The role of methylglyoxal in the non-enzymatic conversion of tryptophan, its methyl ester and tryptamine to 1-acetyl-β-carbolines

Article abstract of DOI:10.1016/j.bmc.2008.02.039

Non-enzymatic modification of l-tryptophan (1) and its metabolites and derivatives with aldehydes, via the Pictet-Spengler reaction, affords β-carbolines. Here we demonstrate that methylglyoxal (2) generates 1-acetyl-β-carbolines from tryptophan (1), from its methyl ester (6) and from tryptamine (4); however, 2 did not generate 1-(1-hydroxyethyl)-β-carboline derivates. HPLC analysis of model reaction systems showed formation of 1-acetyl-β-carboline (3) and 1-acetyl-β-carboline-3-carboxylic acid (5) during incubation of 1 and 2, at pH 5.7 and 7.4, at 100 °C, and only 5 at 37 °C, at the same pH values, with limited access of oxygen. Aerobic conditions caused higher formation of 3 at 37 °C at both pH values, while, at higher temperature, the same effect was only observed at pH 5.7. Lack of oxygen did not much influence the formation of 3 or 5 at both pH and temperature values, in comparison with the formation at limited access of oxygen. Incubation of 2 and 6 generated methyl-1-acetyl-β-carboline-3-carboxylate (7) together with 3 and 5 as a result of hydrolysis of 6 into 1 and, partially, 7 into 5, while in incubation mixtures of 2 and 4 only unstable 1-acetyl-3,4-dihydro-β-carboline (8) was observed. Incubation of 1 with d-glucose as well as incubation of tryptophan with Amadori product 18 under similar conditions did not generate carbolines 3 or 5. For the first time, we were able to demonstrate the presence of 1-acetyl-β-carboline-3-carboxylic acid (5) in some commercially available ketchups and in previously heated tomato concentrate.

Full text of DOI:10.1016/j.bmc.2008.02.039

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4551–4562
The role of methylglyoxal in the non-enzymatic conversion
of tryptophan, its methyl ester and tryptamine
to 1-acetyl-b-carbolines
Ina Nemet and Lidija Varga-Defterdarovi c´ *
-
Division of Organic Chemistry and Biochemistry, Ruder Bo sˇ kovi c´ Institute,
Bijeni cˇ ka cesta 54, P.O. Box 180, HR-10 002 Zagreb, Croatia
Received 2 January 2008; revised 6 February 2008; accepted 12 February 2008
Available online 15 February 2008
Abstract—Non-enzymatic modification of L-tryptophan (1) and its metabolites and derivatives with aldehydes, via the Pictet–Spengler
reaction, affords b-carbolines. Here we demonstrate that methylglyoxal (2) generates 1-acetyl-b-carbolines from tryptophan (1), from
its methyl ester (6) and from tryptamine (4); however, 2 did not generate 1-(1-hydroxyethyl)-b-carboline derivates. HPLC analysis of
model reaction systems showed formation of 1-acetyl-b-carboline (3) and 1-acetyl-b-carboline-3-carboxylic acid (5) during incubation
of 1 and 2, at pH 5.7 and 7.4, at 100 ꢀC, and only 5 at 37 ꢀC, at the same pH values, with limited access of oxygen. Aerobic conditions
caused higher formation of 3 at 37 ꢀC at both pH values, while, at higher temperature, the same effect was only observed at pH 5.7. Lack
of oxygen did not much influence the formation of 3 or 5 at both pH and temperature values, in comparison with the formation at
limited access of oxygen. Incubation of 2 and 6 generated methyl-1-acetyl-b-carboline-3-carboxylate (7) together with 3 and 5 as a
result of hydrolysis of 6 into 1 and, partially, 7 into 5, while in incubation mixtures of 2 and 4 only unstable 1-acetyl-3,4-dihydro-b-
carboline (8) was observed. Incubation of 1 with D-glucose as well as incubation of tryptophan with Amadori product 18 under similar
conditions did not generate carbolines 3 or 5. For the first time, we were able to demonstrate the presence of 1-acetyl-b-carboline-3-
carboxylic acid (5) in some commercially available ketchups and in previously heated tomato concentrate.
ꢁ
2008 Elsevier Ltd. All rights reserved.
1
. Introduction
bound, Amadori rearrangement products. The latter un-
dergo further rearrangements to form irreversibly linked
adducts called advanced glycation end products
Tryptophan, an essential nutrition ingredient, is a build-
ing block of proteins and the precursor, not only of a
variety of biologically active compounds (serotonin,
melatonin, tryptamine, quinolinic, and kynurenic acids),
2
(AGEs). In the Pictet–Spengler reaction the Schiff base
undergoes ring closure resulting in the formation of tet-
3
rahydro-b-carbolines and b-carbolines, a group of com-
pounds with a large array of pharmacological activities.
1
4
but also of the coenzymes, NAD and NADP. Under
physiological conditions, and during thermal food pro-
cessing or storing, tryptophan, like other amino acids,
undergoes non-enzymatic modifications with reducing
carbohydrates (non-enzymatic glycation) via the Mail-
lard and/or the Pictet–Spengler reactions (Scheme 1).
The Maillard reaction is initiated by nucleophilic attack
of the amino group of an amino acid at a sugar carbonyl
group, leading to a reversible Schiff base which sponta-
neously rearranges to form more stable, covalently
These compounds have recently been detected in a vari-
5
ety of food and biological matrices.
We recently showed that methylglyoxal, a physiological
metabolite and a key intermediate in the Maillard reac-
6
–9
tion, and about 20,000 times more reactive than glu-
1
0,11
cose in glycation reactions,
could spontaneously
modify tryptophan, its methyl ester and tryptamine via
the Pictet–Spengler reaction to yield the corresponding
1
2
b-carbolines.
To gain more insight into the possible formation of
these b-carbolines during food processing and storage,
as well as under physiological temperature and pH
Keywords: Methylglyoxal; 1-Acetyl-b-carboline; Tryptophan.
Corresponding author. Tel.: +385 1 456 11 57; fax: +385 1 468 01
95; e-mail: lidija@irb.hr
*
0
968-0896/$ - see front matter ꢁ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.039

4552
I. Nemet, L. Varga-Defterdarovi c´ / Bioorg. Med. Chem. 16 (2008) 4551–4562
COOH
HN
O
N
H
HO
H
H
OH
OH
A
B
H
CHO
OH
H
OH
OH
COOH
NH2
CH OH
H
HO
H
2
glucose-tryptophan Amadori product
COOH
+
N
H
H
CH OH
2
NH
N
N
L-tryptophan (1)
D-glucose
N
H
H
O
H
HO
H
OH
H
OH
OH
H
OH
H
OH
OH
HO
H
H
H
CH OH
CH OH
2
2
1-pentahydroxypentyl-1,2,3,4-tetrahydro-β-carboline 1-pentahydroxypentyl-β-carboline
Scheme 1. Tryptophan in the Maillard (Path A) and Pictet–Spengler (Path B) reaction.
value, here we describe methods for the laboratory
synthesis of b-carbolines derived from tryptophan (1),
tryptamine (4) and tryptophan methyl ester (6) with
methylglyoxal (2), together with a detailed study of their
generation in different model systems and identification
of 1-acetyl-b-carboline-3-carboxylic acid (5) in a ketch-
up and a tomato concentrate after thermal treatment.
diately following the ring closure. This concept is corrob-
orated by experiments on the reduction of tryptamine-
derived b-carboline 8 with sodium cyanoborohydride.
TLC of the crude reaction mixture confirmed complete
consumption of 8, accompanied by the formation of a
new compound. However, after removal of the sodium
cyanoborohydride, only starting compound 8 was iso-
lated in almost quantitative yield.
2
. Results
In addition to the formation of 1-acetyl-b-carbolines, in
the above reactions, the corresponding 1-(1-hydroxy-
ethyl)-b-carbolines were expected as by-products, as it
was already shown that direct condensation of phenyl-
glyoxal or methylglyoxal with L-tryptophan affords
1-(1-hydroxybenzyl)- or 1-(1-hydroxyethyl)-b-carbo-
2
.1. Standards synthesis
Figure 1 summarizes the syntheses performed reacting
methylglyoxal (1) with L-tryptophan (2), tryptamine
4) or L-tryptophan methyl ester (6) under conditions
1
4,15
(
lines, respectively.
Considering the complexity of
characteristic for the Pictet–Spengler reaction (acidic
aqueous medium), along with a number of alternative
procedures used to prepare the desired b-carbolines.
the previously described reaction mixtures, we
attempted to synthesize the hydroxyethyl derivates using
the following alternative approaches. First we resorted
to the Bischler–Napieralski reaction. Amides 10 and
12 required for this purpose were prepared by N,N-dicy-
clohexylcarbodiimide (DCC) mediated condensation of
lactic acid (9) with tryptamine (4) or L-tryptophan
methyl ester (6), in yields of 34 and 36.4%. Subsequent
acetylation of the hydroxyl groups in 10 and 12 afforded
11 and 13 in quantitative yield. Amides 10, 11, and 13
were subjected to Bischler–Napieralski cyclization.
As we recently communicated, acid-catalyzed condensa-
tion of methylglyoxal (2) with L-tryptophan (1) afforded
a mixture of 1-acetyl-b-carboline (3) and 1-acetyl-b-
carboline-3-carboxylic acid (5). Replacing 1 by its
methyl ester (6) led to the formation of methyl-1-ace-
tyl-b-carboline-3-carboxylate (7). Replacement by trypt-
amine (4) afforded partially saturated 1-acetyl-3,4-
dihydro-b-carboline (8), which required exposure to
alkaline conditions to convert to the fully aromatized
1
6
However, refluxing amide 11 in POCl3, afforded
1-acetyl-3,4-dihydro-b-carboline (8) (g = 13.4%), instead
of the expected 1-hydroxyethyl analogue. Refluxing
1
2
product 3.
1
7
amide 10 in TFA, gave the same (8) product, but in
much lower yield (g = 2%). Even though the reaction
mixtures were treated with saturated aqueous NaHCO3,
the formation of 8 was most likely the result of deacet-
ylation and subsequent oxidation of the deprotected hy-
droxyl group induced by acid and heating during the
As it is well known that all the above b-carbolines arise
via tetrahydro-b-carbolines as intermediates, we postu-
lated that the latter should be present in the described
reaction mixtures. However, addition of potassium
dichromate, as a known initiator of tetrahydro-b-carbo-
line oxidation into the corresponding b-carbolines, to
reaction mixtures containing L-tryptophan (1) and meth-
ylglyoxal (2) did not increase the yield of 1-acetyl-b-carb-
oline (3), likely because 1-acetyl-1,2,3,4-tetrahydro-b-carboline
underwent spontaneous oxidative aromatization imme-
1
3
reaction of POCl or TFA with water. Submission of
3
amide 13 to the Bischler–Napieralski reaction in POCl3
resulted in reductive dehydration and formation of
methyl-1-ethyl-b-carboline-3-carboxylate (14) (g = 33%).
1
8
Using PCl5 in the same reaction scheme starting with

I. Nemet, L. Varga-Defterdarovi c´ / Bioorg. Med. Chem. 16 (2008) 4551–4562
4553
NH
O
N
H
?
NaCNBH3
R
N
O
H+
NH2
N
O
O
+
N
N
H
O
N
H
H
OH-
8
4
2
3
(R: H)
R
5 (R: COOH)
7
(R: COOCH3)
NH2
O
O
H+
+
N
H
NaBH4
TFA
NaBH4
POCl3
2
1
6
(R: COOH)
(R: COOCH3)
R
O
O
POCl3, PCl5
or TFA
N
H
acetanhydride
N
DCC
O
OH
N
OAc
H
OH
4
+
HOBt
N
N
H
OH
HO
N
H
H
9
1
1
10
1
1
5 (R: H)
6 (R: COOCH3)
OH-
COOCH3
O
COOCH3
O
POCl3, PCl5
or TFA
N
acetanhydride
N
DCC
O
OH
COOH
N
OAc
H
OH
H
6
+
N
H
N
H
HOBt
HO
12
9
OH
13
N
H
17
POCl3
COOCH3
N
N
H
1
4
Figure 1. Synthesis of 1-acetyl- and 1-(1-hydroxyethyl)-b-carbolines.
L-tryptophan (1), its methyl ester 6 or its metabolite 4
with methylglyoxal (2) under acidic conditions. Thus
amines 1, 4, and 6 were incubated with methylglyoxal
amides 11 or 13 did not result in b-carboline formation
(
Fig. 1).
(
3
2) in 50 mM phosphate buffer, at pH 5.7 and 7.4, at
7 and 100 ꢀC. In addition, with the aim to examine
Because the Bischler–Napieralski reaction, under the
previously described conditions, failed for 1-(1-hydroxy-
ethyl)-b-carbolines 15–17, compounds 15 and 16 were
prepared by reduction of the corresponding acetyl deri-
vates 3 and 7 with sodium borohydride, in yields of 45
and 79%, respectively, while 17 was obtained by alkaline
hydrolysis of ester 16. However, HPLC and TLC analy-
sis of the respective incubation mixtures showed that
the role of oxidative processes in the formation of
b-carbolines 3 and 5 and their distribution following
interaction of 1 and 2, incubations were also conducted
under conditions with different availability of oxygen.
When L-tryptophan (1) and methylglyoxal (2) were re-
acted in airtight closed vessels (limited oxygen availabil-
ity; Fig. 2), at 37 ꢀC, 1-acetyl-b-carboline-3-carboxylic
acid (5) was detected at both examined pH values, while
1
3
1
-(1-hydroxyethyl)-b-carbolines cannot be generated
by direct condensation of L-tryptophan (1), tryptamine
4), and L-tryptophan methyl ester (6) with methylgly-
oxal (2) (Fig. 1).
(
-acetyl-b-carboline (3) was found only at pH 5.7, after
days (Fig. 2A and B). After the first day of incubation,
the same amount of b-carboline 5 was obtained at both
pH values; further accumulation of 5 was only observed
at pH 7.4. At 100 ꢀC, 3 and 5 were formed at both pH val-
ues, (Fig. 2C and D), but while the amount of 5 was the
same, in both cases, the formation of b-carboline 3 was
enhanced at the higher pH (Fig. 2D).
2
carbolines in model systems under physiological and food
storage or processing temperatures and pH values
.2. Formation of methylglyoxal (2) derived 1-acetyl-b-
As shown in Section 2.1, 1-acetyl-b-carbolines can be
formed in spontaneous non-enzymatic reactions of

4554
I. Nemet, L. Varga-Defterdarovi c´ / Bioorg. Med. Chem. 16 (2008) 4551–4562
0
0
0
0
0
0
0
0
.14
.12
.10
.08
.06
.04
.02
.00
0.14
A
B
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0
2
4
6
8
0
2
4
6
8
t / days
t / days
0
0
0
0
0
0
0
0
.14
.12
.10
.08
.06
.04
.02
.00
0.14
0.12
0.10
C
D
0
0
0
0
0
.08
.06
.04
.02
.00
0
1
2
3
4
5
0
1
2
3
4
5
t / h
t / h
Figure 2. Formation of 1-acetyl-b-carboline (3; n) and 1-acetyl-b-carboline-3-carboxylic acid (5; j) during incubation of L-tryptophan (1) with
methylglyoxal (2) in phosphate buffer at pH 5.7 (A and C) and 7.4 (B and D) at 37 ꢀC (A and B) and 100 ꢀC (C and D), under limited availability of
oxygen.
When the incubations were carried out under aerobic
conditions, at 37 ꢀC, (Fig. 3A and B) 1-acetyl-b-carbo-
line (3) was formed at both pH values, but an approxi-
mately two times higher amount accumulated at pH 5.7
ready described. The hydrolysis occurred faster at
higher pH and temperature (Fig. 5B and D). At the
same time 2 and 6 also reacted directly. Thus, at 37 ꢀC
(Fig. 5A and B) 1-acetyl-b-carboline-3-carboxylic acid
(5) and methyl-1-acetyl-b-carboline-3-carboxylate (7)
were formed at both pH values while 1-acetyl-b-carbo-
line (3) was not detected in any incubation mixture. At
pH 7.4 and 37 ꢀC b-carboline 7 reached its maximum
after the first day of incubation and then rapidly disap-
peared (Fig. 5B). As the concentration of b-carboline 5
constantly increased during the incubation period, but
its amounts were the same as those detected when
L-tryptophan (1) and methylglyoxal (2) were incubated
(Fig. 2B), it may be concluded that hydrolysis of 7 into
5 had non-considerable influence on the final concentra-
tion of 5. At pH 5.7 (Fig. 5A), the amount of b-carbo-
line 5 formed was lower than during the incubation of
1 and 2 under similar conditions (Fig. 2A) which is
probably the result of slower hydrolysis of ester 6, that
is, smaller concentration of 1. b-Carboline 7 reached
its maximum after 24 h of incubation at acidic pH, at
both temperatures (Fig. 5A and C). However, although
(
Fig. 3A) than at pH 7.4 (Fig. 3B). On the other hand
the trend of 1-acetyl-b-carboline-3-carboxylic acid (5)
formation was almost identical to that with the limited
oxygen availability (Fig. 2A and B). A similar trend in
the distribution of 3 in spite of oxygen availability was
also observed at 100 ꢀC and pH 7.4 (Figs. 2D and
3
D). Additional oxygen (Fig. 3 vs Fig. 2) also increased
the formation of the examined b-carbolines, at both
temperatures, but considerably only at pH 5.7 in which
case the amounts of 5 were three times and those of 3
even ten times higher (Fig. 3A and C vs Fig. 2A and
C), while at pH 7.4 the amounts of 3 and 5 were similar
to those obtained with limited amounts of oxygen
(
Fig. 3B and D vs Fig. 2B and D).
The results presented in Figure 4 showed that b-carbo-
lines 3 and 5 can be formed even under oxygen
deficiency.
7
was degraded during incubation it was constantly
During the incubation of L-tryptophan methyl ester (6)
with methylglyoxal (2) at 37 ꢀC and 100 ꢀC (Fig. 5), part
of the ester (6) was hydrolyzed and the free L-trypto-
phan (1) formed reacted with methylglyoxal (2) as al-
formed as a result of slower ester 6 hydrolysis in an
acidic medium. As no parallel increase of b-carboline 5
was observed, it was assumed that b-carboline 7 was
consumed in other processes which were beyond the

I. Nemet, L. Varga-Defterdarovi c´ / Bioorg. Med. Chem. 16 (2008) 4551–4562
4555
0
0
0
0
0
0
0
0
.14
.12
.10
.08
.06
.04
.02
.00
0.14
A
B
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0
2
4
6
8
0
2
4
6
8
t / days
t / days
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
C
D
0
1
2
3
4
5
0
1
2
3
4
5
t / h
t / h
Figure 3. Formation of 1-acetyl-b-carboline (3; n) and 1-acetyl-b-carboline-3-carboxylic acid (5; j) during incubation of L-tryptophan (1) with
methylglyoxal (2) in phosphate buffer at pH 5.7 (A and C) and 7.4 (B and D) at 37 ꢀC (A and B) and 100 ꢀC (C and D), under aerobic conditions.
scope of this study (Fig. 5A). Similar observations were
made at 100 ꢀC (Fig. 5C and D), although, under these
conditions, 1-acetyl-b-carboline (3) was also detected. In
addition, comparison of the amounts of b-carboline 5
formed from 6 in alkaline medium (Fig. 5D), with those
observed in incubations of 1 and 2 under similar condi-
tions (Fig. 2D), suggested that 5 may also arise by
hydrolysis of 7. During the incubations of tryptamine
the incubation mixtures did not show formation of
1-acetyl-b-carbolines 3 or 5. 1-Acetyl-b-carboline-3-car-
boxylic acid (5) was, however, detected in some commer-
cially available ketchups and in a tomato concentrate,
after heating at 100 ꢀC, for 1 h, while no 5 was found be-
fore heating. Detection, in this case, was by a highly sen-
sitive and specific LC–MS/MS method (Fig. 6). Unlike
5, 1-acetyl-b-carboline (3) was not detected in any of
the analyzed samples of food or beverage.
(
4) and methylglyoxal (2) (data not shown), 1-acetyl-
,4-dihydro-b-carboline (8) was formed under all exam-
3
ined conditions (pH 5.7 and 7.4, at 37 and 100 ꢀC), and
during all incubation periods, excluding pH 7.4 at
3. Discussion
1
00 ꢀC, in which case 8 disappeared after 2 h of incuba-
tion. 1-Acetyl-b-carboline (3) was not detected in any of
those incubations presumably because the pH was not
alkaline enough and/or the degradation rate was faster
than complete aromatization.
b-Carbolines represent a large group of alkaloids widely
distributed in nature, occurring in plants, marine organ-
isms, insects and, most importantly, in foods and in hu-
man tissues and body fluids. The compounds are of
great interest due to their diverse biological activities,
such as inhibition of topoisomerase and monoamine
oxidase, binding to benzodiazepine and serotonin recep-
tors and intercalating into DNA. In addition, these
alkaloids also exhibit a broad spectrum of pharmacolog-
ical activities including sedative, anxiolytic, hypnotic,
The possible formation of 1-acetyl-b-carbolines 3 and 5
from D-glucose and L-tryptophan (1) under the above
conditions were examined by incubation of D-glucose
and L-tryptophan (1) in different molar ratios (10:1
and 20:1), as well as by incubation of previously synthe-
sized N-(1-deoxy-D-fructos-1-yl)-L-tryptophan (18), the
Amadori product of L-tryptophan (1), with and without
the presence of L-tryptophan (1) in phosphate buffer at
pH 5.7 and 7.4, at 37 ꢀC, for 21 days, and at 100 ꢀC,
for 5 h. Under these conditions, RP HPLC analysis of
4
anticovulsant, antitumor, and antimicrobial effects.
In the current study, it was demonstrated that the
endogenous metabolite and intermediate in the Maillard
6
–9
reaction, methylglyoxal (2), reacts with L-tryptophan

4556
I. Nemet, L. Varga-Defterdarovi c´ / Bioorg. Med. Chem. 16 (2008) 4551–4562
0
0
0
0
0
0
0
0
.14
.12
.10
.08
.06
.04
.02
.00
0.14
A
B
0.12
0.10
0.08
0
0
0
0
.06
.04
.02
.00
0
2
4
6
8
0
2
4
6
8
t / days
t / days
0
0
0
0
0
0
0
0
.14
.12
.10
.08
.06
.04
.02
.00
0.12
C
D
0
0
0
0
0
0
.10
.08
.06
.04
.02
.00
0
1
2
3
4
5
0
1
2
3
4
5
t / h
t / h
Figure 4. Formation of 1-acetyl-b-carboline (3; n) and 1-acetyl-b-carboline-3-carboxylic acid (5; j) during incubation of L-tryptophan (1) with
methylglyoxal (2) in phosphate buffer, pH 5.7 (A and C) and 7.4 (B and D) at 37 ꢀC (A and B) and 100 ꢀC (C and D), under nitrogen.
(
1), its metabolite 4 and derivate 6, under physiological
line 5 can also be formed under physiological pH and
temperature (pH 7.4, 37 ꢀC), in contrast to the restricted
reactivity of some other physiological aldehydes, such as
glucose, which can generate corresponding b-carbolines
from L-tryptophan (1) (regardless of the temperature)
and during food storing or processing temperatures and
pH values, to yield 1-acetyl-b-carbolines 3, 5, 7, and 8.
Some of them were previously isolated from a number
of plants
affinity for the brain benzodiazepine recognition site
1
9–21
22
and microorganisms, and their high
4
only in acidic solution. The different distribution of
2
3
received particular attention.
products at different pH values, together with beneficial
influence of oxygen on the decarboxylation step, in par-
ticular under acidic conditions, suggests that oxidation
of the respective intermediate 1,2,3,4-tetrahydro-b-carb-
olines does not involve only one, but likely a combination
of several mechanisms. L-Tryptophan methyl ester (6)
and tryptamine (4) formed b-carbolines 7 and 8, under
the examined model conditions. Under certain condi-
tions (100 ꢀC, pH 7.4), incubation of 6 and 2 also afforded
carbolines derived from L-tryptophan (1), due to partial
hydrolysis of 6 into 1 and 7 into 5. The overall reactivity
of the amino group toward methylglyoxal (2) decreased
in the order 6 > 1 > 4 (details not shown). This can be
While some dicarbonyl compounds were previously
shown to condense with L-tryptophan (1) to yield
1
tion,
ethyl)-derivates 15–17 in mixtures of methylglyoxal (2)
and 1, 4, or 6, under the conditions adopted in this work.
-hydroxylcarbolines via the Pictet–Spengler reac-
1
4,15
we failed to observe formation of 1-(1-hydoxy-
The systematic study outlined above showed that, at
1
00 ꢀC, methylglyoxal (2) and L-tryptophan (1) gener-
ated 1-acetyl-b-carboline (3) at both pH 5.7 and 7.4,
regardless of oxygen availability. At 37 ꢀC, this hap-
pened, at a comparable rate, only under slightly acidic
conditions, else only after prolonged incubation or in
the presence of oxygen. Lower temperatures favored con-
servation of the carboxyl group at the newly formed ring,
why 1-acetyl-b-carboline-3-carboxylic acid (5), on the
other hand, was detected in the incubation mixtures of
rationalized by comparison of the pK values of the three
a
bases which decrease in the same order. The tryptophan
methyl ester imine intermediate (Schiff base), formed be-
fore the attack of electrons from the indole moiety and
3
ring closure, appears to be more electrophilic than the
imine formed from tryptamine (4).
1
able influence of oxygen on its formation. These results
clearly demonstrate that methylglyoxal-derived b-carbo-
and 2 under all examined conditions with non-consider-
The presence of 1-acetyl-b-carboline-3-carboxylic acid
(5) in tomato concentrate after heating at 100 ꢀC, for

I. Nemet, L. Varga-Defterdarovi c´ / Bioorg. Med. Chem. 16 (2008) 4551–4562
4557
1
.2
.0
.8
.6
.4
.2
.0
0.06
0.05
0.04
0.03
0.02
0.01
0.00
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0.06
0.05
0.04
0.03
0.02
0.01
0.00
A
B
1
0
0
0
0
0
0
2
4
6
8
0
2
4
6
8
t / days
t / days
1
.0
0.06
1.2
1.0
0.8
0.06
0.05
0.04
0.03
0.02
0.01
0.00
C
D
0
.05
.04
.03
.02
.01
0.00
0.8
0
0.6
0
0
.6
.4
.2
.0
0.4
0
0
0.2
0
0
0.0
0
0
1
2
3
4
5
0
1
2
3
4
5
t / h
t / h
Figure 5. Formation of 1-acetyl-b-carboline (3; n; solid line), 1-acetyl-b-carboline-3-carboxylic acid (5; j; solid line), methyl-1-acetyl-b-carboline-3-
carboxylate (7; ꢀ; dotted line) and L-tryptophan (1; •; dashed line) during incubation of L-tryptophan methyl ester (6; Æ; dashed line) with
methylglyoxal (2) in phosphate buffer at pH 5.7 (A and C) and 7.4 (B and D) at 37 ꢀC (A and B) and 100 ꢀC (C and D).
demonstration of 5 as a food contaminant. Its possible
origin could be explained in accord with the model sys-
tems outlined above.
0
12007_karbolini_33
.06
MRM of 4 Channels ES+
255.08 > 237
3
100
Occurrence of b-carbolines, primarily b-carboline (nor-
harman) and 1-methyl-b-carboline (harman), was dem-
onstrated in different foodstuffs and biological fluids
4
.55
2
4
3.50 14.91 18.96
and tissues. Recently Herraiz and Galisteo demon-
strated the presence of sugar-derived tetrahydro-b-
carbolines in fruit juices and jams, which during heat-
1
27.77
2
4.94
37.50
0
5
1
0.00
12007_karbolini_33
.02
20.00
30.00
ing could be rearranged into 1-acetyl-b-carbolines. Be-
cause of that and because of the possible formation of
methylglyoxal (2) during the Maillard reaction of sug-
ars (glucose) with amino acids, formation of 1-acetyl-
b-carboline 3 should be expected in systems containing
free L-tryptophan (1). Moreover, R o¨ nner et al. showed
that heating of D-glucose and L-tryptophan (1) (5:1
molar ratio) in 0.1 M phosphate buffer, at pH 5, gen-
0
MRM of 4 Channels ES+
3
255.08 > 209.1
1
00
1
84
3.95
6
.47
21.00
33.31 37.36
7
.88 13.90 18.74
30.65
30.00
2
5
erated 1-acetyl-b-carboline (3). In the model systems
described above compounds 3 and/or 5 were detected.
The b-carboline 3 formed from D-glucose and 1 was
observed only at higher temperatures, that is, as R o¨ n-
0
Time
1
0.00
20.00
Figure 6. Detection of 1-acetyl-b-carboline-3-carboxylic acid (m/z
55.08) by LC–ESI/MS/MS analysis in a tomato concentrate previ-
2
5
2
ner et al. demonstrated above 120 ꢀC. Also, the
methylglyoxal formed in the reaction systems may be
consumed in additional possible pathways in which
the formation of compounds similar to the already
isolated and characterized methylglyoxal amine
ously heated 1 h at 100 ꢀC. Multiple reaction monitoring (MRM) with
m/z 237.0 and 209.1 as characteristic fragment ions.
1 h, while no 5 was detected prior the thermal treatment
and to the best of our knowledge, represents the first
2
6
derivates, such as carboxyethyl lysine (CEL)
or

4558
I. Nemet, L. Varga-Defterdarovi c´ / Bioorg. Med. Chem. 16 (2008) 4551–4562
2
7
methylglyoxal-derived lysine dimer (MOLD), can be
expected.
acetate/methanol (1:1, v/v) (F), and ethyl acetate/etha-
nol/water/acetic acid (7:1:1:1, v/v/v/v) (G). The identifi-
cation of b-carbolines in biological samples was
accomplished by LC–MS/MS using a Waters 2695 Sep-
aration Module with a Micromass Quatromicro triple
quadropole mass spectrometry detector (Waters-Micro-
mass, Manchester, UK). For the chromatographic sepa-
ration, an Atlantis dC-18 column (3 lm, 2.1 · 50 mm;
Waters, Milford, MA, USA) (flow rate: 0.2 mL/min)
was used. Ammonium formate (26 mM, pH 3.8) (sol-
vent H) and 90% aqueous acetonitrile (solvent I) were
used in gradient 3 according to the following program:
60% H and 40% I to 40% H and 60% I in 10 min; 40%
H and 60% I to 10% H and 90% I in 2 min; isocratic
10% H and 90% I for 7 min; 10% H and 90% I to 60%
H and 40% I in 3 min; and isocratic 60% H and 40% I
for 9 min to return to the initial conditions.
4. Conclusion
The described results and interpretations show that
methylglyoxal (2) and L-tryptophan (1) as well as its
metabolite 4 and derivate 6 generate 1-acetyl-b-carbo-
lines in model systems mimicking in vivo and/or food
processing and storage pH values and temperatures.
These results, together with the detection of one of the
described b-carbolines in some foods, corroborate the
proposed chemical pathways based on the Maillard
reaction.
Further studies focusing on the quantification of the
described b-carbolines and their biological activity are
required to demonstrate the distribution of these
b-carbolines and their physiological impact.
5.2. Chemicals
Reagents of the highest quality available were obtained
from Sigma (St. Louis, MO, USA); only ammonium for-
mate and acetonitrile were from Burdick & Jackson
5. Experimental
(Muskegon, MI, USA). Aqueous solutions of
methylglyoxal were prepared as previously described.
2
8
5
.1. General methods
Aqueous solutions of glyoxal, methylglyoxal, and
butan-2,3-dione were standardized using Friedmann’s
Melting points were determined in open capillaries on a
T o¨ ttoli (B u¨ chi) apparatus and are uncorrected. NMR
spectra were recorded on a Bruker AV 600 spectrometer
2
9
volumetric method. 1-Acetyl-b-carboline (3), 1-acet-
yl-b-carboline-3-carboxylic acid (5), and methyl-1-acet-
yl-b-carboline-3-carboxylate (7) were prepared as
1
3
operating at 150.91 MHz for C and 600.13 MHz for
H. The spectra were measured in D O and DMSO-d
1
12
previously described.
2
6
solution at 25 ꢀC. Chemical shifts in parts per million
ppm) were referenced to TMS in DMSO-d and to
dioxane in D O. Spectra were assigned based on 1D
(
5.2.1. Synthesis of 1-acetyl-b-carboline (3). The title com-
pound was prepared in three different ways, of which the
12
following two approaches were previously described :
6
2
1
(
H and APT) an 2D homonuclear (COSY) and hetero-
nuclear (HMQC, HMBC) experiments. Preparative
HPLC was performed on a Varian ProStar HPLC sys-
tem equipped with a Eurospher 100 C-18 semiprepara-
tive column (5 lm; 8 · 250 mm) (flow rate: 1.0 mL/
min), while analytical HPLC was performed on a HP
from L-tryptophan (1) and methylglyoxal (2) (Method
A) or from 1-acetyl-3,4-dihydro-b-carboline (8) (Meth-
od B).
Method C. L-Tryptophan (1) (200 mg; 0.98 mmol) dis-
solved in aqueous HCl (0.1 M; 5 mL) was stirred with
methylglyoxal (2) (288.5 mM; 6.8 mL; 1.96 mmol) at
80 ꢀC, for 2 h. An aqueous solution of potassium
dichromate (10%, 6 mL) was then added. The precipi-
tate formed was filtered off and purified by RP HPLC
1
090 system equipped with a diode-array detector using
an Eurospher 100C-18 analytical column (5 lm;
· 250 mm) (flow rate: 0.5 mL/min). The solvents used
for HPLC were 40% aqueous methanol containing
.1% TFA (solvent A); 75% aqueous methanol contain-
4
0
ing 0.1% TFA (solvent B); 72% aqueous methanol con-
taining 0.1% TFA (solvent C) and 35% aqueous
methanol containing 0.1% TFA (solvent D). Elution
was programmed as follows: Gradient 1: isocratic A
for 10 min; 100% A to 100% B in 10 min; isocratic B
for 10 min; 100% B to 100% A in 5 min, and isocratic
A for 5 min to return to the initial conditions; Gradient
(solvent B, t = 14.1 min) to yield 1-acetyl-b-carboline
(3) (2 mg; g = 2%).
R
5.2.2. Synthesis of 1-acetyl-3,4-dihydro-b-carboline (8).
The title compound was prepared in four different ways,
of which the condensation of tryptamine hydrochloride
(4) with methylglyoxal (2) (Method A) was previously
1
2
2
: isocratic D for 10 min; 100% D to 100% B in 10 min;
described.
isocratic B for 10 min; 100% B to 100% D in 5 min, and
isocratic D for 5 min to return to the initial conditions.
Thin layer chromatography was carried out with TLC
glass plates precoated with silica gel 60 F254 (Merck,
Darmstadt, Germany). Visualization was accomplished
using ninhydrin (2% in ethanol), the chlorine–iodine re-
agent, or by heating with H SO (10% in water). Flash
Method B. To a flask containing tryptamine hydrochlo-
ride (4) (200 mg; 1.02 mmol) dissolved in a mixture of
aqueous HCl (0.1 M; 2.5 mL) and methanol (4 mL),
an aqueous solution of methylglyoxal (2) (482 mM;
4.2 mL; 2.04 mmol) was added. The mixture was stirred
at 40 ꢀC overnight, neutralized with saturated aqueous
2
4
column chromatography was performed on silica gel
0.040–0.063 mm; Merck, Darmstadt, Germany). The
following solvents were used: ethyl acetate (E); ethyl
NaHCO , and extracted with chloroform (3· 20 mL).
3
(
The combined organic layers were washed with water,
dried over anhydrous Na SO , and evaporated. Purifica-
2
4

I. Nemet, L. Varga-Defterdarovi c´ / Bioorg. Med. Chem. 16 (2008) 4551–4562
4559
tion by flash column chromatography on silica gel (sol-
vent E; R = 0.92) afforded 1-acetyl-3,4-dihydro-b-carb-
oline (8) (17 mg; g = 7.9%).
was added. The mixture was stirred at room tempera-
ture, for 2 h, poured into ice cold water (40 mL) and
then stirred for an additional 2 h, at room temperature.
The mixture was evaporated as an azeotropic mixture
with toluene. N-(O-Acetyl-lactoyl)tryptamine (11) was
f
Method C. N-Lactoyltryptamine (10) (50 mg;
0
.22 mmol) was dissolved in concd TFA (3 mL) and
obtained as a colorless transparent oil (118 mg;
1
heated under reflux for 45 min. After cooling, the mix-
ture was neutralized with saturated aqueous NaHCO3
and extracted with chloroform (3· 20 mL). The com-
bined organic layers were washed with water, dried over
anhydrous Na SO , and evaporated. Purification by
g = 100%; Rf (E) = 0.93). H NMR (DMSO-d ) d
6
(ppm): 1.31 (d, J = 6.8 Hz, 3H, CH ); 2.06 (s, 3H,
COCH ); 2.83 (t, J = 7.4 Hz, 2H, 4-H); 3.34 (m, 2H, 3-
3
3
H); 4.92 (q, J = 6.8 Hz, 1H, CHO); 6.98 (m, 1H, 7-H);
7.07 (m, 1H, 6-H); 7.14 (s, 1H, 2-H); 7.34 (d,
J = 8.1 Hz, 1H, 5-H); 7.55 (d, J = 7.9 Hz, 1H, 8-H);
8.09 (t, J = 5.6 Hz, 1H, NHCO); 10.83 (s, 1H, NH).
2
4
flash column chromatography on silica gel (solvent E;
R = 0.92) afforded 1-acetyl-3,4-dihydro-b-carboline (8)
f
1
3
(
0.9 mg; g = 2%).
C NMR (DMSO-d ) d (ppm): 17.78 (CH ); 20.88
6 3
(
COCH ) 25.12 (C-4); 39.64 (C-3); 69.81 (CHO);
3
Method
(
D. N-(O-Acetyl-lactoyl)tryptamine
(11)
160 mg; 0.58 mmol) was dissolved in POCl3 (4 mL)
111.45 (C-5); 111.75 (C-4a); 118.32 (C-7); 118.34 (C-8),
121.00 (C-6); 122.80 (C-2); 127.32 (C-4b); 136.35 (C-
8a); 169.80 (OCO); 169.94 (NHCO). Anal. Calcd for
C H N O · 0.1H O: C, 65.25; H, 6.64; N, 12.06.
and heated under reflux for 2 h. After cooling the mix-
ture was added dropwise to ice-cooled, saturated aque-
ous NaHCO (40 mL). Final neutralization was done
1
5
18
2
3
2
Found: C, 65.07; H, 6.07; N, 10.31.
3
by adding solid NaHCO . The resulting mixture was
3
extracted with chloroform (3· 50 mL). The combined
organic layers were washed with water, dried over anhy-
drous Na SO , and evaporated. The crude product was
5.2.5. Synthesis of N-lactoyl-L-tryptophan methyl ester
(12). A solution of L-tryptophan methyl ester (6)
(500 mg; 2.08 mmol) and NEM (262 lL; 2.08 mmol) in
THF/water (10 mL, 3:2, v/v) was added dropwise to a
cooled (ꢁ15 ꢀC) solution of lactic acid (9) (310 lL;
4.16 mmol), HOBt (561 mg; 4.16 mmol), and DCC
2
4
purified by flash column chromatography on silica gel
(
solvent E; R = 0.92) to yield 1-acetyl-3,4-dihydro-b-
f
carboline (8) (16.6 mg; g = 13.4%).
(1.03 g; 4.57 mmol) in THF (30 mL). The resulting mix-
5.2.3. Synthesis of N-lactoyltryptamine (10). Tryptamine
hydrochloride (4) (300 mg; 1.53 mmol) was dissolved in
THF/water (6 mL; 3:2, v/v) and neutralized with NEM
ture was stirred overnight at room temperature. The
dicyclohexylurea was filtered off, and the filtrate was
evaporated. The residue was dissolved in ethyl acetate
(50 mL) and extracted with saturated aqueous NaHCO3
(2· 50 mL), 10% aqueous citric acid (2· 50 mL) and
brine (2· 50 mL). The combined organic layers were
dried over anhydrous Na SO and evaporated. The
(N-ethyl morpholine) (196 lL; 1.53 mmol). The solution
was added dropwise to a solution of lactic acid (9)
(228 lL; 3.05 mmol), 1-hydroxybenzotriazole (HOBt)
(412 mg; 3.05 mmol) and DCC (686 mg; 3.33 mmol) in
2
4
THF (10 mL), at ꢁ15 ꢀC. The mixture was stirred
overnight at room temperature. Dicyclohexylurea was
filtered off, and the filtrate was evaporated. The residue
was dissolved in ethyl acetate (40 mL) and extracted
crude product was purified by flash column chromatog-
raphy on silica gel (solvent E; R = 0.45) to yield N-lac-
toyl-L-tryptophan methyl ester as a colorless transparent
f
1
oil (12) (416 mg; g = 36.4%). H NMR (DMSO-d ) d
6
with saturated aqueous NaHCO (2· 40 mL), 10% aque-
(ppm): 1.15 (d, J = 6.9 Hz, 3H, CH ); 3.19 (d, 2H, 4-
H); 3.61 (s, 3H, COOCH ); 3.97 (m, 1H, CHOH); 4.59
3
3
ous citric acid (2· 40 mL), and saturated brine (2·
3
4
0 mL). After drying over anhydrous Na SO , the or-
(m, 2H, 3-H); 5.60 (br, 1H, CHOH); 6.99 (m, 1H, 7-
H); 7.07 (m, 1H, 6-H); 7.12 (s, 1H, 2-H); 7.34 (d,
J = 8.1 Hz, 1H, 5-H); 7.46 (d, J = 7.9 Hz, 1H, 8-H);
7.74 (t, J = 7.9 Hz, 1H, NHCO); 10.90 (s, 1H, NH).
2
4
ganic phase was evaporated and the residue purified
by flash column chromatography on silica gel (solvent
E; R = 0.45) to yield N-lactoyltryptamine (10)
f
1
3
(
122.7 mg; g = 34%). Mp (ethyl acetate/n-hexane): 94–
C NMR (DMSO-d ) d (ppm): 20.83 (CH ); 26.96 (C-
6 3
1
9
J = 6.7 Hz, 3H, CH ); 2.83 (t, J = 7.5 Hz, 2H, 4-H);
7 ꢀC; H NMR (DMSO-d ) d (ppm): 1.20 (d,
4); 51.90 (COOCH ); 52.20 (C-3); 66.98 (CHOH);
6
3
109.01 (C-4a); 111.40 (C-5); 118.06 (C-8); 118.41 (C-7),
120.98 (C-6); 123.67 (C-2); 127.16 (C-4b); 136.07 (C-
8a); 172.08 (COOCH ); 174.27 (NHCO). Anal. Calcd
3
3
.15 (m, 1H, CHOH); 3.33 (m, 2H, 3-H); 5.49 (d,
J = 4.6 Hz, 1H, CHOH); 6.97 (m, 1H, 7-H); 7.06 (m,
H, 6-H); 7.15 (s, 1H, 2-H); 7.33 (d, J = 8.0 Hz, 1H, 5-
H); 7.56 (d, J = 7.9 Hz, 1H, 8-H); 7.77 (t, J = 5.6 Hz,
3
1
for C H N O4 · 0.2H O: C, 61.30; H, 6.31; N, 9.53.
1
5
18
2
2
Found: C, 61.32; H, 6.21; N, 9.45.
1
3
1
H, NHCO); 10.81 (s, 1H, NH). C NMR (DMSO-
d ) d (ppm): 21.03 (CH ); 25.23 (C-4); 38.90 (C-3);
5.2.6. Synthesis of N-(O-acetyl-lactoyl)-L-tryptophan
methyl ester (13). To N-lactoyl-L-tryptophan methyl es-
ter (12) (200 mg; 0.69 mmol), dissolved in abs pyridine
(5 mL), acetanhydride (2 mL) was added. The reaction
mixture was stirred at room temperature for 2 h, poured
into ice cold water (40 mL), and then stirred at room
temperature, for an additional 2 h. The mixture was
evaporated as an azeotropic mixture with toluene. N-
(O-Acetyl-lactoyl)-L-tryptophan methyl ester (13) was
obtained as a colorless transparent oil (118 mg;
6
3
6
7
4
7.26 (CHOH); 111.31 (C-5); 111.73 (C-4a); 118.17 (C-
); 118.31 (C-8), 120.89 (C-6); 122.54 (C-2); 127.19 (C-
b); 136.24 (C-8a); 174.25 (NHCO). Anal. Calcd for
C H N O : C, 67.22; H, 6.94; N, 12.06. Found: C,
1
3
16
2
2
6
7.14; H, 6.78; N, 11.88.
5.2.4. Synthesis of N-(O-acetyl-lactoyl)tryptamine (11).
To N-lactoyltryptamine (10) (100 mg; 0.43 mmol), dis-
solved in abs pyridine (4 mL), acetanhydride (2 mL)

4560
I. Nemet, L. Varga-Defterdarovi c´ / Bioorg. Med. Chem. 16 (2008) 4551–4562
1
g = 100%; R (E) = 0.82). H NMR (DMSO-d ) d (ppm):
5.2.9. Synthesis of methyl-1-(1-hydroxyethyl)-b-carbo-
f
6
1
3
4
6
.26 (d, J = 6.9 Hz, 3H, CH ); 2.03 (s, 3H, COCH );
0
line-3-carboxylate (16). NaBH (20 mg; 0.52 mmol) was
3
3
4
.14 (m, 2H, 4-H and 4 -H); 3.58 (s, 3H, COOCH );
3
added to a methanol suspension (20 mL) of methyl-1-
acetyl-b-carboline-3-carboxylate (7) (140 mg; 0.52) and
stirred at room temperature for 2 h. The reaction mix-
ture was poured into distilled water (50 mL) and ex-
tracted with ethyl acetate (3· 50 mL). The combined
organic layers were washed with water, dried over anhy-
drous Na SO , and evaporated. The crude product was
.52 (m, 2H, 3-H); 4.98 (q, J = 6.9 Hz, 1H, CHO);
.99 (m, 1H, 7-H); 7.08 (m, 1H, 6-H); 7.16 (s, 1H, 2-
H); 7.33 (d, J = 8.0 Hz, 1H, 5-H); 7.49 (d, J = 7.9 Hz,
1
1
H, 8-H); 8.39 (d, J = 7.5 Hz, 1H, NHCO); 10.90 (s,
C NMR (DMSO-d ) d (ppm): 17.31
1
3
H, NH).
6
(
CH ); 20.64 (COCH ); 26.73 (C-4); 51.83 (COOCH );
3 3 3
2
4
5
1
1
2.84 (C-3); 69.23 (CHO); 109.24 (C-4a); 111.36 (C-5);
17.97 (C-8); 118.38 (C-7), 120.93 (C-6); 123.67 (C-2);
27.09 (C-4b); 136.04 (C-8a); 169.58 (OCO); 170.04
purified by flash column chromatography on silica gel
(solvent F; R = 0.19) to yield methyl-1-(1-hydroxy-
f
ethyl)-b-carboline-3-carboxylate
(16)
(120 mg;
H
1
(
NHCO); 171.96 (COOCH3). Anal. Calcd for
g = 79%). Mp (CHCl /n-hexane): 136–137 ꢀC;
3
C H N O · 0.2H O: C, 60.78; H, 6.12; N, 8.34.
Found: C, 60.74; H, 6.01; N, 8.46.
NMR (DMSO-d ) d (ppm): 1.56 (d, J = 6.7 Hz, 3H,
1
7
20
2
5
2
6
CH ); 3.91 (s, 3H, COOCH ); 5.24 (q, J = 6.7 Hz, 1H,
3
3
CHOH); 5.90 (br, 1H, CHOH); 7.28 (m, 1H, 7-H);
7.57 (m, 1H, 6-H); 7.77 (d, J = 8.2 Hz, 1H, 5-H); 8.36
(d, J = 7.9 Hz, 1H, 8-H); 8.83 (s, 1H, 4-H); 11.69 (s,
5
ylate (14). N-(O-Acetyl-lactoyl)-L-tryptophan methyl es-
ter (13) (100 mg; 0.30 mmol) dissolved in POCl (4 mL)
3
.2.7. Synthesis of methyl-1-ethyl-b-carboline-3-carbox-
1
3
1H, NH).
(CH ); 51.58 (COOCH ); 69.73 (CHOH); 112.92 (C-5);
C NMR (DMSO-d ) d (ppm): 22.61
6
was refluxed for 2 h and then added dropwise to stirred
and cooled (ꢀC) aqueous NaHCO (40 mL). Final neu-
3
3
116.58 (C-4); 119.96 (C-8); 120.10 (C-4a); 121.67 (C-7);
128.28 (C-1); 128.31 (C-6); 133.91 (C-9a); 135.08 (C-3);
140.95 (C-4b); 148.72 (C-8a); 166.05 (COOCH ). Anal.
3
tralization was done by adding solid NaHCO . The
3
reaction mixture was extracted with chloroform (3·
3
5
water, dried over anhydrous Na SO , and evaporated.
0 mL). The combined organic layers were washed with
Calcd for C H N O · 0.8H O: C, 63.28; H, 5.52; N,
1
5
14
2
3
2
9.84. Found: C, 63.74; H, 5.14; N, 10.15.
2
4
The crude product was purified by flash column chro-
matography on silica gel (solvent E; R = 0.65) to yield
5.2.10. Synthesis of 1-(1-hydroxyethyl)-b-carboline-3-car-
boxylic acid (17). To a solution of methyl-1-(1-hydroxy-
ethyl)-b-carboline-3-carboxylate (16) (40 mg; 0.15
mmol) in dioxane (3 mL) aqueous NaOH (33%; 1 mL)
and several drops of water were added. The mixture
was stirred at room temperature, overnight, and then
acidified with aqueous HCl (6 M). The dioxane was
evaporated and the resulting aqueous solution of 1-(1-
hydroxyethyl)-b-carboline-3-carboxylic acid (17) was al-
lowed to stand for several days at +4 ꢀC. The precipitate
was filtered off and dried in a desiccator (14.5 mg;
f
unstable methyl-1-ethyl-b-carboline-3-carboxylate (14)
1
(
J = 7.6 Hz, 3H, CH ); 3.11 (q, J = 7.6 Hz, 2H, CH );
25 mg; g = 33%). H NMR (CDCl ) d (ppm): 1.29 (t,
3
3
2
4
2
.00 (s, 3H, COOCH ); 7.33 (m, 1H, 7-H); 7.50 (m,
3
H, 5-H and 6-H); 8.15 (d, J = 7.9 Hz, 1H, 8-H); 8.78
1
3
(
s, 1H, 4-H); 9.82 (s, 1H, NH). C NMR (CDCl ) d
3
(
ppm): 12.94 (CH ); 27.45 (CH ); 52.65 (COOCH );
3
2
3
1
1
1
12.06 (C-5); 116.51 (C-4); 120.71 (C-7); 121.50 (C-8);
21.94 (C-4a); 128.37 (C-1); 128.60 (C-6); 135.68 (C-3);
35.74 (C-4a); 140.61 (C-9a); 147.29 (C-8a); 167.00
(
COOCH3).
g = 38%; R (G) = 0.39). Mp: 298–300 ꢀC (decompg);
f
1
H NMR (DMSO-d ) d (ppm): 1.63 (d, J = 6.6 Hz,
6
5
NaBH (18 mg; 0.48 mmol) was added to a methanol
solution (25 mL) of 1-acetyl-b-carboline (3) (100 mg;
0.48) and stirred at room temperature for 1.5 h. The
reaction mixture was poured into distilled water
.2.8. Synthesis of 1-(1-hydroxyethyl)-b-carboline (15).
3H, CH ); 5.73 (q, J = 6.6 Hz, 1H, CHOH); 7.43 (m,
1H, 7-H); 7.74 (m, 1H, 6-H); 7.88 (d, J = 8.2 Hz, 1H,
5-H); 8.58 (d, J = 7.9 Hz, 1H, 8-H); 9.20 (s, 1H, 4-H);
3
4
1
3
12.99 (s, 1H, NH). C NMR (DMSO-d ) d (ppm):
6
22.81 (CH ); 65.94 (CHOH); 113.40 (C-5); 117.54 (C-
3
(
50 mL) and extracted with ethyl acetate (3· 50 mL).
4); 120.24 (C-4a); 121.55 (C-8); 123.08 (C-7); 129.95
(C-1); 130.86 (C-6); 131.42 (C-9a); 132.59 (C-3); 143.14
(C-4b); 147.04 (C-8a); 163.19 (COOH). Anal. Calcd
for: C H N O · HCl · 0.8H O: C, 55.73; H, 4.68;
The combined organic layers were washed with water,
dried over Na SO , and evaporated. The crude product
was purified by flash column chromatography on silica
2
4
1
4
12
2
3
2
gel (solvent F; R = 0.72) to yield 1-(1-hydroxyethyl)-b-
N, 9.28. Found: C, 55.94; H, 4.99; N, 9.68.
f
carboline (15) (45 mg; g = 45%). Mp (methanol/ethyl
acetate (18:2, v/v)): 194–195 ꢀC (decompg) (lit.:
5.2.11. Synthesis of N-(1-deoxy-D-fructos-1-yl)-L-trypto-
phan (18). A suspension of L-tryptophan (1) (1 g;
4.90 mmol) and D-glucose (9 g; 49 mmol) in abs metha-
nol (150 mL) was refluxed for 5 h. The mixture was
3
0
1
1
J = 6.5 Hz, 3H, CH ); 5.22 (q, J = 6.5 Hz, 1H, CHOH);
97 ꢀC ); H NMR (DMSO-d ) d (ppm): 1.56 (d,
6
3
5
.73 (s, 1H, CHOH); 7.21 (m, 1H, 7-H); 7.51 (m, 1H, 6-
H); 7.70 (d, J = 8.2 Hz, 1H, 5-H); 7.99 (d, J = 5.2, 1H, 4-
H); 8.19 (d, J = 7.7 Hz, 1H, 8-H); 8.23 (d, J = 5.2 Hz,
evaporated, dissolved in redistilled water (50 mL), and
applied to
(10 · 2 cm). The excess of glucose was removed with
water (200 mL), and the remaining product was then
eluted with aqueous ammonia (1 M; 150 mL). The elu-
ate was evaporated and the obtained crude product
was purified by RP HPLC (solvent D; t = 11.6 min).
R
The fractions containing the pure N-(1-deoxy-D-fruc-
tos-1-yl)-L-tryptophan (19) were pooled and evaporated.
+
a
column of Dowex 50W-X8
H
1
3
1
H, 3-H); 11.26 (s, 1H, NH). C NMR (DMSO-d ) d
6
(
ppm): 22.87 (CH ); 69.28 (CHOH); 112.35 (C-4);
3
1
1
1
13.43 (C-5); 118.93 (C-8); 120.44 (C-4a); 121.31 (C-7);
27.75 (C-6); 128.19 (C-1); 132.26 (C-9a); 136.59 (C-3);
40.49 (C-4b); 148.65 (C-8a). Anal. (C H N O · 0.2
1
3
12
2
CH OH) C, H, N; Calcd for C, 72.51; H, 5.90; N,
3
1
2.81. Found: C, 72.66; H, 5.84; N, 12.31.

I. Nemet, L. Varga-Defterdarovi c´ / Bioorg. Med. Chem. 16 (2008) 4551–4562
4561
The resulting colorless transparent oil was dissolved in
methanol and precipitated with diisopropyl ether
50 mM; pH 5.7 and 7.4), with or without the presence
of L-tryptophan (1) (1 mM). Samples (400 lL) were
incubated in duplicate at 37 and 100 ꢀC in airtight closed
vessels. Aliquots from the samples kept at 37 ꢀC were
collected after 1, 3, 4, 7, 10, 14, 16, 18, and 21 days of
the incubation period; for samples kept at 100 ꢀC, this
was done after 0.5; 1.0; 1.5; 2.0; 3.0; and 5.0 h. All ali-
quots were stored at ꢁ20 ꢀC and thawed immediately
before RP HPLC analysis.
(
1
130 mg; g = 7%). Mp: 135–137 ꢀC (decompg) (lit.:
3
1
13
43 ꢀC (dec) ); C NMR (D O) d (ppm): 25.69 (Trp
2
0
0
CH ; C-11 , b-p); 25.75 (Trp CH ; C-11 , a-f); 25.87
2
2
0
0
(
Trp CH ; C-11 , b-f); 52.89 (Trp CH; C-10 , b-p);
2
5
0.43 (Fru C-1; b-f); 51.80 (Fru C-1; a-f); 52.87 (Fru
C-1; b-p); 60.90 (Fru C-6; a-f); 61.54 (Fru C-6; b-f);
0
0
6
6
2.87 (Trp CH; C-10 , b-f); 62.88 (Trp CH; C-10 , a-f);
3.82 (Fru C-6; b-p); 68.80 (Fru C-5; b-p); 69.29 (Fru
C-4; b-p); 70.28 (Fru C-3; b-p); 73.99 (Fru C-4; b-f);
6.17 (Fru C-4; a-f); 78.08 (Fru C-3; b-f); 80.92 (Fru
C-5; b-f); 82.51 (Fru C-3; a-f); 95.10 (Fru C-2; b-p);
Each incubation mixture was analyzed in duplicate by
RP HPLC using gradient 2, monitoring the absorbance
at 280, 254, 290 nm (for 1 and 18) or at 280, 254, 290,
352, and 380 nm (for 3 and 5). Quantification of each
compound was done by determining peak areas on chro-
matograms followed by comparison with the corre-
sponding calibration curves. The latter were linear in
the concentration range of 0.025–1.0 mM, for all com-
pounds, at all examined wavelengths.
7
0 0
06.97 (Trp C , C-3 ); 111.99 (Trp CH, C-5 ); 118.53
q
1
0 0
Trp CH, C-8 ); 119.57 (Trp CH, C-7 ); 122.24 (Trp
(
CH, C-6 ); 125.13 (Trp, CH, C-2 ); 126.66 (Trp C ,
C-4 ); 135.41 (Trp C , C-9 ); 172.69 (Trp COOH, C-12 ).
q
0
0
q
0
0
0
5
.2.12. Incubation of L-tryptophan (1), its derivate
methyl ester) (6) and its metabolite (tryptamine) (4) with
methylglyoxal (2). To determine the dependence of
-acetyl-b-carboline formation on temperature, pH,
(
5.3. LC–MS/MS analysis of b-carbolines in food samples
1
and oxygen availability during the incubation of L-tryp-
tophan (1) and methylglyoxal (2), 1 and 2 (1.0 mM final
concentrations) were incubated in phosphate buffer
1
3
3
-Acetyl-b-carboline-3-carboxylic acid (5) (tR (gradient
) = 3.0 min) and 1-acetyl-b-carboline (3) (tR (gradient
) = 4.5 min) were analyzed by electronspray positive
(
Na HPO /NaH PO ; 50 mM; pH 5.7 and 7.4). The
2 4 2 4
ionization-mass spectrometric multiple reaction moni-
toring (ESI+ MRM). The ionization source temperature
was 130 ꢀC and the desolvation gas temperature was
mixtures (400 lL) were incubated in duplicate at 37 ꢀC
individual vial for each day) and at 100 ꢀC (individual
(
vial for each hour) in airtight closed vessels (limited oxy-
gen availability) and in HPLC vials under nitrogen (no
oxygen availability) to avoid any influence of the oxygen
during sample preparation for HPLC analysis. Unlim-
ited oxygen availability was achieved when incubations
were performed by intensive stirring of incubation mix-
ture in open flasks with condenser. From the samples
kept at 37 ꢀC aliquots were withdrawn daily for 9 days,
for samples kept at 100 ꢀC, this was done after 0.5; 1.0;
400 ꢀC. The cone gas and desolvation gas-flow rates
were 850 and 150 L/h, respectively. The capillary voltage
was 3.60 kV and the cone voltages for 1-acetyl-b-carbo-
line-3-carboxylic acid (5) (m/z 255.08) and 1-acetyl-b-
carboline (3) (m/z 211.13) were 20.0 and 35.0 V, respec-
tively. The collision energies for the 1-acetyl-b-carbo-
line-3-carboxylic acid (5) fragment ions at m/z 209.1
and m/z 237.0 were 20.00 and 12.00 eV, respectively.
The collision energies for the 1-acetyl-b-carboline (3)
fragment ions at m/z 169.0 and m/z 193.1 were 28.00
and 22.00 eV, respectively. Argon gas was in the colli-
sion cell. The detection limits for fragment ions m/z
1
.5; 2.0; 3.0, and 5.0 h of incubation. All aliquots were
stored at ꢁ20 ꢀC and thawed immediately before RP
HPLC analysis.
2
09.1 and m/z 237.0 of 5 were 0.36 and 0.37 pmol,
Tryptophan methyl ester (6) and tryptamine (4) were
also incubated with methylglyoxal (2) in airtight closed
vessels under conditions previously described.
respectively, while, for fragment ions m/z 169.0 and
m/z 193.1 of 3, were 0.40 and 0.54 pmol, respectively.
Programmed molecular ion and fragment ion masses
were optimized to ±0.1 Da for multiple reaction moni-
toring detection of the analyte.
Each incubation mixture was analyzed in duplicate by
RP HPLC using gradient 1, monitoring the absorbance
at 280, 254, 290 nm (for 1, 6, and 4) or at 280, 254,
2
90, 352, and 380 nm (for 3, 5, and 7). Quantification
Acknowledgments
was done by determining peak areas on chromatograms,
followed by comparison with the corresponding calibra-
tion curves. The latter were linear in the concentration
range of 0.025–1.0 mM, for all compounds, at all exam-
ined wavelengths. Analysis of 1-acetyl-3,4-dihydro-b-
carboline (8) was done only qualitatively due to its par-
ticular instability.
This work was supported by the Croatian Ministry of
Science, Education and Sports Grant No. 098-
0982933-2936. We thank Dr. Vincent M. Monnier for
access to his HPLC-MS/MS instrument. Visiting re-
search of I.N. in Dr. Monnier’s laboratory was
supported by
Organization.
a Fellowship from the Fulbright
5
.2.13. Incubations of L-tryptophan (1) with D-glucose and
N-(1-deoxy-D-fructos-1-yl)-L-tryptophan (18) with and
without L-tryptophan (1). L-Tryptophan (1) (1.0 mM)
and D-glucose (10.0 or 20.0 mM) as well as N-(1-
deoxy-D-fructos-1-yl)-L-tryptophan (18) (1 mM) were
incubated in phosphate buffer (Na HPO /NaH PO ;
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