Thermal reactions of malic acid benzylamine salts

Article abstract of DOI:10.14233/ajchem.2013.14815

Malic acid, a component present in fruit, is known to be an important precursor for the prebiotic formation of polyaspartic acid. Malic acid is a dicarboxylic acid, possessing one hydroxyl group. It yields many types of crystalline salts with amino compounds. Although the thermal reactions of the amino salts have been reported, their dehydration reaction pathway has not been studied. This paper describes the dehydration process using thermal gravimetry and differential thermal analysis. The results show that the malic acid monobenzylamine and the dibenzylamine salts release water molecules during an endothermic reaction to afford malic acid benzylimide and malic acid dibenzylamide, respectively. The latter compound is stable up to 230 °C.

Full text of DOI:10.14233/ajchem.2013.14815

Asian Journal of Chemistry; Vol. 25, No. 13 (2013), 7451-7456
http://dx.doi.org/10.14233/ajchem.2013.14815
Thermal Reactions of Malic Acid Benzylamine Salts
3
3
T. MUNEGUMI1,2,*, H. TOCHINO and K. HARADA
1Department of Science Education, Naruto University of Education, Naruto, Tokushima 772-8502, Japan
2Department of Materials Chemistry and Bioengineering, Oyama National College of Technology, Oyama, Tochigi 323-0806, Japan
3Department of Chemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8571, Japan
*Corresponding author: Fax: +81 88 6876022; Tel: +81 88 6876418; E-mail: tmunegumi@naruto-u.ac.jp
(Received: 10 November 2012;
Accepted: 1 July 2013)
AJC-13727
Malic acid, a component present in fruit, is known to be an important precursor for the prebiotic formation of polyaspartic acid. Malic acid
is a dicarboxylic acid, possessing one hydroxyl group. It yields many types of crystalline salts with amino compounds. Although the
thermal reactions of the amino salts have been reported, their dehydration reaction pathway has not been studied. This paper describes the
dehydration process using thermal gravimetry and differential thermal analysis. The results show that the malic acid monobenzylamine
and the dibenzylamine salts release water molecules during an endothermic reaction to afford malic acid benzylimide and malic acid
dibenzylamide, respectively. The latter compound is stable up to 230 ºC.
Key Words: Thermal analysis, Malic acid, Benzylamine salt.
This paper describes the amide formation mechanism
using N-benzyl-ammonium salts (3 and 4 in Fig. 2) of malic
acid (1). Thermal analysis of the resulting dibenzylamides
showed unexpected thermal stability at elevated temperatures.
We found some similarity and some difference between the
ammonium salts and N-benzyl-ammonium salts of malic acid
in terms of their thermal reactions.
INTRODUCTION
Malic acid is known as a component in wine and in
extracts of grape and other fruits. This bifunctional organic
compound, possessing one hydroxyl and two carboxyl groups,
forms the monoammonium salt1-3 and other N-substituted
ammonium salts4. Upon heating, the monoammonium salt
affords polyaspartic acid3,5,6. This has been suggested as an
EXPERIMENTAL
important pathway to the prebiotic polyaspartic acid3,5,6
.
The thermal analyzer used was a Shimadzu DT-40
(Shimadzu, Kyoto, Japan). Infrared spectra were recorded
using a Hitachi Model 260-50 infrared spectrophotometer
(Hitachi, Tokyo, Japan). 1H NMR spectra were measured with
a JOEL 400MHz 1H NMR instrument (Joel, Tokyo, Japan).
DL-Malic acid (1) was supplied by Fuso Chemical Co.,
Ltd. (Osaka, Japan). Benzylamine (2) was purchased from
Wako Pure Chemical Industries Co., Ltd. (Osaka, Japan).
General procedure of heating reaction: DL-Malic acid
(1.34 g, 10 mmol) and benzylamine (1.07 g, 10 mmol or 2.14
g, 20 mmol) were mixed in a test tube (20 cm × 2 cm ID) and
stood for several minutes under nitrogen flow until the test
tube cools down to room temperature. The test tube was
immersed into an oil bath controlled at 160 ºC and the reaction
was carried out for 4 h under a nitrogen flow.
Microwave activation of ammonium malate can also afford
polyaspartic acid7-9. One of the proposed mechanisms for the
thermal peptide formation is as follows. Dehydration from the
C2 and C3 positions proceeds first to afford maleic acid and
fumaric acid monoammonium salts, which themselves react
to afford their monoamides (Fig. 1). Monoammonium salt and
monoamide with α-carboxylate are supported as the superior
structure rather than those with β-carboxylate due to stronger
acidity of α-carboxylate. The monoamide nitrogen attacks the
C=C double bond to afford polyaspartic acid. Another
possible mechanism involves first (similarly) the formation of
the monoamide, followed by the nucleophilic substitution of
the ammonium nitrogen to the C2 position to afford poly-
aspartic acid.
Although thermal reactions using N-substituted ammo-
nium salts of malic acid have been reported4, their dehydration
mechanism has not been reported and N-substituted
polyaspartic acids have not been obtained.
Sample preparation for thermal analysis
DL-Malic acid mono-(N-benzyl-ammonium) salt (3):
DL-Malic acid (13.4 g, 100 mmol) was dissolved in ethanol

7452 Munegumi et al.
Asian J. Chem.
COONH
4
NH
COO
H
H
4
H
H
H
HO
C
COO NH
COOH
HO
C
CONH
2
4
C
C
C
C
and/or
H C
2
H C
2
COOH
COOH
HOOC
OH
H
O
C
O
C
C
H
H
CONH
2
H
CONH
2
OH
H C
2
H O
2
-{l(m+n)-1}
C
C
C
H
C
2
H
N
H
C
H
C
O
N
C
OH
C
O n
H
H
COOH
HOOC
H
m
l
O
O
C
C
O
H C
2
C
C
O
OH
OH
H C
2
N
C
H N
2
C
H
H
l(m+n)-1
Fig. 1. Formation of aspartic acid by heating DL-malic acid mono ammonium salt
Procedure of thermal analysis: Samples of DL-malic
acid dibenzylamine salt (8-13 mg, weighed to within 0.1 mg)
were placed in the aluminum sample pans of the differential
thermal analyzer. The α-aluminum reference disk was placed
in the center of the second aluminum pan. The temperature of
the heating chamber was programmed from 27-400 ºC using
a heating rate of 10 ºC/min, under a nitrogen flow (50 mL/
min).
O
C
HO
HO
COOH
COOH
H
N
2
2
O
H N
3
OH
3
C
O
1
O
C
2
H N
2
HO
H
N
O
O
3
2
RESULTS AND DISCUSSION
H
N
3
C
O
Thermal reaction of DL-malic acid (1): DL-Malic acid
(1.34 g, 10 mmol) in a test tube (20 cm × 2 cm ID) was heated
under a nitrogen flow for 4 h at 160 ºC and at 180 ºC to afford
fumaric acid (5) (Scheme-I). The yields were 81 and 88 %,
respectively.Analytical data were as follows. 1H NMR (CD3OD):
δ = 6.93 (2H, s). IR (KBr, νmax, cm-1): 3050-3100 (-COOH),
1670 (C=C), 1000 (C=C), 900 (-COOH). Calcd. (%) for
C4H4O4: C, 41.39; H, 3.47. Found (%): C, 41.91; H, 3.65.
4
Fig. 2. Preparation of DL-malic acid N-benzyl-ammonium salts (3, 4)
(50 mL) and the resulting solution was cooled in an ice bath.
Benzylamine (10.7 g, 100 mmol) was added dropwise to the
cooled solution to yield a precipitate (21 g, 87 %). The preci-
pitate (5 g) was recrystallized to yield 2.2 g (44 %; m.p. 145-
146 ºC). Elemental analysis of the resulting crystals was
consistent with the calculated value. Calcd. (%) for C11H15NO5:
C, 54.76; H, 6.26; N, 5.80. Found (%): C, 54.75; H, 6.27; N,
5.80. The higher acidity of the carboxylic group at the C2
position (α) (α: pKa = 3.4; β: pKa = 5.13) suggests that the α-
carboxyl group preferentially forms a salt with benzylamine.
DL-Malic acid di-(N-benzyl-ammonium) salt (4): DL-
Malic acid (13.4 g, 100 mmol) was dissolved in ethanol (50
mL) and the resulting solution was cooled in an ice bath.
Benzylamine (21.4 g, 200 mmol) was added dropwise to the
cooled solution to yield a precipitate (30.4 g, 87 %). The
precipitate (5 g) of the total amount was recrystallized to yield
3.6 g (72 %; m.p. 140-142 ºC). Elemental analysis of the
resulting crystals was consistent with the calculated value, as
the half hydrate. Calcd. (%) for C18H24N2O5·1/2H2O: C, 60.49;
H, 7.05; N, 7.84. Found (%): C, 60.22; H, 6.78; N, 7.49.
HO
COOH
H
COOH
COOH
H
HOOC
5
1
Scheme-I
Thermal analysis of DL-malic acid (Fig. 3) shows the
dehydration. The DTA curve shows a sharp endothermic peak,
which corresponds to the melting point of DL-malic acid,
because the sample weight did not reduce during the endo-
thermic peak revealed in DTA curve.
Thermal reaction of DL-malic acid mono-(N-benzyl-
ammonium) salt (3): DL-Malic acid (1) (1.34 g, 10 mmol)

Vol. 25, No. 13 (2013)
Thermal Reactions of Malic Acid Benzylamine Salts 7453
0
24
48
Time (min)
72
96
120
0
100
200 300
Temperature (ºC)
400
500
Fig. 3. Thermal analysis of DL-malic acid (1). Temperature was programed
from 27-160 ºC at 10 ºC/min under a nitrogen flow (50 mL/min).
Until 40 min after start of heating, the temperature seems to reach
160 ºC
Fig. 4. Thermal analysis of DL-malic acid mono-benzylamine salt (3).
Temperature was programed from 27-440 ºC at 10 ºC /min under a
nitrogen flow (50 mL/min)
and benzylamine (2) (1.07 g, 10 mmol) were mixed and then
heated under a nitrogen flow at 160 ºC for 4 h (Scheme-II).
The resulting reaction mixture showed several spots on a thin
layer chromatogram, run using chloroform (CHCl3): ethyl
acetate (EtOAc) (3:1) as developing solvent (Rf = 0.81, 0.35
and other values).After the extraction of a chloroform solution
of the reaction mixture with 0.1 M HCl, followed by drying
with anhydrous magnesium sulfate, the solution was evapo-
rated to dryness and then the resulting residue was loaded onto
a silica gel column, for chromatographic separation (developing
solvent, CHCl3-AcOEt (3:2)). N-Benzyl-maleimide (6) (57 %)
and N-benzyl-maleimide (9) (8 %) were isolated.
occurring at the same time as the melting. When the endother-
mic peak revealed, the weight loss reduced to 10.8 % of the
sample weight. The value (10.8 %) is due to a little larger
value than the theoretical weight loss by dehydration of one
molecule (7.46 %).
The first dehydration can be explained by an amidation
between benzylamine and one carboxyl group of malic acid.
Another possible explanation is dehydration from the hydroxyl
group at the C2 position of malic acid and hydrogen at the C3
position. However, the dominant formation of N-benzyl-
maleimide (7) (57 %) compared with N-benzyl-maleimide (9)
(8 %) explains the preferential amide formation rather than
dehydration from the C2 and C3 positions.
H
CONH
HO
CONH Bzl
Bzl
The second larger peak emerges after the first peak. The
sequential dehydration reaction is suggested to proceed until
the second peak emerges. However, until the end of the second
endothermic peak, a much greater weight loss (35 %) than the
theoretical value (7.46 %) is observed. The second larger peak
may include the second dehydration accompanied by degra-
dation to show 35 % weight loss. In the second dehydration
step, it is proposed that dehydration takes place with the forma-
tion of N-benzyl-malimide (7) by cyclization of malic acid
mono-N-benzylamide (6a, 6b) as shown in Scheme-II. N-
Benzyl-malimide (7) seems to be formed by dehydration from
the C2 and C3 positions of malic acid mono-N-benzylamide
(8a, 8b). The remaining carboxyl group may catalyze the
dehydration. The later emerging, two smaller peaks in Fig. 4
may indicate a thermal degradation.
6a
8a
HO
COO
Bzl
H3N
-H2O
-H2O
COOH
COOH
HOOC
H
H
HO
COOH
COOH
8b
6b
3
CONH Bzl
H
CONH Bzl
160 οC, 4h
-H2O
O
O
HO
N
Bzl
N
Bzl
9
7
O
O
Scheme-II
Analytical data were as follows. N-Benzyl-maleimide (6):
1
Thermal stability of N-benzyl-malimide (7):The thermal
reaction of isolated N-benzyl-malimide (7) was carried out at
160 and 200 ºC for 4 h and 240 ºC for 1 h, as shown in Scheme-
II. N-Benzyl-maleimide (9) did not form by dehydration from
N-benzyl-malimide (7).
m.p. 116-117 ºC. Rf = 0.35 (CHCl3:AcOEt (3:1)). H NMR
(CD3Cl): δ = 7.40 (5H, m), 4.68 (2H, s), 3.48 (1H, s), 2.85
(2H, dd, J = 6.0, 7.9Hz). Calcd. (%) for C11H11NO3: C, 64.38;
H, 5.40; N, 6.81. Found (%): C, 64.29; H, 5.36; N, 6.81.
N-Benzyl-maleimide (9): m.p. 60-62 ºC, Rf = 0.81 (CHCl3:
AcOEt (3:1)). 1H NMR (CD3Cl): δ = 7.32 (5H, m), 6.67 (2H,
s), 4.65 (2H, s).
Thermal reaction of DL-malic acid di-(N-benzyl-
ammonium) salt (4): DL-Malic acid (1) (1.34 g, 10 mmol)
and benzylamine (2) (2.25 g, 21 mmol) were mixed and heated
at 160 ºC for 4 h under nitrogen flow. The resulting residue
was recrystallized from chloroform to afford malic acid di-N-
benzylamide (10) (1.52 g, 48 %), as shown in Scheme-III
Fig. 4 shows the thermal analysis curve of the mono-(N-
benzyl-ammonium) salt (3) of DL-malic acid. The DTA curve
shows two larger and two smaller endothermic peaks. The first,
larger, sharp peak corresponds to the endothermal dehydration

7454 Munegumi et al.
Asian J. Chem.
(m.p. 147-148 ºC). 1H NMR (CD3Cl): δ = 7.62 (2H, br), 7.35
(10H, br), 5.22 (1H, br), 4.38 (6H, m), 2.80 (2H, m); 1H NMR
(CD3Cl/D2O): δ = 7.35 (10H, br), 4.38 (5H, m), 2.80 (2H, dd,
J = 4.0, 7.0, 15Hz). IR (KBr, νmax, cm-1): 3300 (NH), 1650
(amide I), 1550 (amide II), 730,710 (Ph). Calcd. (%) for
C18H24N2O5: C, 69.21; H, 6.45; N, 8.96. Found (%): C, 69.14;
H, 6.43; N, 8.91.
Thermal analysis of the DL-malic acid dibenzylamine salt
(4) was carried out under conditions similar to those used in
the thermal reaction (temperature programmed 27-160 ºC at
5 ºC/min). Results are shown in Figs. 6 and 7. Fig. 6 shows a
sharp peak and a following broad endothermic absorption on
the DTA curve and fast weight loss from the starting point of
melting on the TG curve. Fig. 7 shows a magnified range of
Fig. 6. Two endothermic peaks, which seem to be due to melting
and dehydration, are evident. The weight loss until this point
(15.2 %) almost corresponds to the dehydration of 2.5 mole-
cules (12.6 %).
HO
CONH Bzl
HO
COO
COO
Bzl
H3N
-H2O
H3N
Bzl)
(-COO
160 οC, 4h
COO H3N Bzl
(-CONH-Bzl)
4
H N Bzl
3
8a-b
HO
CONH Bzl
-H2O
10
CONH Bzl
Scheme-III
Fig. 5 shows the thermal analysis of the DL-malic acid
di-(N-benzyl- ammonium) salt. The first peak is a sharp peak,
followed by two broad peaks. The former almost corresponds
to the melting of the DL-malic acid dibenzylamine salt.
However, a weight loss that commenced before the end of the
first peak means that dehydration began during the melt of
the crystal. The dehydration corresponds to the first amide
formation (Scheme-IV).
0
26
52
Time (min)
78
104
130
Fig. 6. Thermal analysis of malic acid di-benzylamine salt (4). Temperature
was programed from 27-160 ºC at 50 ºC/min under a nitrogen flow
(50 mL/min)
O
HO
N
Bzl
7
H2N Bzl
O
160 οC, 4h
HO
-H2O
HO
CONH
Bzl
CONH Bzl
-H2O
H3N
Bzl)
(-COO
H N Bzl
CONH Bzl
COO
(-CONH-Bzl)
8a-b
Scheme-IV
3
10
0
8
16
Time (min)
24
32
40
Fig. 7. Thermal analysis of malic acid di-benzylamine salt (4). Temperature
was programed from 27-160 ºC at 50 ºC/min under a nitrogen flow
(50 mL/min)
Thermal reaction of N-benzyl-malimide (7) and
benzylamine (2): N-Benzyl-malimide (7) (1.03 g, 5.0 mmol)
and benzylamine (2) (0.47 g, 5.0 mmol) were mixed and heated
at 160 ºC for 4 h under nitrogen flow to afford malic acid
dibenzylamide (10) (1.66 g, 70 %; m.p. 147 ºC). Results show
that the ring of the N-benzyl-imide (7) was opened by nucleo-
philic attack of benzylamine.
0
100
200
Temperature (ºC)
300
400
Thermal stability of DL-malic acid di-N-benzyl-amide
(10): Fig. 8 shows the thermal analysis of a standard sample
of DL-malic acid di-N-benzyl-amide (10). Two sharp peaks
Fig. 5. Thermal analysis of malic acid di-benzylamine salt (4). Temperature
was programed from 27-370 ºC at 10 ºC/min under a nitrogen flow
(50 mL/min)

Vol. 25, No. 13 (2013)
Thermal Reactions of Malic Acid Benzylamine Salts 7455
TABLE-1
RESULTS OF THERMAL REACTIONS OF DL-MALIC ACID (1), BENZYLAMINE (2) AND RELATED COMPOUNDS
Benzylamine Temperature
Reaction
time (h)
Yield
(%)
m.p.
(ºC)
Substrate
Product
(equivalent)
(ºC)
160
180
160
160
160
160
160
160
200
240
160
160
190
Non
Non
1
4
4
80
88
57
8
–
Malic acid (1)
Malic acid (1)
Malic acid (1)
Malic acid (1)
Malic acid (1)
Malic acid (1)
Fumaric acid (5)
–
Fumaric acid (5)
4
N-Benzyl-malimide (7)
116-117
1
4
60-62
N-Benzyl-maleimide) (9)
2
4
48
79
70
0
147-148
Malic acid di-N-benzylamide (10)
3
4
147-148
Malic acid di-N-benzylamide (10)
1
4
147
–
N-Benzyl-malimide (7)
Malic acid di-N-benzylamide (10)
Non
Non
Non
1
4
Non
Non
Non
Non
Non
Non
N-Benzyl-malimide (7)
4
0
–
N-Benzyl-malimide (7)
4
0
–
N-Benzyl-malimide (7)
4
0
–
Malic acid di-N-benzylamide (10)
Malic acid di-N-benzylamide (10)
Malic acid di-N-benzylamide (10)
Non
Non
12
4
0
–
0
–
for the melting of DL-malic acid di-N-benzyl-amide (10)
appear on the DTA curve. This compound (10) is stable, with
no weight loss up to ca. 230 ºC.
This research has demonstrated that thermal reactions of
DL-malic acid N-benzyl-ammonium salts (3, 4) pass through
direct amidation between a carboxyl group and benzylamine
(2). Results of the thermal reaction are summarized in Table-1.
The reaction pathway demonstrated by this research is shown
in Fig. 9. The reaction pathway is supported by thermal analysis
data of starting materials and predicted intermediates.
Conclusion
0
100
200 300
Temperature (ºC)
400
500
Thermal reactions of DL-malic acid mono-N-benzylamine
and di-N-benzylamine salts (3, 4) were carried out to afford
the imide and di-N-benzylamide (10), respectively. Once the
Fig. 8. Thermal analysis of malic acid di-benzylamide (10). Temperature
was programed from 27-460 ºC at 10 ºC/min under a nitrogen flow
(50 mL/min)
H
CONH BBzl
HO
CONH Bzl
6a
HO
COO
8a
H3N Bzl
H2N Bzl
-H2O
-H2O
2
H
COOH
COOH
HOOC
H
160 οC
160 οC
HO
COOH
8b
COOH
3
6b
CONH Bzl
H
CONH BBzl
160 οC
-H2O
O
O
O
HO
COOH
COOH
HO
H
COOH
-H2O
N
Bzl
N
Bzl
160 οC, 4h
HOOC
HO
H
7
9
5
H2N Bzl
O
1
160 οC, 4h
-H2O
HO
CONH Bzl
H2N Bzl
2
COO
COO
H3N Bzl
HO
CONH Bzl
-H2O
-H2O
2
H3N
Bzl)
(-COO
160 οC, 4h
CONH
Bzl
4
H N Bzl
3
H N Bzl
3
COO
(-CONH-Bzl)
8a-b
Fig. 9. Summary of plausible pathways in thermal reactions of DL-malic acid (1), benzylamine (2) and related compounds

7456 Munegumi et al.
Asian J. Chem.
two carboxyl groups of malic acid are lost upon thermal
reaction with benzylamine, dehydration from malic acid does
not occur. This was confirmed by the thermal stability of the
imide (7) and dibenzylamide (10). The approach to studying
thermal reactions by using thermal analysis will be useful for
explaining many organic reaction pathways.
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4. C. Lutz, Ber., 37, 2127 (1904).
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ACKNOWLEDGEMENTS
7. K. Harada, H. Mizumoto, K. Ikeda and N. Fujii, Origins Life, 14, 492
(1986).
The authors thank Mr. Saito for his technical assistance.
8. K. Harada, A. Shimoyama and H. Mizumoto, Method of Manufacturing
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