Synthesis and characterization of a dehydrogenation product arising from the oxidation of aminoethylcysteine ketimine decarboxylated dimer

Article abstract of DOI:10.1021/np060641t

While investigating the antioxidant properties of aminoethylcysteine ketimine decarboxylated dimer (1) (a natural substance occurring in biological fluids such as human urine and plasma and in bovine cerebellum), a previously unreported oxidation product was obtained. This compound was identified and characterized through comparison with an authentic sample prepared via Pd-catalyzed dehydrogenation of 1. This molecule is an example of an alternative oxidation pathway involving 1.

Full text of DOI:10.1021/np060641t

1046
J. Nat. Prod. 2007, 70, 1046-1048
Synthesis and Characterization of a Dehydrogenation Product Arising from the Oxidation of
Aminoethylcysteine Ketimine Decarboxylated Dimer
Alberto Macone,†, Aldo Caiazzo,‡, Antonio Antonucci,† Igor Fochi,§ Mirella Nardini,| Silvestro Dupre`,† and
Rosa Marina Matarese*,†
Department of Biochemical Sciences “A. Rossi-Fanelli” and National Research Council Institute of Molecular Biology and Pathology,
UniVersity of Rome “La Sapienza”, Piazzale Aldo Moro 5, 00185 Rome, Italy, Van’t Hoff Institute for Molecular Sciences, Faculty of Science,
UniVersity of Amsterdam, Nieuwe Achtergracht 129, 1018 WS Amsterdam, The Netherlands, Toxicological Chemistry Unit of the Department of
EnVironment and Primary PreVention, Italian National Institute for Health, Viale Regina Elena 299, 00161 Rome, Italy, and National Institute
for Food and Nutrition Research, Via Ardeatina 546, 00178 Rome, Italy
ReceiVed December 30, 2006
While investigating the antioxidant properties of aminoethylcysteine ketimine decarboxylated dimer (1) (a natural substance
occurring in biological fluids such as human urine and plasma and in bovine cerebellum), a previously unreported
oxidation product was obtained. This compound was identified and characterized through comparison with an authentic
sample prepared via Pd-catalyzed dehydrogenation of 1. This molecule is an example of an alternative oxidation pathway
involving 1.
Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD,
Figure 1) is a natural sulfur-containing compound present in human
plasma and urine1,2 and detected in mammalian cerebellum.3
Recently the presence of AECK-DD in some edible vegetables
has been detected.4 As of present, no biosynthetic pathway for the
formation of AECK-DD has been established, which suggests a
possible external intake route via the dietary supply for the presence
of this molecule in mammals. Concerning its biological role, AECK-
DD has strong antioxidant activity and has been reported to interact
in Vitro with both reactive oxygen and nitrogen species (e.g.,
Figure 1.
hydrogen peroxide, superoxide anion, hydroxyl radical, peroxyni-
trite, and its derivatives).5,6 In addition, this molecule has been
shown to protect human low-density lipoprotein and a human
monocytic cell line against the oxidative stress induced by CuCl2
and tert-butyl hydroperoxide.7,8 Currently, the only products arising
from the oxidation mixtures of AECK-DD that have been unam-
biguously characterized are a sulfoxide species9 and its dimeric
species.10
To further characterize the antioxidant properties of AECK-DD,
a set of experiments were carried out with AECK-DD in the
presence of CuCl2/tert-butyl hydroperoxide and 2,2′-azo-bis-2-
amidinopropane hydrochloride. The GC/MS analysis of the corre-
sponding reaction mixtures showed the presence of a new species,
2a, which had a mass of 226.
The decrease by two units with respect to the mass of the starting
material (mass 228) suggested that the new species formed as a
consequence of oxidative dehydrogenation. Indeed, the high-
resolution mass spectra performed on this sample showed a fine
mass value of 226.023788, which indicated a rough formula
(C9H10S2N2O) compatible with the formation of a new double bond
in the tricyclic structure. To confirm this hypothesis, AECK-DD
was submitted to metal-mediated dehydrogenation in the presence
of palladium on activated carbon (10% loading), which is a well-
known method for the introduction of double bonds in aromatic
and heterocyclic systems (Scheme 1).11-14
Scheme 1
226 (GC/MS analysis). This new species was isolated from the
reaction mixture by column chromatography (SiO2, hexane/ethyl
acetate ) 7:3 v/v), and it was obtained as a bright yellow solid
(40% isolated yield). The retention time of 2b on GC/MS was
identical to that of 2a observed in the oxidation of 1. This
observation was confirmed by analyzing by GC/MS an oxidation
mixture spiked with 2b, which resulted in the appearance of just
one peak with the corresponding retention time of 2a. In addition,
the GC/MS and the HRMS analyses of two distinct samples of 2a
and 2b revealed an identical fragmentation pattern. On the basis
of these results, we concluded that 2a and 2b were the same species.
In order to elucidate the structure of this new compound and to
determine the position of the newly formed double bond in the
molecule, a series of spectroscopic analyses were carried out. The
UV analysis of 2b dissolved in acetone and diluted with water
(water/acetone, 280:1 v/v, 44 µM final concentration) showed a
maximum absorbance at 338 nm, which corresponded to a shift of
30 nm with respect to that observed for 1 (308 nm). This difference
correlates well with the Woodward-Fieser rule for the expected
spectral shift observed following an extension of the conjugation
The reaction was carried out in refluxing mesitylene for 3 days,
leading to the appearance of a new product, 2b, whose mass was
in a UV-active molecule.15,16 The H NMR analysis of 2b carried
1
* Corresponding author. E-mail: rosamarina.matarese@uniroma1.it.
† University of Rome La Sapienza.
out in CDCl3 showed an evident simplification of the spectra with
respect to that reported for 1.10 The signal at 3.97 ppm caused by
the tertiary hydrogen on carbon 10a in 1 disappeared, while a new
signal at 5.58 ppm arose, suggesting the presence of an olefinic
proton. The spectrum also showed the presence of four structured
‡ University of Amsterdam.
§ Italian National Institute for Health.
| National Institiute for Food and Nutrition Research.
These authors contributed equally to this work.
10.1021/np060641t CCC: $37.00
© 2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/01/2007

Notes
Journal of Natural Products, 2007, Vol. 70, No. 6 1047
argon atmosphere. Purifications via column chromatography were
performed on silica gel (Biosolve, 60 Å, 0.063-0.200 mm).
signals in the alkyl region, each of them accounting for two protons.
This indicates that there are four methylene groups in the molecule,
one less than in 1. Out of all the possible isomers resulting from
the introduction of a double bond into 1, only the one reported in
Scheme 1 is compatible with this spectroscopic data.
Instruments. GC/MS analyses were performed on an Agilent 6850A
gas chromatograph coupled to a 5973N quadrupole mass selective
detector (Agilent Technologies, Palo Alto, CA). Gas chromatographic
separations were carried out on an Agilent HP-5MS fused-silica
capillary column (30 m × 0.25 mm i.d., film thickness 0.25 µm).
Injection mode: splitless at a temperature of 260 °C. Column
temperature program: 70 °C (1 min) then to 280 °C at a rate of 10
°C/min and held for 15 min. The carrier gas was helium at a constant
flow of 1.0 mL/min. The spectra were obtained in electron impact mode
at 70 eV ionization energy and a mass range scan from m/z 30 to 500;
ion source temperature 280 °C, ion source vacuum 10-5 Torr.
The HMRS analysis was perforned on a VG AutoSpec spectrometer.
The sample (10 ng/µL in methanol) was analyzed through direct
injection via a septum set at 260 °C. The spectra were obtained in
electron impact mode at 70 eV ionization energy and a mass range
scan from m/z 80 to 335, 0.3 s + 0.3 s delay time, centroid at 5000
RP; gas reference PFK; ion source at 250 °C, 500 µA.
This conclusion was supported by more detailed NMR analyses.
The 13C NMR spectrum of 2b (CDCl3) showed the disappearance
of the peak at 59.8 ppm (due to a tertiary carbon in R-position to
the tertiary nitrogen in 1 and of one of the alkyl carbons in the
20-45 ppm region). On the other hand, two new carbon signals
appear, one at 98.2 ppm and another in the 130s ppm region, highly
indicative of the presence of two new olefinic carbons. In the
heteronuclear multiple-quantum coherence spectrum (HMQC), each
of the four carbons in the alkyl region (20-40 ppm) is correlated
to one of the four triplets in the alkyl region of the proton spectrum
(showing their secondary structure), while the peak at 98.2 ppm is
correlated to the olefinic proton at 5.58. The remaining carbons
(>100 ppm) are not detected, accounting for their quaternary
structure. These findings are confirmed by the attached proton test
spectrum (APT); whereas the quaternary carbons at >100 ppm and
the secondary carbons in the alkyl region are positive, the signal at
98.2 ppm is the only negative one, making it a primary carbon.
NMR spectroscopy was performed using a Varian Unity Inova
spectrometer, operating at 500 MHz for 1H. All experiments were
1
carried out at 296 K in CDCl3. H and 13C shifts were referenced to
internal CDCl3 (7.26 and 77.25 ppm, respectively).
UV-vis spectra were performed on a Varian Cary 50 spectropho-
tometer.
1
The two-dimensional H-1H COSY spectrum of 2b shows two
main spin systems; the signal at 3.91 ppm is correlated to the one
at 3.04 ppm, while the signal at 3.64 ppm is correlated to the one
at 2.99 ppm. This accounts for the presence of two couples of CH2-
CH2 systems, which is compatible with the structure of 2b reported
in Scheme 1.
These findings also provide an explanation for why the 1H NMR
spectrum of 2b is much simpler than that reported for 1.10 Indeed,
the introduction of a new double bond between carbons 10 and
10a destroys the chiral center on position 10a, lowers the number
of hydrogens in the molecule, and makes the two six-membered
rings more similar in terms of structure.
Melting points were determined using a Buchi B545 melting point
apparatus and they should be considered uncorrected.
Oxidation Experiments. To a solution 200 µM AECK-DD (1) in
phosphate buffer saline (PBS) at pH 7.2 were added CuCl2 (1 mM
final) and t-BuOOH (1 mM final) or ABAP (1 mM final). The mixtures
were kept at 37 °C for 60 min. Aliquots of 0.5 mL volume were taken
after 5, 15, 30, and 60 min of incubation time; each of them was added
with 10 µL of 10 µM DTPA and 20 µL of 2% BHT. The aliquots
were extracted with chloroform (3 × 2 mL), and the organic fractions
were combined and concentrated under reduced pressure. The residues
were redissolved in a mixture of acetone-hexane (1:4 v/v) and the
resulting solutions analyzed directly by GC/MS.
The palladium-catalyzed dehydrogenation reaction on 1 occurs
selectively on the bond between carbons 10 and 10a. From what is
known about the mechanism of dehydrogenation involving cyclo-
hexane,17,18 it is possible that the process is triggered by an
interaction between the hydrogen atoms involved and the Pd center,
similar to a hydrogen bond interaction. The hydrogen atom on
carbon 10a is the only tertiary one in the molecule, and the
corresponding C-H bond is likely more susceptible to undergo a
metal-mediated cleavage. These findings suggest that the oxidative
dehydrogenation of AECK-DD (1) in the presence of peroxides
might follow a similar path and that the reactivity of the molecular
region involved in the metal-catalyzed dehydrogenation process
could be very important in understanding its well-known antioxidant
properties.
In conclusion, a new product following the oxidation of
aminoethylcysteine ketimine decarboxylated dimer (1) has been
observed, synthesized, and characterized through HRMS, UV, and
NMR techniques. This product, which shows an additional unsat-
uration with respect to 1, may arise from an oxidative dehydroge-
nation path that involves the carbons 10 and 10a of the tricyclic
structure. Further studies are under investigation to clarify the
mechanism of formation of 2b under oxidative conditions also in
ViVo.
Palladium-Catalyzed Dehydrogenation of Aminoethylcysteine
Ketimine Decarboxylated Dimer (1). A 100 mg (0.438 mmol) sample
of aminoethylcysteine ketimine decarboxylated dimer (1) and 100 mg
of 10% palladium on activated carbon were inserted in a Schlenk flask
under argon atmosphere. The system was then briefly evacuated and
backfilled with argon (3 cycles). Mesitylene (3 mL) was added, and
the resulting mixture was stirred at the solvent reflux temperature for
3 days. The mixture was cooled down and filtered through a pad of
Celite. The filtrate was concentrated in Vacuo and the oily residue
purified on silica gel (hexane/ethyl acetate ) 7:3 v/v) to provide 39.5
mg of 2b as a bright yellow solid (40% isolated yield). According to
IUPAC nomenclature, the name of 2b is 3,4,7,8-tetrahydro-2H,5H-
pyrrolo[2,1-c:3,4-b′]bis[1,4]thiazin-5-one. The numbering scheme is
reported in Figure 1, following the guidelines of the Ring Systems
Handbook, Chemical Abstract Service (1993), RF 29858.
1H NMR (500 MHz, CDCl3): δ 5.58 (H10, s, 1H), 3.91 (H7, m, 2H),
3.70 (NH, bs, 1H), 3.64 (H3, m, 2H), 3.04 (H8, m, 2H), 2.99 (H2, m,
2H). 13C NMR (125.7 MHz, CDCl3): δ 162.8 (C5), 132.1, 130.2, 103.1
(C10b), 98.2 (C10), 42.3 (C3), 39.2 (C7), 26.4 (C8), 25.5 (C2). GC/MS
(EI, m/z): 226 (100%), 211 (28%), 198 (11%), 180 (18%). HRMS:
calcd for C9H10N2OS2 226.023457; found 226.023788 (-1.5 ppm).
Mp: 146 °C.
Acknowledgment. We would like to thank P. W. Wills, Ph.D for
his help in revising the manuscript.
Supporting Information Available: GC/MS analysis of 1 and 2a;
NMR, MS, and UV spectra of 2b. This material is available free of
charge via the Internet at http://pubs.acs.org.
Experimental Section
General Experimental Procedures. Aminoethylcysteine ketimine
decarboxylated dimer (1) was synthesized as previously reported.19 tert-
Butyl hydroperoxide was purchased from Sigma Chemicals Co., CuCl2
was purchased from Fluka (Buchs, Switzerland), and 2-2′-azo-bis(2-
amidinopropane hydrochloride) (ABAP) was purchased from Poly-
science (Warrington, PA). Palladium on activated carbon (10% loading),
diethylenetriaminepentaacetic acid (DTPA) and 2,6-di-tert-butyl-4-
methylphenol (BHT) were purchased from Aldrich. Mesitylene was
distilled over sodium and stored over 4 Å molecular sieves under an
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1048 Journal of Natural Products, 2007, Vol. 70, No. 6
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NP060641T