Characterization of 2-methyl-4-amino-5-(2-methyl-3-furylthiomethyl)pyrimidine from thermal degradation of thiamin

Article abstract of DOI:10.1021/jf011591v

Thiamin hydrochloride was thermally degraded in phosphate buffer (pH 6.5) at 110 °C for 2 h. A major decomposition product was isolated by column chromatography and structurally identified by spectrometric techniques (¹H NMR, ¹³C NMR

Full text of DOI:10.1021/jf011591v

J. Agric. Food Chem. 2002, 50, 4055−4058 4055
Characterization of
-Methyl-4-amino-5-(2-methyl-3-furylthiomethyl)pyrimidine from
Thermal Degradation of Thiamin
2
†
†
†
‡
JIN-WOO JHOO, MING-CHI LIN, SHENGMIN SANG, XIAOFANG CHENG,
†
‡
,†
NANQUN ZHU, RUTH E. STARK, AND CHI-TANG HO*
Department of Food Science, Rutgers University, 65 Dudley Road,
New Brunswick, New Jersey, 08901, and Department of Chemistry, College of Staten Island,
City University of New York, New York, New York 10314
Thiamin hydrochloride was thermally degraded in phosphate buffer (pH 6.5) at 110 °C for 2 h. A
major decomposition product was isolated by column chromatography and structurally identified by
spectrometric techniques ( H NMR, 13C NMR, 2D NMR, and MS) as 2-methyl-4-amino-5-(2-methyl-
1
3-furylthiomethyl)pyrimidine (MAMP). The possible formation pathway of MAMP was studied using
two model systems. It is proposed that MAMP is formed by nucleophilic attack of 2-methyl-3-furanthiol
on the thiamin.
KEYWORDS: Thiamin; thermal degradation; 2-methyl-4-amino-5-(2-methyl-3-furylthiomethyl)pyrimidine
INTRODUCTION
acid and 6 (4, 5). The substitution of thiamin by sulfite ion,
which gave 6 as the leaving group, was proposed as a multistep
process rather than an SN2 mechanism (5). Zoltewicz et al.
demonstrated that in methanol solution, several amine nucleo-
philes could replace the 6 in thiamin through a multistep
mechanism (6). During the thermal degradation of thiamin,
nucleophilic substitution to a bridged methylene carbon is rarely
reported. The current study will discuss the isolation and
characterization of a nucleophilic substituted novel compound,
Thiamin is one of the important precursors for odor-active
sulfur-containing compounds. Flavor generation by thermal
degradation of thiamin has been extensively studied (1-3).
Upon heating, thiamin first forms reactive intermediates such
as 5-hydroxy-3-mercapto-2-pentanone, which further reacts and
leads to many sulfur-containing compounds that have a cooked
and roasted meaty aroma. These reactive intermediates produce
aroma compounds not only through transformation and interac-
tion but also by reaction with other components of food, such
as Maillard reaction products (1-3). 4-Methyl-5-(2-hydroxy-
ethyl)thiazole (6), 4-amino-5-(aminomethyl)-2-methylpyrimi-
dine, and 3-mercaptopropanol, along with hydrogen sulfide,
ammonia, acetic acid, formic acid, formaldehyde, and acetal-
dehyde, have been reported as major compounds from thermally
degraded thiamin (1, 2). Additionally, many furans and
thiophenes, including the character-impact meaty aroma com-
pound 2-methyl-3-furanthiol (7), were identified from thermally
degraded thiamin (7) (3). Although numerous volatile thermal
degradation compounds have been identified from thiamin,
limited information is available on nonvolatile or semivolatile
compounds from thermally degraded thiamin.
2
-methyl-4-amino-5-(2-methyl-3-furylthiomethyl)pyrimidine
(MAMP; 1), from thermally degraded thiamin in an aqueous
solution and its possible formation pathway.
MATERIALS AND METHODS
Materials and General Procedures. Thiamin hydrochloride was a
gift from Takeda Pharmaceuticals North America, Inc. (Lincolnshire,
IL). 2-Methyl-3-furanthiol, all chemicals used for synthesis exper-
iments, silica gel (130-270 mesh) for column chromatography,
and TLC plates (250 µm thickness, 2-25 µm particle size) were
purchased from Sigma-Aldrich Chemical Co. (Milwaukee, WI). All
solvents used for chromatographic isolation were of analytical grade
and purchased from Fisher Scientific (Springfield, NJ). Thin-layer
chromatography was performed on silica gel TLC plates with com-
Thiamin consists of two subunits, a pyrimidine unit, 4-amino-
pounds visualized by spraying with 5% (v/v) H
solution.
2 4
SO in an ethanol
5-hydroxymethyl-2-methylpyrimidine, and a thiazole unit, 4-meth-
yl-5-(2-hydroxyethyl)thiazole (6). Early studies reported that
treatment of thiamin with sulfite led to the cleavage of thiamin
and afforded 2-methyl-4-amino-5-pyrimidylmethanesulfonic
Thermal Reaction of Thiamin. Thiamin hydrochloride (30 g) was
dissolved in 0.1 M potassium phosphate buffer (300 mL, pH 6.5). The
solution was heated, in a 1000 mL flask fitted with a condenser, at
1
10 °C in an oil bath for 2 h. After thermal reaction, the reaction mixture
*
Author to whom correspondence should be addressed [telephone (732)
was cooled to room temperature and extracted with methylene chloride
(300 mL) three times. The solvent was removed from the extract under
reduced pressure.
9
32-9611, ext. 235; fax (732) 932-6776; e-mail ho@aesop.rutgers.edu].
†
Rutgers University.
College of Staten Island, City University of New York.
‡
1
0.1021/jf011591v CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/31/2002

4056 J. Agric. Food Chem., Vol. 50, No. 14, 2002
Jhoo et al.
Table 1. 13C and H NMR Chemical Shift of MAMP and Thiamina
1
δC
δH
C
MAMP
thiamin
MAMP
thiamin
2
4
5
6
7
8
167.1 s
163.1 s
112.0 s
154.8 d
24.9 q
164.1 s
164.0 s
106.9 s
145.8 d
22.0 q
7.23 s
2.32 s
3.59 s
8.21 s
2.75 s
5.62 s
9.86 s
34.3 t
51.0 t
155.5 d
2
′
158.3 s
110.1 s
116.6 d
142.3 d
11.4 q
1
1
Figure 1. Significant HMBC (H−C) and H− H COSY correlations of
MAMP.
3
′
4
′
143.8 s
137.4 s
30.2 t
61.2 t
12.2 q
6.24 br s
7.29 br s
1.90 s
5
′
Reaction with Thiamin and Cysteine. Two model systems were
prepared. For model system III, thiamin monochloride (1 g) was
dissolved in water (15 mL). Model system IV was prepared with thiamin
monochloride (1 g) and cysteine (1 g) in water (15 mL). Each reaction
mixture was refluxed in an oil bath at 80 °C for 70 min. After cooling,
mL of 1000 ppm of tridecane (in methylene chloride) was added to
each reaction mixture as internal standard. The solutions were then
extracted with methylene chloride (15 mL). After drying with anhydrous
6
′
3.32 t
4.03 t
2.85 s
7
′
8′
a
MAMP was measured in CD3OD. Assignments were based on HMQC, HMBC,
and COSY experiments. Thiamin was measured in D2O.
1
Purification of Thermally Degraded Compound. The methylene
chloride extract (700 mg) was subjected to silica gel column chroma-
tography with an ethyl acetate/methanol (10:0, 10:0.2, 10:0.4, 10:1,
each 300 mL) solvent system, which afforded four fractions. Fraction
4
MgSO , the organic fraction was concentrated by a stream of nitrogen
and analyzed by GC-MS.
RESULT AND DISCUSSION
1
was further purified with RP-18 (MeOH/water, 3:2) to afford
compound 1 (20 mg) as an amorphous powder: positive-ion HRFAB-
+
After thermal degradation of thiamin hydrochloride in aque-
ous solution, a major degradation compound was isolated with
chromatographic methods. The structure was elucidated by
MS, m/z 236.0865 [M + H] , calcd for C11
H
14ON
3
+
S, 236.0858; EI-
•
MS spectral data, m/z (relative intensity) 235 (M , 11), 122 (100),
1
1
13 (5), 81 (28), 80 (9), 59 (2), 54 (15), 42 (27), 28 (8); H NMR (600
13
1
13
4
MHz, in methanol-d ) see Table 1 ; C NMR (150 MHz, in methanol-
interpretation of H NMR, C NMR, 2D NMR, and MS spectra.
Compound 1 was isolated as an amorphous solid having a
molecular formula of C11H13ON3S, which was determined by
4
d ) see Table 1.
NMR, APCI-MS, and GC/GC-MS Analysis. H NMR and 13C
1
+
NMR and all 2D NMR spectra were obtained on a Varian 600
instrument (Varian Inc., Palo Alto, CA). The compound was analyzed
in CD OD, with TMS as the internal standard. HRFAB-MS was run
3
positive-ion HRFAB-MS ([M + H] at m/z 236.0865), as well
1
3
13
as by its C NMR data and EI-MS spectral data. The C NMR
spectrum showed 11 carbon signals. After careful comparison
on a JEOL HX-110 double-focusing mass spectrometer. Gas chroma-
tography (GC) was performed on an HP 5790A gas chromatograph
equipped with a flame ionization detector (FID) and a nonpolar fused
silica capillary column (30 m, 0.32 mm i.d., 0.25 µm film thickness,
DB-1, J&W Scientific Inc.). The column temperature was programmed
from 40 to 260 °C at a rate of 2 °C/min. The injector temperature and
FID temperature were set at 270 and 300 °C, respectively. The flow
rate of the helium carrier gas was 1 mL/min. GC-MS was performed
on an HP 5790A GC, which was coupled to an HP 5970A mass
spectrometer. The ionization of the mass spectrometer was set at 70
eV.
13
with the C NMR spectrum of authentic thiamin and published
data (8), one methyl carbon signal (δ 24.9) and four olefinic
carbon signals (δ 112.0, 154,8, 163.1, and 167.1) were in
agreement with the spectral characteristics of a pyrimidine
moiety in thiamin. In addition, one methyl proton signal at δ
2
.32 (3H, s, CH3) and one olefinic proton signal at δ 7.23 (1H,
s, CH) also agreed with a pyrimidine moiety when compared
1
with the H NMR spectrum of authentic thiamin and published
data (9). Interestingly, bridged methylene carbon (C-8) was
13
shifted upfield by ∼16.7 ppm compared with the C NMR
Synthesis of 2-Methyl-4-amino-5-(2-methyl-3-furylthiomethyl)-
pyrimidine (MAMP; 1). Thiamin hydrochloride (6 g) was dissolved
in methanol (50 mL). Triethylamine (4 g) was slowly added into the
solution with gentle shaking until the reaction mixture became clear.
It was then concentrated under reduced pressure to approximately its
half volume and left to stand overnight at room temperature. Thiamin
monochloride was obtained by filtration from the reaction mixture.
Subsequently, thiamin monochloride (1 g) and 7 (1 g) were dissolved
in methanol (15 mL) and reacted in an oil bath at 80 °C for 2 h. After
cooling, the solution was concentrated to dryness under reduced
pressure. The residue was extracted with ethyl ether. The extract was
then purified by column chromatographic methods to obtain compound
spectrum of thiamin. This suggests that C-8, which is connected
to the nitrogen atom of 6 in the thiamin, would be displaced
with others.
The remaining five carbon peaks showed one methyl carbon
(
1
δ 11.4) and four olefinic carbons (δ 110.1, 116.6, 142.3, and
58.3) (Table 1). The H NMR spectrum also gave one methyl
1
proton signal at δ 1.90 (3H, s, CH3) and two olefinic protons at
δ 6.24 (1H, s, CH) and δ 7.29 (1H, s, CH) (Table 1). This
spectral information suggests that the remaining moiety has a
furan ring structure having one methyl group. According to the
1
1
H- H COSY spectrum, two olefinic proton signals have
1
as described in the purification of compound 1 from thermal reaction
correlated with each other. Along with the NMR interpretation,
the m/z 113 and 81 peaks in the mass spectrum suggest the
presence of a 2-methyl-3-furanthiyl moiety. As we previously
mentioned, the C-8 in the pyrimidine moiety shifted upfield.
The evidence strongly supports the assumption that the pyri-
midine unit and 2-methyl-3-furanthiol (7) connected with each
other through the methylene bridge. The HMBC spectrum
decisively showed a cross-peak between H-8 and C-3′ (Figure
1). Consequently, the compound was determined to be 2-methyl-
4-amino-5-(2-methyl-3-furylthiomethyl)pyrimidine (MAMP; 1)
of thiamin. This synthetic method was modified from the method of
Shimahara et al. (7).
MAMP Formation Pathway Studies. Two model reactions were
carried out to understand the formation pathway of 1. The composition
of model systems is described below. Model system I was prepared
with thiamin monochloride (1 g) in methanol (15 mL). For model
system II, the mixture of thiamin monochloride (1 g) and 7 (1 g) was
dissolved in methanol (15 mL). Each reaction model system was heated
at 80 °C for 2 h in an oil bath. The reaction mixtures were then extracted
with methylene chloride and analyzed by GC-MS.

Pyrimidine Derivative from Thiamin Degradation
J. Agric. Food Chem., Vol. 50, No. 14, 2002 4057
Table 2. Thermal Degradation Products from Different Model Systems
for MAMP Formation
Table 3. Thermal Degradation Products from Reaction with Thiamin
Monochloride and Cysteine
a
a
a
(1) thiamin monochloride; (2) thiamin monochloride and cysteine.
a
(1) thiamin monochloride; (2) thiamin monochloride and 2-methyl-3-furanthol.
(Figure 1). The structure suggested for compound 1 was
ultimately confirmed by synthesis, which was performed ac-
cording to modified methods of Shimahara et al. (7). The
spectral characteristics of compound 1 were identical with those
of the compound isolated from synthesis.
Formation of MAMP from Thiamin. It is of interest to
understand the formation pathway of the novel compound 1.
Torward this end, we propose two possible formation pathways.
One is formation of 1 through thiamin rearrangement during
thermal degradation. The other possible pathway is substitution
of 6 by 7. Two model systems were, therefore, carried out to
differentiate these two pathways. Model systems I and II consist
of thiamin monochloride in methanol and thiamin monochloride
and 7 in methanol, respectively. After the thermal reaction of
each model system, thermal degradation products were deter-
mined by GC-MS analysis. As shown in Table 2, the formation
of 1 was dramatically increased when thiamin monochloride
was thermally degraded in the presence of 7. These results favor
the pathway that compound 1 results from the substitution of 6
by 7 rather than by the rearrangement of thiamin itself.
Nucleophilic substitution of thiamin by sulfite ion has been
kinetically proven to be a multistep process (4, 5). Zoltewicz
et al. also reported that amine nucleophiles such as pyridine,
Figure 2. Proposed formation pathway of MAMP.
of thiamin as a nucleophile, reacts with the stabilized cation
intermediate (5), which leads to the formation of compound 1
4
-aminopyridine, aniline, and diazabicyclo[2.2.2]octane could
(Figure 2).
substitute the thiazole portion of the thiamin molecule in
methanol with a multistep mechanism (6). Zoltewicz et al. (5,
2-Methyl-3-furanthiol (7) is one of the important meaty aroma
compounds from the thermal degradation of thiamin (10).
According to the present study, formation of 7 would be
decreased during the thermal degradation of thiamin because
of its participation in the formation of 1. However, 1 had only
a very weak sulfury odor; however, its formation may affect
the meatlike flavor profile during the thermal degradation
of thiamin. Because thiamin is considered to be one of the
most important ingredients for savory process flavors, the
formation of 1 at the expense of 7 would be unfavorable. To
develop an improved method for the production of meaty
6
) proposed that after the protonation of a pyrimidine ring,
followed by a nucleophile attack on the pyrimidine ring by a
hydroxy ion, the resulting group (6) is dissociated (Figure 2).
The pyrimidine ring then forms a stabilized cation intermediate.
Subsequently, the cation intermediate reacts with nucleophiles
and gives nucleophilic substituted compounds (5, 6). We
presumed the formation pathway of 1 would be similar to that
proposed by Zoltewicz et al. (5, 6) as a multistep mechanism.
Here, compound 7, which is generated from thermal degradation

4058 J. Agric. Food Chem., Vol. 50, No. 14, 2002
Jhoo et al.
aroma from the thermal reaction of thiamin, it is possible
that the addition of another compound, having a nucleophilic
character similar to that of 7, into the reaction mixture
may result in increased formation of 7. Therefore, we carried
out the analysis of two model systems. Model systems III and
IV consist of thiamin monochloride in methanol and thiamin
monochloride and cysteine (which was added as a nucleo-
philic competitor of 7), respectively. After thermal reaction
of each model system, degradation products were analyzed
using GC-MS. As expected, when thiamin was thermally
degraded in the presence of cysteine, the formation of 7 was
increased, with a decreased amount of 1 (Table 3). The results
strongly support the assumption that cysteine has the same
nucleophilic character and can compete with 7 for reaction with
thiamin.
(4) Williams, R. R.; Waterman, R. E.; Keresztesy, J. C.; Buchman,
1
E. R. Studies of crystalline vitamin B . III. Cleavage of vitamin
with sulfite. J. Am. Chem. Soc. 1935, 57, 536-537.
(
5) Zoltewicz, J. A.; Kauffman, G. M. Kinetics and mechanism
of the cleavage of thiamin, 2-(1-hydroxyethyl)thiamin, and
a derivative by bisulfite ion in aqueous solution. Evidence
for an intermediate. J. Am. Chem. Soc. 1977, 99, 3134-
3
142.
6) Zoltewicz, J. A.; Baugh, T. D. A multistep mechanism of
nuclophilic substitution of vitamin B in methanol. Bioorg. Chem.
985, 13, 209-218.
(
1
1
(
7) Shimahara, N.; Nakajima, N.; Hirano, H. Decomposition of
thiamin in alcohol solution. I. Chem. Pharm. Bull. 1974, 22,
2081-2085.
(8) Himmeldirk, K.; Sayer, B. G.; Spenser, I. D. Comparative
biogenetic anatomy of vitamin B
1
: A 13C NMR investigation
of the biosynthesis of thiamin in Escherichia coli and in
Saccharomyces cereVisiae. J. Am. Chem. Soc. 1998, 120, 3581-
LITERATURE CITED
3
589.
(
(
(
1) G u¨ ntert, M.; Br u¨ ning, J.; Emberger, R.; Hopp, R.; K o¨ psel, M.;
Surburg, H.; Werkhoff, P. Thermally degraded thiamin. In FlaVor
Precursors; American Chemical Society: Washington, DC,
(9) Otagiri, Y.; Uekama, K.; Ikeda, K. Kinetics and mechanism of
transamination of thiamine in the presence of bisulfite. Chem.
Pharm. Bull. 1975, 23, 13-19.
(10) Hartman, G. J.; Carlin, J. T.; Scheide, J. D.; Ho, C.-T. Volatile
products formed from the thermal degradation of thiamin at high
and low moisture levels. J. Agric. Food Chem. 1984, 32, 1015-
1018.
1
992; pp 140-163.
2) G u¨ ntert, M.; Bertram, H.-J.; Emberger, R.; Hopp, R.; Sommer,
H.; Werkhoff, P. Thermal degradation of thiamin (vitamin B ).
1
In Sulfur Compounds in Foods; American Chemical Society:
Washington, DC, 1994; pp 199-223.
3) G u¨ ntert, M.; Br u¨ ning, J.; Emberger, R.; K o¨ psel, M.; Kuhn, W.;
Thielmann, T.; Werkhoff, P. Identification and formation of
some selected sulfur-containing flavor compounds in various
meat model systems. J. Agric. Food Chem. 1990, 38, 2027-
Received for review December 4, 2001. Revised manuscript received
April 26, 2002. Accepted April 29, 2002.
2
041.
JF011591V