The possible production of natural flavours by amino acid degradation

Article abstract of DOI:10.1007/s00706-010-0331-3

This work describes the degradation of phenylalanine and tryptophane catalysed by their complexes with Fe(II), Co(II), and Cu(II). The influences of the central atom and of the reaction conditions on the degradation of the amino acids were observed. The necessary condition of the degradation is the possibility of a redox reaction on the central atom between M(II) and M(III). Moreover, the coordination sphere of the central cation of the transition metal must not be sterically shielded. The necessary conditions are fulfilled only in the Fe(II) complexes. The degradation is strictly anaerobic because due to the influence of oxygen, an irreversible oxidation of Fe(II) to Fe(III) proceeds. This reaction results in 5-hydroxy-1H-indol instead of the mixture of the degradation products, such as benzaldehyde, phenylacetaldehyde, and phenylacetic acid. The influence of the temperature on the catabolism is very important because the reaction accelerates with temperature increase. The phenylalanine anion acts as a reducing agent, and Fe(II) is spontaneously reduced to Fe(0). Springer-Verlag 2010.

Full text of DOI:10.1007/s00706-010-0331-3

Monatsh Chem (2010) 141:823–828
DOI 10.1007/s00706-010-0331-3
ORIGINAL PAPER
The possible production of natural flavours
by amino acid degradation
Katar ´ı na Kla cˇ anov a´
Michal Rosenberg
•
Peter Fodran
•
Received: 11 February 2010 / Accepted: 11 May 2010 / Published online: 9 June 2010
Ó Springer-Verlag 2010
Abstract This work describes the degradation of phen-
ylalanine and tryptophane catalysed by their complexes
with Fe(II), Co(II), and Cu(II). The influences of the central
atom and of the reaction conditions on the degradation of
the amino acids were observed. The necessary condition of
the degradation is the possibility of a redox reaction on the
central atom between M(II) and M(III). Moreover, the
coordination sphere of the central cation of the transition
metal must not be sterically shielded. The necessary con-
ditions are fulfilled only in the Fe(II) complexes. The
degradation is strictly anaerobic because due to the influ-
ence of oxygen, an irreversible oxidation of Fe(II) to
Fe(III) proceeds. This reaction results in 5-hydroxy-1H-
indol instead of the mixture of the degradation products,
such as benzaldehyde, phenylacetaldehyde, and phenyla-
cetic acid. The influence of the temperature on the
catabolism is very important because the reaction accel-
erates with temperature increase. The phenylalanine anion
acts as a reducing agent, and Fe(II) is spontaneously
reduced to Fe(0).
Keywords Amino acids Á Catabolism Á Redox reaction Á
Metal complexes Á Aromatic carbonyls Á Aromatic acids
Introduction
The degradation of amino acids is an important process by
which various components of natural flavour complexes in
foodstuffs are produced. The most important amino acid
degradation catalysts are nonheme metalloproteins. They
are oxidative catalysts in which the centre of the enzyme
contains the coordinated cation of a transition metal.
Metalloproteins are able to exist in more than one oxidation
degree and because of this they are useful catalysts for the
biological processes that demand electron transport [1].
From the preparative point of view of the possible
production of many interesting products, the simulation of
the metalloprotein reaction centers, where water is used as
a reaction medium, is extremely important. In the case of
the implementation of these reaction types in industrial
production, the problems with energetic demands of pro-
duction and problems with the waste disposal of the
reaction solvents will be eliminated. Because of this, many
experiments on the degradation of amino acids using var-
ious reactants were realised. Ameta et al. [2] realised the
oxidative decarboxylation of amino acids by potassium
permanganate.
K. Kla cˇ anov a´ (&)
Institute of Biochemistry, Nutrition and Health Protection,
Faculty of Chemical and Food Technology,
Slovak University of Technology in Bratislava,
Radlinsk e´ ho 9, 812 37 Bratislava, Slovak Republic
e-mail: katarina.klacanova@stuba.sk
Itoh et al. [3] reported the catalytic oxidative decar-
boxylation reaction of amino acids with coenzyme PQQ
(Methoxantin; Fig. 1). The reaction is followed by the pH
change from 7.21 to 8.58. The products of degradation are
shown in Table 1. The yields and also the individual
products are very interesting. Consequently, it is possible
to prepare natural phenylacetaldehyde from natural phen-
ylalanine. Phenylacetaldehyde is indispensable for the food
P. Fodran
FLOP, Mandlov a´ 37, 85110 Bratislava, Slovak Republic
ˇ
M. Rosenberg
Institute of Biotechnology and Food Science,
Faculty of Chemical and Food Technology,
Slovak University of Technology in Bratislava,
Radlinsk e´ ho 9, 812 37 Bratislava, Slovak Republic
123

8
24
K. Kla cˇ anov a´ et al.
COOH
acid oxidase from T. variabilis and peroxidase from
C. cinereus. Although they used D-amino acid as a starting
material, they supposed that their results most probably
could be transferred to the systems based on L-phenylalanine
and the less available enzymes L-amino acid oxidase or
L-aminotransferase [5, 7]. They established several mecha-
nisms of D-phenylalanine transformation into benzaldehyde.
On the basis of their results, they proposed the mechanistic
pathway: a non-oxidative decarboxylation of phenylpyru-
vate (or its enol form) into phenylacetate, and then an
oxidative deformylation of the enol form of phenylacetal-
dehyde leading to benzaldehyde and formic acid, most
probably through a dioxetane ring [5].
HOOC
HN
O
N
HOOC
O
Fig. 1 Coenzyme PQQ (methoxantin) [3]
industry as a flavour compound, and it is largely deficient
on the market. Another product of this degradation is
phenylacetic acid, the esters of which are necessary for the
production of honey flavours.
The metabolism of L-phenylalanine has been studied in
several white rot fungi [8, 9]. Among the potential aroma
producers, white rot basidiomycetes were probably the
most versatile microorganisms. These fungi were able to
produce a wide variety of volatile aryl metabolites of
commercial interest, such as vanillin, benzaldehyde (bitter
almond aroma), and cinnamaldehyde [10].
Amino acids are also interesting ligands for the prepa-
ration of transition metal complexes. These are common
objects for the study of the structure and especially redox
attributes.
For the production of flavour compounds, the biotrans-
formation of amino acids, mainly phenylalanine, by the
cultures of microorganisms or moulds, and by the crude
extracts of enzymes is important as well. The biotransfor-
mation has previously been shown to produce variable
amounts of benzaldehyde, which is the next most important
flavour compound after vanillin and is used in cherry and
other fruit aromas [4–6].
Lapadatescu et al. [11] studied aryl metabolite biosyn-
thesis in the white rot fungus Bjerkandera adusta by using
1
4
13
C- and C-labelled L-phenylalanine as precursors. The
metabolite formation required de novo protein biosynthe-
sis. The results have shown that L-phenylalanine was
deaminated to trans-cinnamic acid by a phenylalanine
ammonia lyase, and trans-cinnamic acid was in turn con-
verted to aromatic acids (phenylpyruvic, phenylacetic,
mandelic, and benzoylformic acids); benzaldehyde was a
metabolic intermediate. These acids were transformed into
benzaldehyde, benzyl alcohol, and benzoic acid [11].
The metabolism of phenylalanine is extremely compli-
cated and can have an atypic course in many cases. One of
the reasons for starting this investigation was the possi-
bility that the defect in the metabolic hydroxylation of
phenylalanine to tyrosine, which is known to be the
primary fault in phenylketonuria, might be associated
with a similarly abnormal hydroxylation of tryptophane.
Researchers have found a ‘‘natural’’ benzaldehyde pro-
duction to be possible through these biotransformations as
an alternative to the extraction from vegetable sources. In
these studies, benzaldehyde was a product of complex
metabolic pathways that were more or less elucidated.
Moreover, different enzymes were involved in these bio-
transformations, which led to different intermediates of
reactions [5].
Okrasa et al. [5] studied a novel bi-enzymatic transfor-
mation of D-phenylalanine into benzaldehyde using D-amino
Table 1 Oxidative
decarboxylation of the a-amino
acids with coenzyme PQQ [3]
Amino acids
Initial pH
7.21
Final pH
8.28
Product [yields (%)]
COOH
CHO
(16)
COOH
NH2
(20)
COOH
7.20
8.58
CHO
N
H
NH2
N
H
(37)
1
23

The possible production of natural flavours
825
Table 2 Products of the
Compound
Retention
time (min)
Area (%)
reaction of phenylalanine in the
coordination sphere of Fe(II) in
the complex with phenylalanine
at room temperature under 24 h
stirring in an inert atmosphere
isolated from the reaction
mixture before and after
acidification
Before acidification
Benzaldehyde
3.3
4.4
3.2
4.4
7.5
6.9
38.9
8.3
Phenylacetaldehyde
Benzaldehyde
After acidification
Phenylacetaldehyde
Phenylacetic acid
25.1
40.8
Table 3 Products of the reaction of tryptophane in the coordination sphere of Fe(II) in the complex with tryptophane at room temperature under
2
4 h stirring in an inert atmosphere isolated from the reaction mixture before and after acidification
Compound
Retention time (min)
Area (%)
Before acidification
After acidification
1H-Indole-3-carboxaldehyde
14.0
12.8
14.0
31.5
11.3
7.7
Indole-3-acetaldehyde
1
H-Indole-3-carboxaldehyde
5
-Hydroxyindol is an atypic metabolite in this case [12].
Table 4 Products of the reaction of phenylalanine in the coordination
sphere of Co(II) in the complex with phenylalanine at room tem-
perature under 24 h stirring in an inert atmosphere isolated from the
reaction mixture
Although it has been suggested by Harley-Mason that
phenylalanine could serve as a precursor of 5-hydroxyin-
dole compounds, the results of the experiments with
1
4
Compound
Retention time (min)
3.2
Area (%)
4.5
C-labelled amino acids make this unlikely [13].
Benzaldehyde
Results and discussion
Table 5 Products of the reaction of phenylalanine in the coordination
sphere of Co(II) in the complex with phenylalanine at 52 and 64 °C
under stirring in air isolated from the reaction mixture
Tables 2, 3, 4, and 5 contain the results of the GC/MS
analysis of the amino acid degradation products that were
catalysed by transition metal complexes.
Compound
-Hydroxy-1H-indol
Retention time (min)
30.1
Area (%)
100.0
This work describes a procedure of the possible flavour
compound preparation on the basis of natural precursors. It
is based on the reaction simulation, which uses a catalyst as
the active center of the metalloprotein. Most metallopro-
teins contain the coordinated cation of a transition metal.
We have chosen Cu(II), Co(II), and Fe(II) because these
cations belong to the most active ones in metalloproteins.
For the case of simplicity, we used amino acid anions as
ligands which were further transformed. We used the
tryptophane and phenylalanine amino acids because of the
eventual creation of interesting degradation products, such
as benzaldehyde, phenylacetaldehyde, phenylacetic acid,
and indol or its derivatives. We used the GC/MS method
for product verification. Because of the supposition that
some products of the amino acid degradation should be
organic acids and because the reaction runs in alkaline
medium, we used a two-stage method for isolation of the
reaction products. Alkali indifferent substances were iso-
lated by direct extraction into diethylether in the first stage.
In the second stage, the rest of the reaction products was
isolated after acidification to pH 2–3 by extraction into
5
diethylether. The results of all product isolations are pre-
sented in Tables 2, 3, 4, and 5.
Comparison of yields of the reactions catalysed by the
complexes containing various central atoms is very inter-
esting. It was observed that the reaction catalysed by the
Fe(II) complex has the most rapid progress, whereas the
reaction catalysed by the Cu(II) complex has the slowest
progress (practically zero), which is suggested also by the
reaction kinetics. In the case of the Fe(II) complex, the
products of the reaction have been isolated in excellent
yield unlike the Cu(II) complex, where no product of the
reactant transformation has been isolated. After the eval-
uation of the reaction products and yields, we proposed the
mechanism of the destructive amino acid transformation
catalysed by transition metal complexes (Scheme 1).
The mechanism is based on the redox reaction between
water and the amino acid anion. It is compatible with the
123

8
26
K. Kla cˇ anov a´ et al.
H
H
COO Na
NH2
O
O
C
+
H O
2
H2
N
O
Na
O
O
Ph
O
FeII
NH2
H2
N
O
N
Ph
Ph
O
O
H2
FeII
O
N
Ph
O
H
O
H2
HCOONa
O
oxidation
H
NH2
FeII
H2
N
O
Ph
O
OH
O
N
Ph
O
H2
HCOONa
O
H
O
OH
oxidation
H2 NH3
H
N
2
O
O
Ph
O
N
Ph
N
O
FeII
FeIII
- NH , CO
3
- NaOH
O
O
N
Ph
Ph
O
H
2
O
H2
Scheme 1
3
0
20
10
0
mechanism suggested by Okrasa et al. [5]. The original
supposition of Lapadatescu et al. [11] that the reaction in
the in vivo experiments is connected with the eliminated
amino acid deamination leading to the formation of the
substituted propenoic acid was not confirmed.
1
9
9
8
00
5
0
-10
-20
The presented facts are the consequence of kinetically
controlled reactions. The reaction was dramatically chan-
ged if the reaction was thermodynamically controlled. The
influence of increased temperature in the case of the Fe(II)
complex led to the spontaneous reduction of Fe(II) to
metallic iron, which precipitated in the form of a metallic
mirror.
5
-
-
-
-
30
40
50
60
80
7
5
0
100
200
300
400
500
600
700
800
900
Temperature/°C
DSC analysis indicates that there is a substance with an
atypic degradation in two stages. The degradation is more
penetrative in the presence of oxygen than in an inert
atmosphere. The first stage of degradation has the maxi-
mum under inert atmosphere at a temperature of about
Fig. 2 Thermogravimetry (TG) and differential thermal analysis (DTA)
of the reaction mixture containing reduced metal under inert atmosphere
On the basis of the experimental results, the negative
influence of oxygen on the reaction was detected. We have
observed the oxidation of Fe(II) to Fe(III) accompanied by
a colour change of the reaction mixture and with the
inactivation of the oxidative catalyst. This reaction resulted
in 5-hydroxy-1H-indol instead of the mixture of degrada-
tion products such as benzaldehyde, phenylacetaldehyde,
and phenylacetic acid. It is very interesting that the
2
3
25 °C, and the second stage has the maximum at about
80 °C (Fig. 2). The analogous temperatures in the pres-
ence of oxygen are moved to lower values (Fig. 3). From
the loss of weight, which is comparable in both cases, it is
possible to deduce that a release of a fixed CO might be
2
observed.
1
23

The possible production of natural flavours
827
cobalt(II) sulfate heptahydrate, and copper(II) sulfate
pentahydrate were purchased from Lachema. Sodium
hydroxide, diethylether, hydrochloric acid, and anhydrous
sodium sulfate were purchased from Microchem.
1
00
1
00
5
8
6
4
2
0
0
0
0
0
9
90
Apparatus
85
80
75
-
-
-
20
40
60
Products were analysed by GC/MS and TG/DTA methods.
Gas chromatography (GC) was carried out using an Agilent
Technologies 6890 gas chromatograph equipped with an
Agilent Technologies 5973 inert mass selective spectrom-
eter and with chromatographic column model no. J&W
0
100
200
300
400
500
600
700
800
900
Temperature/°C
1
22-503
E
DB-5, 30 m 9 0.25 mm 9 0.5 lm. The
Fig. 3 Thermogravimetry (TG) and differential thermal analysis
DTA) of the reaction mixture containing reduced metal in the
presence of oxygen
(
records of thermogravimetry (TG) and differential thermal
analysis (DTA) were performed on the DTG-60 (Shima-
dzu, Japan) with a mass range TG ± 500 mg, apparatus
sensitivity 0.001 mg, and range of DTA measurement
± 1,000 lV.
phenylalanine metabolism in the case of some genetic
defects (such as phenylketonuria and Down syndrome) is
comparable with the process of metabolism in our sug-
gested mechanism [12–15].
Synthesis
From all our experiments, we can deduce that the most
active catalyst of the amino acid degradation is the Fe(II)
complex. The reason is that the Fe(II) complexes have a
square-planar configuration of the coordination sphere
unlike the Co(II) complexes with a tetrahedral coordination
sphere. It implies that in the case of the Fe(II) complexes,
the axial positions are not shielded and so a charge transfer
starts between the central atom and other donor group. In
the case of the Co(II) complex, mainly in the coordination
with large ligands, other positions around the central atom
are strongly sterically shielded. For successful degradation
in the coordination sphere of the complex, the ability of a
redox reaction between M(II) and M(III) of the central atom
is necessary. Strongly electron-deficient groups originate in
another incoming ligand on the entering of a donor group
into the coordination sphere of the metal cation. These
groups have a tendency to attract electrons from the metal
cation while the metal oxidation degree increases (see
Scheme 1). After the reactions between ligand and water,
the reverse reduction of the metal cation to the original
oxidation degree proceeds. This idea is supported by the
finding that in the case of the Cu(II) complex, the reaction in
the coordination sphere of the complex under any condi-
tions does not run because under any described conditions,
the change from Cu(II) to Cu(III) is not possible.
Transition metal complexes with phenylalanine and tryp-
tophan were synthesised by a modification of the reaction
described in Ref. [14]. Accordingly, 1 mmol aqueous
solution of the metal sulfate hydrate at pH 5.0 was added to
an aqueous solution of the sodium salt of amino acids
containing 2 mmol of the ligand (molar proportion
metal:ligand of 1:2) at pH 9.0.
These reactions were carried out:
1.
at room temperature under stirring for 24 h in an inert
atmosphere;
2
.
.
at 52 and 64 °C under stirring in air;
at 70 °C under heating in an inert atmosphere.
3
In all experiments, a mixture of 10 mmol amino acid
3
3
with 10.5 cm 1 M solution of NaOH and 10 cm distilled
water was added to the prepared complexes. After adding
the mixture, greyish-green Fe(II) solution was spontane-
ously oxidised to Fe(III) (brick red solution) in the second
experiment, whereas in the third experiment it was spon-
taneously reduced to Fe(0) (black precipitate). After
finishing the reaction, the mixture was extracted by
3
1 9 100 cm diethylether and dried by anhydrous sodium
sulfate. In the first experiment, the water part of the first
extraction was acidified by 10% HCl to pH 2–3, extracted
3
by 1 9 100 cm diethylether, and dried by anhydrous
sodium sulfate. The ethereal solutions of the extracts in all
3
experiments were concentrated to 5 cm and analysed by
Experimental
GC/MS.
Materials
In the third experiment the reduced iron was filtered at
usual laboratory conditions. Consequently, the spontaneous
oxidation of iron to the mixture of oxides and carbonates
Both amino acids, phenylalanine, and tryptophane, were
purchased from Merck. Iron(II) sulfate heptahydrate,
3
was observed. The filtrate was extracted by 2 9 50 cm
123

8
28
K. Kla cˇ anov a´ et al.
5. Okrasa K, Guibe-Jampel E, Plenkiewicz J, Therisod M (2004) J
´
Mol Catal B Enzym 31:97
toluene and methanol at the reflux after drying. The
product was dried at the normal temperature and analysed
by TG/DTA methods.
6
7
. Krings U, Berger RG (1998) Appl Microbiol Biotechnol 49:1
. Alexandre FR, Pantaleone DP, Taylor PP, Fotheringham IG,
Ager DJ, Turner NJ (2002) Tetrahedron Lett 43:707
Acknowledgments This work was supported by the Slovak
Research and Development Agency (contract no. APVV-20-005605)
and Slovak Grant Agency VEGA (project no. 1/0845/08).
8. Krings U, Hinz M, Berger RG (1996) J Biotechnol 51:123
9. Jensen KA, Evans KMC, Kirk TK, Hammel KE (1994) Appl
Environ Microbiol 160:709
1
1
0. Janssens L, De Pooter HL, Schamp NM, Vandamme EJ (1992)
Process Biochem 27:195
1. Lapadatescu C, Gini e´ s Ch, Le Qu e´ r e´ JL, Bonnarme P (2000)
Appl Environ Microbiol 66:1517
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