1. Synthesis and some properties of phosphatidylhydroxyacetone
as à new phospholipid formed by free-radical fragmentation of
cardiolipin
Vasilkevich Alexey, Kisel Mikhail
Minsk, Belarus
vasilkevich@iboch.bas-net.by
alex.vasilkevich@gmail.com
O
OO
C
RP
O
O
O
C
R
O
O
O
A number of biochemical processes in body as well as external physical and chemical condition is accompanied by the formation of reactive
oxygen and nitrogen intermediates, which may afterwards cause cellular damage. Determination of the mentioned processes pathways and the
role of the newly formed substances is an important biochemical subject.
P
O
O
O O
R
OH
O
P
O O
O
R'
OH H2O
P
O
O
O O
R
OH
O
P
O O
O
R'
O
O
O
H
P
OH
O
O O
R
H
O
O
P
O O
O
R'
LH L
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O
P
O O
O
R'
Phosphatidylhydroxyacetone (PHA) is the phospholipid
along with phosphatidic acid (PA) found in mouse liver
mitochondria subjected to oxidative stress (Yurkova,
2008).
PA and PHA are detected as cardiolipin fragmentation
products by thin-layer chromatography combined with
MALDI-TOF mass spectrometry in oxidized samples, but
not in unperturbed ones. The formation of PAand PHAin
copper-treated model membrane correlats well with the
decrease of cardiolipin (Yurkova, 2011).
We have been first to synthesize PHA from phosphatidylcholine at preparative amount by the means of microbial phospholipase D from
Streptomyces netropsis. The yield comprised about 80 %. Reaction time: 30 min, temperature 37 °Ñ, 1 ml reaction mixture chloroform:water
2:1 contained PC (35 mg), hydroxyacetone (20 µl), phospolipase D and NaAc buffer (pH 5,5).
O
OO
C
C13H27
P
O
O
O
C
C13H27
O
O
OO
C
C13H27
P
O
O
OPhospholipase D
C
C13H27
O
O
OH HO N+
O
O
ON+
Dimyristoylphosphatidylcholine Dimyristoylphosphatidylhydroxyacetone
-1
1622 cm
PHA
PC
30
40
50
60
70
80
90
100
110
120
130
140
0 20 40 60 80 100
d, nm
PHA percentage, %
Liposome diameter
Calculation by liposome number Calculation by liposome size
-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.5
31P NMR
4.71
4.93
PC
PHA
Reference:
Triphenylphosphine
Equipment:
NMR: Avance 500 (Bruker)
IR: FTIR-spectrophotometer Bomem Michelson 100
Liposome size determination: 90Plus Particle Size Analyzer,
Brookhaven Instruments Corporation
References:
Yurkova et al. Archives of Biochemistry and Biophysics 480 (2008)
17–26,
Yurkova et al. Chemistry and Physics of Lipids 164 (2011) 393– 400
Phosphatidylhydroxyacetone
(PHA)
Why to study?
?-The new synthetic phospholipid
having carbonyl group may exhibit
new biologically active properties
?-Allows to study the role of
cardiolipin fragmentation
processes in body more deeply.
http://iboch.bas-net.by/
Laboratory of Lipid
Chemistry
Head of laboratory - Prof. Mikhail Kisel, D.Sc.
tel. +375 (17) 263-71-31
e-mail:
Research Areas
?Chemical and enzymatic synthesis of physiologically
active lipids and related compounds;
?Study of enzymatic and free-radical reactions
mechanisms leading to the signal lipids formation and
the addressed regulation of such reactions by lipid
effectors;
?Construction of liposomal drug-delivery systems;
?Synthesis and investigation of lipoid compounds for
plant protection.
kisel@iboch.bas-net.by Applied Research
?Technological process for 5-aminolevulinic acid
hydrochloride which is the chemical substance for
"Alamin" – a preparation for diagnostics and therapy of
bladder cancer.
?Technological process for 5-aminolevulinic acid hexyl
ester which is the effective regulator of plant growth.
?Preparative form of systemic fungicide "Baifucide"
(active substance difenoconazole) for plant protection
against fungal disease.
?Fluorescent and chromogenic lipid substrates for
assaying lipolytic enzymes.
-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0
1H NMR
1H NMR
DM PC
DM PHA
(CH
3
)
3
choline
CH
2
hydroxyacetone
CH
3
hydroxyacetone
Modified method:
Samples in D O2
Detergent CHAPS
Reference Triphenylphosphine
0 min 15 min
PHA
PC
PC
Thin-layer chromatography
Eluent: chloroform-methanol-water (65-25-4).
Revealing reagent: molybdate reagent
(Vaskovsky, 1975)