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(8a–e) were desilylated with tetra-n-butylammoniumfluoride in
tetrahydrofuran, yielding desired glyceride building blocks 4a–
e.16–18 All aforementioned products were isolated and purified by
silica gel flash column chromatography and characterized by spec-
troscopic methods as described earlier.9
The introduction of one or two nonbridging sulfur atoms into
lysophosphatitydylcholine molecule was performed using oxathia-
phospholane or dithiaphospholane approach, respectively. These
methods were successfully employed for the synthesis of phosp-
horothioate/phosphorodithioate nucleotide and oligonucleotide
analogues of phosphodiester type (see Scheme 2 and the Supple-
mentary data).10
CH3
CH3
CH3
O
C
+
O
O
O
O
N
HO
O
H
R
P
Figure 1. The structure of natural R-lysophosphatidylcholine (LPC, 1). Substituents
R–C(O)– refer to various fatty acid residues present in lysophospholipids.
increase of glucose stimulated insulin secretion in NIT-1 insuli-
noma cells.4 Nevertheless, the nature of the endogenous ligands
of GPR119 and its physiological role in direct regulation of insulin
secretion by the pancreatic b cells still have to be explained.5
In our previous publication we have described the chemical
synthesis of sulfur analogues of LPA and cPA, in which either one
or two nonbridging phosphate oxygen atoms are substituted by
sulfur to give phosphorothioate or phosphorodithioate analogues,
respectively.9 Intriguing biological properties of natural LPC
prompted us to undertake studies on the synthesis of its sulfur
analogues modified in the same manner. On the basis of our earlier
studies performed on the synthesis of phosphorothioate and phos-
phorodithioate derivatives of nucleotides and oligonucleotides10
we expected that lysophospholipid sulfur analogues should have
similar physicochemical properties as natural lysophospholipids,
yet should be more resistant towards hydrolytic enzymes.11 In or-
der to prevent possible 1?2 acyl migration in LPC analogues, the
oxygen atom in position 2 of glycerol was protected by methyla-
tion.9,12–14 The conditions of chemical synthesis enabled us also
to obtain LPC sulfur analogues as homogenic compounds, each
containing only one fatty acid residue. Thus, LPC sulfur analogues,
both phosphorothioates (2a–e) and phosphorodithioates (3a–e),
were prepared as a series of five different compounds, bearing
the residues of the following fatty acid: (a) lauric (12:0), (b) myris-
tic (14:0), (c) palmitic (16:0), (d) stearic (18:0), (e) oleic (18:1) (see
Fig. 2).
The application of aforementioned approach to the synthesis of
sulfur analogues of 2-methoxy-lysophosphatidylcholine 2 and 3 is
shown on Scheme 3.
For the synthesis of phosphorothioate analogues racemic 1-
acyl-2-methoxyglycerols 4a–e were reacted in anh. dichlorometh-
ane solution with equimolar amount of 2-N,N-diisopropylamino-1,
3,2-oxathiaphospholane in the presence of S-ethylthiotetrazole,
and then with elemental sulfur, to give 3-O-(2-thio-1,3,2-oxathia-
phospholane) lipid derivatives 9a–e (X = O), each showing in 31P
NMR two signals at d ca. 105 ppm. Such chemical shifts were
earlier described as characteristic for various 2-alkoxy-2-thio-1,
3,2-oxathiaphospholane derivatives.10a Compounds 9a–e were
isolated by silica gel flash column chromatography as pale-yellow
oils in 54–78% yield, and characterized by spectroscopic methods.
Due to appearance of two signals in 31P NMR spectra it was
concluded that each of 9a–e is a mixture of two stereoisomers
not separable by chromatography (TLC).
Each of oxathiaphospholane derivatives 9a–e was then treated
in anh. dichloromethane with 3 mol equiv of choline p-toluenesul-
fonate20 and 2 mol equiv of 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU). The ring opening condensation was accompanied by spon-
taneous elimination of episulfide.
Crude products were purified by silica gel flash column chroma-
tography to give 2-methoxy-LPC phosphorothioates (2a–e) as
white solids in 50–60% yield. The products were obtained as a
mixture of stereoisomers (two signals in 31P NMR at d ca.
59 ppm), which could not be separated by chromatography
(column or TLC). Such chemical shifts are characteristic for dialkyl
phosphorothioates.10a The purified products 2a–e were character-
ized by spectroscopic methods (1H and 31P NMR, MALDI TOF
MS). They were soluble in chloroform, methanol and water, and
did not decompose when stored at À20 °C. The details are given
in Supplementary data.
The diversity of biological functions and activity of lysophos-
phatidylcholine allowed us to assume that phosphorothioate/
phosphorodithioate derivatives 2 and 3 may also have interesting
biological or even therapeutic properties. For preliminary biologi-
cal studies the phosphorothioates (2a–e) and phosphorodithioates
(3a–e) were synthesized from racemic glycidol.
The chemical synthesis of 1-acyl-2-methoxyglycerols (4a–e),
that were crucial building blocks for the preparation of 2a–e and
3a–e, was performed exactly as described in our previous paper
(see Scheme 1).9
Thus, all five aforementioned fatty acids were reacted with
commercially available racemic glycidol (5) in the presence of cat-
alytic amounts of n-tributylamine into 1-acylglycerols (6a–e)
according to the procedure described by Lok et al.15 In the follow-
ing step 1-acylglycerols were regioselectively silylated with t-
butyldimethylsilyl chloride in the position 3 of glycerol (primary
hydroxyl group).16–18 The silyl ethers (7a–e) were then methylated
at the central oxygen of glycerol with trimethylsilyldiazomethane
in the presence of 40% fluoroboric acid.19 The 2-methoxy ethers
The presence of two signals in 31P NMR spectra of each 2a–e
(and also 9a–e) can be attributed to the presence of two centers
of asymmetry (at C2 and at phosphorus), leading to the formation
of two pairs of diastereoisomers. Unfortunately, unlike the previ-
ously phosphorothioate cPA analogues,9 these stereoisomers could
not be separated by either column chromatography or thin layer
chromatography.
For the synthesis of phosphorothioate analogues racemic 1-
acyl-2-methoxyglycerols 4a–e containing one of the aforemen-
tioned fatty acid residues were reacted in anh. dichloromethane
solution with 2-N,N-diisopropylamino-1,3,2-dithiaphospholane in
the presence of S-ethylthiotetrazole, and then with elemental sul-
fur, to give 3-O-(2-thio-1,3,2- dithiaphospholane) lipid derivatives
10a–e (X = S), showing in 31P NMR single signals at d ca. 123 ppm).
Such chemical shifts were earlier described as characteristic for
various 2-alkoxy-2-thio-1,3,2-dithiaphospholane derivatives.10b,g
Pure compounds 10a–e were isolated by silica gel flash column
chromatography as pale-yellow oils in 39–50% yield, and
characterized by spectroscopic methods.
CH3
CH3
CH3
+
O
C
X
2N
OCH2CH
R
O
OMe
P
S
O
LPC sulfur analogues 2,3
3a-e X = S phosphorodithioates
2a-e X = O phosphorothioates
a - lauroyl b - myristoyl c - palmitoyl d - stearoyl e - oleoyl
Each of dithiaphospholane derivatives 10a–e was then
treated in anh. dichloromethane with 3 mol equiv of choline
Figure 2. The structure of LPC 2-methoxy-sulfur analogues (phosphorothioates 2a–
e and phosphorodithioates 3a-e).