Formation of 2-Pentylpyridine from the Thermal Interaction of Amino Acids and 2,4-Decadienal

Article abstract of DOI:10.1021/jf950623+

To study the mechanism of 2-pentylpyridine formation in model systems, 2,4-decadienal was reacted with five amino acids (glycine, aspartic acid, asparagine, glutamic acid, and glutamine) at 180°C for 1 h (pH 7.5). In addition to 2-pentylpyridine, 3-pentylpyridine was also tentatively identified from the thermal reactions. The relative yields of alkylpyridine formation from the reactions were asparagine > glutamine > aspartic acid > glutamic acid > glycine. When amide-¹⁵N-labeled glutamine and asparagine were heated with 2,4-decadienal, the relative contribution of amide nitrogens to the formation of alkylpyridine was determined. Approximately half of nitrogen atoms in 2-pentylpyridine formed were contributed by the amide nitrogens of asparagine, whereas almost all of them came from the amide nitrogens in glutamine. The results above may indicate that both free ammonia and α-amino groups bound in amino acids can contribute to the formation of alkylpyridines, but free ammonia does so more effectively.

Full text of DOI:10.1021/jf950623+

3906
J. Agric. Food Chem. 1996, 44, 3906−3908
F or m a tion of 2-P en tylp yr id in e fr om th e Th er m a l In ter a ction of
Am in o Acid s a n d 2,4-Deca d ien a l
Young-Suk Kim,† Thomas G. Hartman,‡ and Chi-Tang Ho*,†
Department of Food Science and Center for Advanced Food Technology, New J ersey Agricultural
Experiment Station, Cook College, Rutgers University, New Brunswick, New J ersey 08903
To study the mechanism of 2-pentylpyridine formation in model systems, 2,4-decadienal was reacted
with five amino acids (glycine, aspartic acid, asparagine, glutamic acid, and glutamine) at 180 °C
for 1 h (pH 7.5). In addition to 2-pentylpyridine, 3-pentylpyridine was also tentatively identified
from the thermal reactions. The relative yields of alkylpyridine formation from the reactions were
asparagine > glutamine > aspartic acid > glutamic acid > glycine. When amide-15N-labeled
glutamine and asparagine were heated with 2,4-decadienal, the relative contribution of amide
nitrogens to the formation of alkylpyridine was determined. Approximately half of nitrogen atoms
in 2-pentylpyridine formed were contributed by the amide nitrogens of asparagine, whereas almost
all of them came from the amide nitrogens in glutamine. The results above may indicate that both
free ammonia and R-amino groups bound in amino acids can contribute to the formation of
alkylpyridines, but free ammonia does so more effectively.
Keyw or d s: Amino acid-lipid interaction; 2-pentylpyridine; 2,4-decadienal; deamidation and
deamination; ammonia generation
INTRODUCTION
identified as one of the major volatiles even though they
could not identify dithiazine or thiadiazine which re-
quires free ammonia for their formation. It might
indicate that free ammonia was not available in those
systems and 2-pentylpyridine was formed without the
free ammonia. An alternative mechanism in the forma-
tion of 2-pentylpyridine as a result of the direct con-
densation of the amino group of amino acids or peptides
with the aldehydic group of 2,4-decadienal is proposed.
In the present study, the mechanism of alkylpyridines
formation was investigated from the thermal interaction
of 2,4-decadienal and each of the five amino acids
(glycine, aspartic acid, asparagine, glutamic acid, and
glutamine), separately. Aspartic acid, asparagine, and
glutamine can produce free ammonia readily, but
glutamic acid and glycine cannot under the conditions
used. The effects of amino acids and their relative
reactivities on the formation of alkylpyridines were also
studied. In addition, we examined the relative contri-
bution of free ammonia to the formation of alkylpy-
ridines compared with that of R-amino groups bound
in amino acids. The free ammonia can be derived from
both amino groups and amide side chains in asparagine
and glutamine. The contribution of amide nitrogens as
well as amino nitrogens to the formation of N-containing
flavor compounds has been reported previously (Hwang
et al., 1993; Izzo and Ho, 1992). We used asparagine
The interaction of primary amines in proteins or
amino acids with carbonyls in lipids may produce
N-containing heterocyclic volatile compounds such as
alkylpyridines. The importance of heterocyclic com-
pounds in food flavors are well-known, but relatively
little attention has been given to pyridine derivatives
even though many of them have been found in a large
number of foods (Maga, 1981; Vernin, 1982; Ishihara
et al., 1992; Thomas and Bassole, 1992). Among the
pyridine derivatives, 2-pentylpyridine deserves special
attention due to its strong fatty and tallow-like flavor
in diluted solution (Buttery et al., 1977) and its low odor
threshold (0.2 ng/L of air) (Schieberle, 1993). 2-Pen-
tylpyridine has also been identified as the major odor-
active component in mutton meat (Buttery et al., 1977;
Cramer, 1983) and was a major product when valine
was heated with linoleic acid or its esters (Henderson
and Nawar, 1981).
It was proposed that 2-pentylpyridine is formed in the
amino acid-linoleic acid system as a result of the
interaction of ammonia with 2,4-decadienal, one of the
major degradation products of linoleic acid (Henderson
et al., 1980; Kimoto and Gaddis, 1969; Patton et al.,
1959). In this mechanism, ammonia condensed with
2,4-decadienal to form a Schiff base intermediate,
followed by ring closure, leading to the formation of a
dihydropyridine which can then be oxidized to 2-pen-
tylpyridine. Therefore, the free ammonia is needed for
the formation of 2-pentylpyridine. This is further
supported by a recent study of Schieberle (1993) who
showed that the amount of 2-pentylpyridine was in-
creased by the addition of ammonium sulfate or grind-
ing before roasting in a commercial roasted sesame oil.
On the other hand, when Zhang et al. (1989) reacted
2,4-decadienal with glutathione, 2-pentylpyridine was
15N at the sites of amide
and glutamine labeled with
side chains to study the effects of amide nitrogens on
the formation of alkylpyridines.
EXPERIMENTAL PROCEDURES
Sa m p le P r ep a r a tion . Mixtures of trans,trans-2,4-deca-
dienal (0.001 mol) (Sigma Chemical Co., St. Louis, MO) and
five amino acids, i.e. glycine, L-aspartic acid, L-asparagine,
L-glutamic acid, and L-glutamine (0.005 mol) (Sigma), were
dissolved in 100 mL of distilled water, and the pH was adjusted
to 7.5 with 0.1 N NaOH or 0.1 N HCl. Each sample solution
was transferred into a 0.3 L Hoke SS-Dot sample cylinder and
then heated at 180 °C in an oil bath for 1 h. After the reaction,
undecane (100 ppm or 1000 ppm) (Polyscience, Niles, IL) was
added as an internal standard into each sample product which
* Author to whom correspondence should be ad-
dressed [fax (908) 932-6776].
† Department of Food Science.
‡ Center for Advanced Food Technology.
S0021-8561(95)00623-6 CCC: $12.00
© 1996 American Chemical Society

Thermal Interaction of Amino Acids and 2,4-Decadienal
J. Agric. Food Chem., Vol. 44, No. 12, 1996 3907
Ta ble 1. Rela tive Ion Abu n d a n ces (P er cen t) of
P en tylp yr id in es F or m ed fr om th e Rea ction of
Asp a r a gin e or Glu ta m in e w ith 2,4-Deca d ien a l
was then extracted with methylene chloride in a separatory
funnel by multiple extraction (3 × 100 mL). The solvent
extract was washed with distilled water (100 mL) three times
and dried over anhydrous sodium sulfate. The extract was
then concentrated with a Kuderna-Danish apparatus to a
volume of 5 mL and further concentrated with nitrogen gas
to a final volume of 0.1 mL. L-Asparagine-amide-15N and
L-glutamine-amide-15N (Isotech, Inc., Miamisburg, OH) were
used for the determination of derived ammonia to pentylpy-
ridine formation.
a
a
a
b
b
amino acid
alkylpyridine
M92
M93
M94
ML93
ML94
asparagine 2-pentylpyridine
7.37 100
7.19 100
9.70 100
89.27
59.77
3-pentylpyridine 74.51 100
glutamine 2-pentylpyridine 9.32 100
7.58
3-pentylpyridine 71.02 100 11.02
12.35 100
71.27 100
a
M92, M93, and M94 are the relative abundances of the ion peaks
Vola tile Sep a r a tion by Ga s Ch r om a togr a p h y. A Vari-
an 3400 gas chromatograph with an FID and a nonpolar fused
silica capillary column [60 m × 0.25 nm (i.d.), 0.25-µm
thickness, DB-1; J &W] was employed to analyze the volatile
compounds produced from the thermal reactions. The GC
equipment was operated with an injector temperature of 270
°C, a detector temperature of 300 °C, and a helium carrier gas
flow rate of 1.0 mL/min. The temperature of the GC column
was increased from 40 to 150 °C at a rate of 2 °C/min, from
150 to 190 °C at a rate of 1 °C/min, and from 190 to 280 °C at
a rate of 2 °C/min, holding at 280 °C for 30 min. For each
sample, 0.2 µL of solution was injected with a split ratio of
50:1.
from the reaction of unlabeled asparagine or glutamine with 2,4-
b
decadienal. ML93 and ML94 are the relative abundances of the ion
peaks from the reaction of asparagine-amide-15N or glutamine-
amide-15N with 2,4-decadienal.
After the GC was run, volatiles were quantified by compar-
ing their relative peak areas to the area of the internal
standard. Linear retention indices for the volatiles were
determined by comparing their retention times with those of
n-paraffin standard (C6-C19; PolyScience, Niles, IL), according
to the method of Majlat et al. (1974).
GC/MS An a lysis. The samples were analyzed by GC/MS
comprising a Varian 3400 gas chromatograph coupled with a
Finnigan MAT 8230 high-resolution, double-focusing magnetic
sector mass spectrometer (TD-GC-MS). Mass spectra were
obtained by using electron ionization of 70 eV and ion source
temperature of 250 °C. All mass spectra were identified by
an on-line computer library (NIST) or published literature.
Ca lcu la tion s for th e Rela tive Con tr ibu tion of 15N in
Am id e Sid e Ch a in s to Alk ylp yr id in es F or m a tion . If one
15N in an amide side chain of asparagine or glutamine is
transferred into a pyridine ring, the molecular weight of the
pyridine will be increased by one mass unit. There will,
therefore, be two kinds of pyridines which have different
molecular weights. The relative ratios to the overall alkylpy-
ridine of the two different pyridines produced can be desig-
nated by W1 and W2. W1 is the relative ratio of pyridines which
have no 15N in the pyridine ring to the overall alkylpyridines,
and W2 is the relative ratio of pyridines which have a 15N in
the pyridine ring to the overall alkylpyridines, respectively.
We compared m/z 92 (due to loss of one hydrogen from 93),
93, and 94 (due to natural isotope) in the mass spectra
obtained from the normal asparagine and glutamine with m/z
93, 94, and 95 increased by one unit from 15N-labeled ones.
The relative abundance of these peaks were selected to reduce
errors in calculation because m/z 93 was the ion which had
the maximum abundance and retained nitrogen in its ring
structure from the reaction of normal asparagine and glutamine
with 2,4-decadienal. When it was assumed that ML93 and ML94
were the experimental relative abundances of the ion peaks
in the reaction of 15N-labeled asparagine or glutamine with
2,4-decadienal and M92, M93, and M94 were the experimental
relative abundances of the ion peaks in the reaction of
unlabeled asparagine or glutamine with 2,4-decadienal, the
following equations could be used to obtain W1 and W2. The
experimental data are given in Table 1.
F igu r e 1. Amount of alkylpyridines formed from the interac-
tions of 2,4-decadienal and amino acids at 180 °C for 1 h (pH
7.5).
Ta ble 2. Ma ss Sp ectr a l Da ta of P en tylp yr id in es
Id en tified in th e Rea ction s of Am in o Acid s w ith
2,4-Deca d ien a l
pentylpyridine
mass spectral data, m/z (rel intensity)
2-pentylpyridine
93 (100), 106 (24), 120 (22), 65 (9), 78 (8),
92 (8), 94 (7), 51 (6), 107 (5); MW 149
93 (100), 92 (73), 39 (49), 149 (42), 65 (41),
41 (36), 106 (31), 51 (14), 57 (14), 78 (10);
MW 149
3-pentylpyridine
asparagine, glutamic acid, and glutamine separately.
The identification of 2-pentylpyridine was accomplished
by comparing its mass spectral and retention index data
with those of the NIST computer library and published
literature data (Zhang and Ho, 1989). In addition to
2-pentylpyridine, 3-pentylpyridine was tentatively iden-
tified from the mass spectral data. The mechanism of
3-pentylpyridine is not understood at the present time.
The mass spectral data of two alkylpyridines are shown
in Table 2.
ML93 ) M93W1 + M92W2
Figure 1 shows the amount of 2-pentylpyridine and
3-pentylpyridine formed from the interaction of 2,4-
decadienal with each of the five amino acids. As
expected asparagine and glutamine showed the most
activity toward the formation of two pentylpyridines.
It is well-established that asparagine and glutamine are
susceptible to nonenzymatic deamidation in the side
chain amide group and lead to aspartic acid and
glutamic acid with the release of a molecule of ammonia
(Wright, 1991). In asparagine and glutamine the rate
ML94 ) M94W1 + M93W2
The percentage contribution of amide nitrogens to the
formation of alkylpyridines were calculated by using W1 and
W2 as follows: % contribution ) (W2/(W1 + W2) × 100
RESULTS AND DISCUSSION
2-Pentylpyridine was identified in the mixtures when
2,4-decadienal was reacted with glycine, aspartic acid,

3908 J. Agric. Food Chem., Vol. 44, No. 12, 1996
Kim et al.
Ta ble 3. Rela tive Con tr ibu tion of Am id e Nitr ogen s to
P en tylp yr id in e F or m a tion fr om th e Rea ction of
Asp a r a gin e-a m id e-15N or Glu ta m in e-a m id e-15N w ith
2,4-Deca d ien a l
However, the reactivity of free ammonia is probably
much higher than that of the R-amino group.
LITERATURE CITED
amino acid
asparagine
pentylpyridine
% contribution
Buttery, R. G.; Ling, L. C.; Teranishi, R.; Mon, T. R. Roasted
fat: basic volatile components. J . Agric. Food Chem. 1977,
25, 1227-1229.
Cramer, D. A. Chemical compounds implicated in lamb flavor.
Food Technol. 1983, 37 (5), 249-260.
2-pentylpyridine
3-pentylpyridine
46.77
47.46
glutamine
2-pentylpyridine
3-pentylpyridine
97.00
99.50
Henderson, S. K.; Nawar, W. W. Thermal interaction of linoleic
acid and its esters with valine. J . Am. Oil Chem. Soc. 1981,
May, 632-635.
Henderson, S. K.; Witchwoot, A.; Nawar, W. W. The autoxi-
dation of linoleates at elevated temperature. J . Am. Oil
Chem. Soc. 1980, December, 409-413.
Hwang, H. I.; Hartman, T. G.; Rosen, R. T.; Ho, C. T.
Formation of pyrazine from the Maillard reaction of glucose
and glutamine-amide-15N. J . Agric. Food Chem. 1993, 41,
2112-2115.
Ishihara, M.; Tsuneya, T.; Shiga, M.; Kawashima, S.; Yam-
agishi, K.; Yoshida, F.; Sato, H.; Uneyama, K. New pyridine
derivatives and basic components in spearmint oil (Mentha
gentilis f. cardiaca) and peppermint oil (Mentha piperita).
J . Agric. Food Chem. 1992, 40, 1647-1655.
Izzo, H. V.; Ho, C. T. Ammonia affects Maillard chemistry of
an extruded autolyzed yeast extract: pyrazine aroma gen-
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657-674.
Kimoto, W. I.; Gaddis, A. M. Precursors of alk-2,4-dienals in
autoxidized lard. J . Am. Oil Chem. Soc. 1969, 46, 403-
408.
Majlat, P.; Erdos, Z.; Takacs, J . Calculation and application
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matography. J . Chromatogr. 1974, 91, 89-103.
Maga, J . A. Pyridines in foods. J . Agric. Food Chem. 1981,
29, 895-898.
Patton, S.; Barnes, I. J .; Evans, L. E. n-Deca-2,4-dienal, its
origin from linoleate and flavor significance in fats. J . Am.
Oil Chem. 1959, 36, 280-283.
Schieberle, P. Studies on the flavor of roasted white sesame
seeds. In Progress in Flavor Precursor Studies; Schreier,
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IL, 1993; pp 343-360.
Schieberele, P.; Grosch, W. Model experiments about the
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Soc. 1981, May, 602-607.
Sohn, M. G.; Ho, C. T. Ammonia generation during thermal
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2243.
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7, 23-35.
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of deamidation is faster than the rate of thermal
deamination of the R-amino groups (Sohn and Ho, 1995).
It was further demonstrated that free ammonia gener-
ated from the deamidation and the deamination of
amino acids may participate in the formation of nitrogen-
containing flavor compounds such as pyrazines, py-
ridines, and pyrroles (Izzo and Ho, 1992; Hwang et al.,
1993). In a study reported by Sohn and Ho (1995), it
has been shown that asparagine released more am-
monia than glutamine under the same conditions. The
asparagine released ammonia from both amide and
R-amino groups, whereas the ammonia released from
glutamine was mainly due to the deamidation of the
amide group. The fact that the interaction of 2,4-
decadienal with asparagine and glutamine generated
more 2-pentylpyridines than other amino acids may
suggest that free ammonia is essential to the formation
of 2-pentylpyridines.
As shown in Figure 1, both aspartic acid and glutamic
acid formed significant amounts of pentylpyridines
when they reacted with 2,4-decadienal. Sohn and Ho
(1995) reported that aspartic acid produced more than
60% of free ammonia by deamination when heated at
180 °C in an aqueous solution. On the other hand,
glutamic acid was shown to be quite stable and released
only 1.3% of ammonia from its R-amino groups. The
difference in the reactivity of asparatic acid and glutam-
ic acid in releasing free ammonia and in forming
pentylpyridine may suggest that pentylpyridines can
indeed be formed from the interaction of 2,4-decadienal
with the R-amino groups of amino acids through the
mechanism suggested by Zhang and Ho (1989).
Glycine generated very small amounts of pentylpy-
ridine as shown in Figure 1. The difference in the
amount of pentylpyridine formed between glycine and
glutamic acid might be due to their difference in the
reactivities of side chains. The larger side chain of
glutamic acids may make it more reactive than glycine.
To study the effects of amide nitrogens on the forma-
tion of pentylpyridines, asparagine and glutamine la-
beled with 15N at the amide side chains were used.
Table 3 shows the relative contribution of amide nitro-
gens to pentylpyridine formation from the reaction of
2,4-decadienal with asparagine-amide-15N and glutamine-
amide-15N. Approximately half of the nitrogen atoms
in pentylpyridines came from the amide side chain in
asparagine whereas almost all of the pyridine nitrogens
came from the amide side chain in glutamine. It is also
interesting to note that there was no difference between
the relative ratios of 2-pentylpyridine and 3-pentylpy-
ridine formation by amide nitrogens in both asparagine
and glutamine. These labeling studies indicated that
in the cases of asparagine and glutamine the major
route for the formation of pentylpyridine was through
the formation of free ammonia by either deamidation
or deamination.
Received for review September 18, 1995. Revised manuscript
received April 15, 1996. Accepted September 16, 1996.X New
J ersey Agricultural Experiment Station Publication D-10205-
1-95 supported by State Funds and Regional NE-116 Project.
J F950623+
In conclusion, 2-pentylpyridine may be formed through
the interaction of 2,4-decadienal with either free am-
monia or the R-amino group of amino acids or peptides.
X Abstract published in Advance ACS Abstracts, No-
vember 15, 1996.