Reaction of acetaldehyde with 5-aminolevulinic acid via dihydropyrazine derivative

Article abstract of DOI:10.1248/cpb.c14-00694

When a solution of 5-aminolevulinic acid (ALA) was incubated with acetaldehyde at neutral pH, a product was generated. This product was identified as 3-ethylpyrazine-2,5-dipropanoic acid (ETPY). ETPY was stable at neutral pH. It has been reported that ALA dimerizes at neutral pH generating 3,6-dihydro-pyrazine-2,5-dipropanoic acid (DHPY), and subsequently resulting in pyrazine-2,5-dipropanoic acid (PY) by autoxidation. In the present reaction, DHPY generated from ALA reacted with acetaldehyde, resulting in ETPY. Preadministration of ALA 3 min prior to acetaldehyde injection supressed the toxicity of acetaldehyde in male mice. These results suggest that ALA may be useful as a scavenger for acetaldehyde.

Full text of DOI:10.1248/cpb.c14-00694

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Chem. Pharm. Bull. 63, 126–129 (2015)
Vol. 63, No. 2
Note
Reaction of Acetaldehyde with 5-Aminolevulinic Acid via
Dihydropyrazine Derivative
Toshinori Suzuki,* Naoki Yasuhara, Takashi Ueda, Michiyo Inukai, and Mitsunobu Mio
School of Pharmacy, Shujitsu University; Okayama 703–8516, Japan.
Received September 28, 2014; accepted November 25, 2014
When a solution of 5-aminolevulinic acid (ALA) was incubated with acetaldehyde at neutral pH, a
product was generated. This product was identified as 3-ethylpyrazine-2,5-dipropanoic acid (ETPY). ETPY
was stable at neutral pH. It has been reported that ALA dimerizes at neutral pH generating 3,6-dihydro-
pyrazine-2,5-dipropanoic acid (DHPY), and subsequently resulting in pyrazine-2,5-dipropanoic acid (PY)
by autoxidation. In the present reaction, DHPY generated from ALA reacted with acetaldehyde, resulting in
ETPY. Preadministration of ALA 3min prior to acetaldehyde injection supressed the toxicity of acetaldehyde
in male mice. These results suggest that ALA may be useful as a scavenger for acetaldehyde.
Key words acetaldehyde; 5-aminolevulinic acid; 3,6-dihydropyrazine-2,5-dipropanoic acid; 3-ethylpyra-
zine-2,5-dipropanoic acid
5
-Aminolevulinic acid (ALA) is an essential molecule for showed a UV spectrum with λmax=280nm (Fig. 1A, inset). An
both animals and plants as a precursor for biosynthesis of ESI-TOF/MS spectrum showed m/z=207 and 251 in the nega-
1,2)
porphyrins including hemes and chlorophylls. Correspond- tive mode (Fig. 1B). High-resolution ESI-TOF/MS (negative)
ing enzymes synthesize a porphyrin from eight molecules of the molecular ion showed m/z=251.103731, which agreed
of ALA. ALA is stable at acidic pH as low as 2.5. However, with the theoretical molecular mass for C H N O composi-
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16
2
4
1
ALA is reactive and can dimerize without enzymes at neutral tion (251.104172) within 2ppm. H-NMR showed an aromatic
13
to mildly basic pH. At physiological pH, high concentrations proton, five methylene groups, and a methyl group. C-NMR
of ALA dimerize in aqueous solution to produce a dihy- showed two carboxyl carbons, four aromatic carbons, and six
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1
dropyrazine derivative, 3,6-dihydropyrazine-2,5-dipropanoic aliphatic carbons. Combining these data with H– H correla-
3
–6)
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13
acid (DHPY).
DHPY is also reactive and can react with tion spectroscopy (COSY) and H– C heteronuclear multiple
dissolved oxygen resulting in a stable pyrazine derivative, quantum coherence (HMQC) data, the product was identi-
pyrazine-2,5-dipropanoic acid (PY). Acetaldehyde (CH CHO) fied as 3-ethylpyrazine-2,5-dipropanoic acid (ETPY), a novel
3
is the first metabolite of ethanol. Long-term exposure to ethyl-substituted derivative of PY at the 3 position (Fig. 1C).
CH CHO may cause various dysfunctions including cardio-
Figure 2A shows the time-dependent changes in HPLC
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–10)
vascular diseases and cancer.
For acute toxicity, CH CHO peak area of ALA, DHPY, PY, and ETPY, while Fig. 2B
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11)
is ten times more toxic than ethanol based on its LD50 value.
It has been reported that LD50 of acetaldehyde in mice is when 10mM ALA and 5mM CH CHO were incubated in
shows the time-dependent changes in concentration of ETPY,
3
12)
500mg/kg (11.4mmol/kg) via intraperitoneal injection.
100 mM potassium phosphate buffer at pH 7.4 and 37°C for
up to 4h. The concentrations of PY and ETPY increased with
increasing incubation time, while the concentration of DHPY
Results and Discussion
A solution of 10mM ALA and 5mM CH CHO were incu- quickly increased in the initial period and then decreased
3
bated in 100mM potassium phosphate buffer at pH 7.4 and slowly with a profile as an intermediate. At 4h, the concentra-
37°C for 2h. When the reaction mixture was analyzed by re- tion of ETPY generated was 1.7mM.
versed phase (RP) HPLC, three product peaks appeared in the Figure 2C shows the ALA dose-dependence of concentra-
chromatogram in addition to a peak including ALA with a re- tions of ETPY, when 0–20mM ALA and 5mM CH CHO were
3
tention time of 2.7min and a UV spectrum with λmax=265nm incubated in 100mM potassium phosphate buffer at pH 7.4
(
Fig. 1A). The products were collected and subjected to spec- and 37°C for 2h. The concentrations of ETPY increased with
trometric measurements. The product eluted at the retention increasing ALA dose. Figure 2D shows the CH CHO dose-
3
time of 3.0min showed a UV spectrum with λmax=240 and dependence of concentrations of ETPY, when 10mM ALA and
3
37nm and an electrospray ionization time-of-flight (ESI- 0–10 mM CH CHO were incubated in 100mM potassium phos-
3
TOF)/MS spectrum with m/z=181 and 225 in the negative phate buffer at pH 7.4 and 37°C for 2h. The concentrations of
mode. The product was identified as 3,6-dihydropyrazine-2,5- ETPY increased with increasing CH CHO dose up to 5mM,
3
dipropanoic acid (DHPY) by referring to spectrometric data after which the yield of ETPY was constant.
3,4)
reported previously. The product eluted at the retention time
Figure 2E shows the pH dependence of the concentrations
of 3.7min showed a UV spectrum with λmax=277nm. An ESI- of ETPY when 10mM ALA and 5mM CH CHO were incubat-
3
TOF/MS spectrum showed m/z=179 and 223 in the negative ed in 100mM potassium phosphate buffer at pH 4–8 and 37°C
mode. The product was identified as pyrazine-2,5-dipropanoic for 2h. At pH 4.2, ETPY was not detected. The concentration
acid (PY) by referring to spectrometric data reported previ- of ETPY increased with increasing pH from 5 to 8.
3,4)
ously.
The product eluted at the retention time of 7.9min
To understand the stability of ETPY, purified ETPY was
*
To whom correspondence should be addressed. e-mail: tsuzuki@shujitsu.ac.jp
©
2015 The Pharmaceutical Society of Japan

Vol. 63, No. 2 (2015)
Chem. Pharm. Bull.
127
Fig. 1. Separation and Identification of ETPY
(
A) RP-HPLC chromatogram of the reaction mixture of ALA with CH CHO. A solution of 10mM ALA and 5mM CH CHO was incubated in 100mM potassium phos-
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3
phate buffer at pH 7.4 and 37°C for 2h. (B) Negative ion electrospray ionization time-of-flight mass spectrometry spectrum of 3-ethylpyrazine-2,5-dipropanoic acid
ETPY). (C) Structure of ETPY.
(
incubated in 100mM potassium phosphate buffer at pH 7.4 and compound ETPY. Figure 4 shows the survival curves of mice
3
7°C for up to 30d. No decomposition of ETPY was observed when 24mmol/kg ALA, 12mmol/kg CH CHO, and 24mmol/
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by RP-HPLC analysis.
kg ALA 3min prior 12mmol/kg CH CHO were injected in-
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To obtain information about the reaction pathway generat- traperitoneally to each group (n=10). ALA alone caused no
ing ETPY, DHPY or PY purified by RP-HPLC was incubated death. Acetaldehyde caused death of 5 mice up to 3d. Admin-
with CH CHO in potassium phosphate buffer of pH 7.4 at istration of ALA prior to CH CHO caused death of only one
3
3
3
7°C for 2h. The incubation of DHPY with CH CHO resulted mouse. The mortality by acetaldehyde of the ALA pre-treat-
3
in ETPY, meanwhile the incubation of PY with CH CHO re- ment group was significantly lower than without ALA pre-
3
sulted in no change in the PY concentration (data not shown). treatment (p<0.05 in Gehan–Breslow analysis with Holm–
This indicated that ETPY was generated via the reaction of Sidak method). However, a similar experiment, in which ALA
CH CHO with DHPY but not PY. It has been reported that was administered 15min prior to acetaldehyde, showed no sig-
3
another dihydropyrazine derivative, 2,5-dimethyl-3,6-dihydro- nificant differences in mortality between CH CHO and ALA/
3
13)
pyrazine, reacts with CH CHO resulting in an ethyl adduct.
CH CHO (data not shown). The results suggest that CH CHO
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3
3
In the present reaction, DHPY, a dihydropyrazine, which was reacts with DHPY generated from ALA in the peritoneal cav-
generated from ALA by spontaneous dimerization, also re- ity. During the period of 15min, the intraperitoneal concen-
acted with CH CHO, resulting in a stable product, ETPY, an trations of ALA and DHPY may be decreased by absorption
3
ethyl adduct. The possible reaction pathways are summarized through the peritoneum and by reactions with biomolecules in
in Fig. 3.
To obtain information about the effect of ALA on the acute
the peritoneal cavity.
In conclusion, the present results show that ALA dimerize
acetaldehyde toxicity, we conducted animal experiments to form a dihydropyrazine derivative which can react with
using mice. A dose of 12mmol/kg (530mg/kg) CH CHO was CH CHO, resulting in a stable adduct ETPY under neutral
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3
used, since the 2 weeks lethal response of this dose was 5 conditions. When 5mM CH CHO was incubated with 10mM
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mice dead out of 10 tested. A dose of 24mmol/kg (3140mg/ ALA at pH 7.4 and 37°C for 4h, the yield of ETPY was
kg) ALA was used, since CH CHO can react with DHPY 1.7 mM. Preadministration of ALA pior to acetaldehyde in-
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generated from two molecules of ALA, resulting in the stable jection supressed the toxicity of acetaldehyde in mice. This

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Chem. Pharm. Bull.
Vol. 63, No. 2 (2015)
Fig. 2. Effects of the Reaction Conditions
(A) The time course of the changes of HPLC peak areas detected at 260nm in ALA (open circle), DHPY (closed triangle), PY (closed square), and ETPY (closed circle).
A solution of 10mM ALA and 5mM CH CHO were incubated in 100mM potassium phosphate buffer at pH 7.4 and 37°C for 2h. (B) The time course of the concentration
3
changes of ETPY in Fig. 2A. (C) ALA dose dependences of the concentration of ETPY in the reaction of ALA with CH CHO. A solution of 0–20mM ALA and 5mM
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CH CHO was incubated in 100mM potassium phosphate buffer at pH 7.4 and 37°C for 2h. (D) CH CHO dose dependences of the concentration of ETPY in the reaction of
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3
ALA with CH CHO. A solution of 10mM ALA and 0–10mM CH CHO was incubated in 100mM potassium phosphate buffer at pH 7.4 and 37°C for 2h. (E) pH dependence
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3
of concentration of ETPY in the reaction of ALA with CH CHO. A solution of 10mM ALA and 5mM CH CHO was incubated in 100mM potassium phosphate buffer at pH
3
3
4
–8 and 37°C for 2h. The concentration was determined by RP-HPLC analysis detected at 260nm. Means± S.D. (n=3) are shown.
suggests that ALA may act as a scavenger of aldehydes in and particle size 5µm (GL Sciences, Tokyo, Japan) was used.
humans.
The eluent was 20mM ammonium acetate (pH 7.0) containing
% methanol. The column temperature was 40°C and the flow
rate was 1mL/min. The RP-HPLC chromatogram was detect-
5
Experimental
Materials 5-Aminolevulinic acid hydrochloride was ed at 260nm.The ESI-TOF/MS measurements were performed
purchased from Nacalai Tesque (Kyoto, Japan). Acetaldehyde on a MicroTOF spectrometer (Bruker, Bremen, Germany) in
(
(
>99.5%) was obtained from Fluka Analytical/Sigma-Aldrich negative mode. The sample isolated by RP-HPLC was directly
MO, U.S.A.). Water was purified with a Millipore Milli-Q infused into the MS system by a syringe pump without a col-
deionizer.
umn.
Spectrometric Data 3-Ethylpyrazine-2,5-dipropanoic acid
HPLC and MS Conditions The HPLC system consisted
of Shimadzu LC-10ADvp pumps and an SCL-10Avp system (ETPY). H-NMR (500MHz, DMSO-d ): δ (ppm/TMS) 8.23
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controller. Online UV spectra were obtained with a Shimadzu (s, 1H, H-6), 2.95 (dd, 2H, H-β), 2.89 (dd, 2H, H-β′), 2.78
SPD-M10Avp UV-Vis photodiode-array detector. For the RP- (ddd, 2H, –CH CH ), 2.61 (dd, 2H, H-α), 2.58 (dd, 2H, H-α′),
2
3
13
HPLC, an Inertsil ODS-3 octadecylsilane column 4.6×250mm 1.20 (dd, 3H, –CH CH ). C-NMR (125MHz, DMSO-d ): δ
2
3
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Vol. 63, No. 2 (2015)
Chem. Pharm. Bull.
129
Fig. 3. Proposed Reaction Pathway for the Reaction of ALA with CH CHO
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and the HPLC peak area detected at 260nm relative to those
of dCyd (ε260nm=7400 M− cm ) in the mixed solution. The es-
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timated ε260nm values were 2600M− cm for ETPY.
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Animal Studies All animal studies were previously ap-
proved by the Animal Care Committee of Shujitsu Univer-
sity and were conducted in accordance with the Principles of
Laboratory Animal Care (NIH Publication #85–23). ICR male
mice (six weeks old) were used in animal studies.
Conflict of Interest The authors declare no conflict of
interest.
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4
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3
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1
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1