and 2-(2-hydroxy-
acetyl)furan. Several minor products were identified. These included furfural, 5-me-
thyl-2-furaldehyde, isomaltol, 2-(2-hydroxyacetyl)furan for-mate, and Phydroxy-2,3,5-
hexanetrione (acetylformoin). Formic acid, acetic acid, and levulinic acid were also
present. A new hydroxyfuranone has been isolated, and evidence is presented support-
ing its structure. Degradation studies at pH 1.0,2.15, and 3.5 showed a variation in
the minor components formed. The mechanism for formation of the furan derivatives
is discussed.

REFERENCES

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Carbohyd. Res., 5 (1967) 266-273

ACID-CATALYZED DEGRADATION OF D-FRUCTOSE 273

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Carbobyd. Res., 5 (1967) 266-273