Synthesis of phospholipids on a glyceric acid scaffold: Design and preparation of phospholipase A2 specific substrates

Article abstract of DOI:10.1016/j.tet.2014.03.054

Synthesis of a new series of phospholipid analogues to serve as activity-based probes of secretory phospholipase A₂ enzymes is reported. The synthesis is based upon (1) preparation of long-chain esters and amides of glyceric acid, followed by (

Full text of DOI:10.1016/j.tet.2014.03.054

Tetrahedron 70 (2014) 3155e3165
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of phospholipids on a glyceric acid scaffold: design
and preparation of phospholipase A2 specific substrates
*
Renato Rosseto, Joseph Hajdu
Department of Chemistry and Biochemistry, and Center for Supramolecular Studies, California State University, Northridge, Northridge, CA 91330, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of a new series of phospholipid analogues to serve as activity-based probes of secretory
phospholipase A2 enzymes is reported. The synthesis is based upon (1) preparation of long-chain esters
and amides of glyceric acid, followed by (2) regioselective derivatization of the diol function of the
molecule to achieve phosphorylation at the primary hydroxyl group, and to introduce the incipient sn-2-
ester group of the target compounds. The sequence has been shown to allow incorporation of fluores-
cent, paramagnetic, and redox-active reporter groups, leading to phospholipid analogues applicable to
detect and measure enzyme activity, to develop highly specific, real-time spectroscopic assay of phos-
pholipase A2 enzymes, as well as to track the metabolic fate of the hydrolysis products. The synthetic
method has a great deal of flexibility to open the way to the design and synthesis of activity-probes for
other phospholipid metabolizing enzymes as well.
Received 31 December 2013
Received in revised form 15 March 2014
Accepted 17 March 2014
Available online 22 March 2014
Keywords:
Phospholipid synthesis
Glyceric acid
Phospholipase A2 activity-probe
Reporter group
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Development of new synthetic methods for the preparation of
phospholipids is one of the key steps in advancing membrane
chemistry and biochemistry today.1 The compounds are needed for
structural and dynamic studies of biomembranes and membrane-
bound enzymes with particular emphasis on establishing struc-
tureeactivity relationships with respect to phospholipidephos-
pholipid and phospholipideprotein interactions, as well as for
mechanistic elucidation of phospholipid metabolizing enzymes.1e3
Specifically, phospholipases A2 (PLA2s) comprise a large group of
intracellular and secreted enzymes that catalyze the hydrolysis of
the sn-2-ester bond of glycerophospholipids (Fig. 1), yielding fatty
acids such as arachidonic acid, and lysophospholipids.4 The prod-
ucts are precursors of signaling molecules with a broad range of
biological functions.5 For example, arachidonic acid is converted to
eicosanoids that have been shown to be involved in immune re-
sponse, inflammation, pain perception and sleep regulation,6 while
lysophospholipids are precursors of lipid mediators such as lyso-
phosphatidic acid (LPA)7 and platelet activating factor (PAF).8
Lysophosphatidic acid has been shown to be involved in cell pro-
liferation, survival and migration, while PAF is particularly involved
in inflammatory processes.8
Fig. 1. Hydrolytic cleavage of naturally occurring phospholipids by the four
phospholipases.
and they have been classified based on their structures, catalytic
mechanisms, localizations, and evolutionary relationships.10 Spe-
cifically, the mammalian sPLA2 family includes 10 catalytically ac-
tive isoforms.9 They are low-molecular weight (14e18 kDa)
secreted proteins, with a compact structure stabilized by six con-
served disulfide bonds and two additional disulfides that are
unique to each member.3 Studies focusing on their mechanism of
action have shown that an active site histidine and a highly con-
served neighboring aspartate form the catalytic dyad involved in
the reaction, with absolute dependence on Ca2þ for activation.4
The synthesis here reported focuses on preparation of phos-
pholipase A2 substrates including phospholipid compounds with
chain-terminal reporter groups, specifically directed at sPLA2 en-
zymes. The synthetic method was designed to provide access to
Secreted phospholipases A2 (sPLA2s) are widespread in nature.9
To date more than 30 isozymes have been identified in mammals,
* Corresponding author. Tel.: þ1 818 677 3377; fax: þ1 818 677 2912; e-mail
address: joseph.hajdu@csun.edu (J. Hajdu).
0040-4020/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2014.03.054

3156
R. Rosseto, J. Hajdu / Tetrahedron 70 (2014) 3155e3165
compounds that will be able (1) to detect and measure phospho-
lipase A2 activity, (2) to develop real-time spectrophotometric as-
say of the enzyme, and (3) to serve as markers tracking the
metabolic fate of the fatty acids and lysophospholipids released on
catalytic hydrolysis by the enzyme. The underlying working hy-
pothesis in the design of the target compounds (Fig. 2) was inspired
by our earlier discovery that structural modification of the sn-1-
position of the natural phospholipid, introducing an inverse ester
function to prevent PLA1 activity, yielded analogues that could
readily be hydrolyzed at the sn-2-ester group by sPLA2 enzymes.11
Indeed, studies in other laboratories have also shown, that secre-
tory PLA2s well tolerate a range of structural modifications at the
neighboring sn-1-substitution12 using analogues that readily un-
dergo catalytic hydrolysis at the sn-2-position by the enzyme. Thus,
we have designed two principal target structures 3 and 4 as plat-
forms for preparation of the PLA2-directed substrates: in-
corporating at the sn-1-position glyceric acid derivatives of an ester
group in 3, and an amide group in 4, to prevent cleavage by PLA1, as
well as by other, non-specific esterase enzymes.13
amine, using DCC/DMAP afforded the desired sn-1-substituted
products 7 and 8 in good overall yields (70e80%). Specifically, we
have found that the carboxylic group of glyceric acid is quite re-
active, in that the corresponding amide 8 could be prepared from
the acid directly, without having to synthesize an active ester, most
likely due to the rapid formation of the corresponding anhydride
intermediate.
Acid-catalyzed hydrolytic cleavage of the isopropylidene pro-
tecting group in the series 7 and 8 was next accomplished using
0.4 M hydrochloric acid solution in aq dioxane. In order to achieve
efficient isopropylidene cleavage, it turned out to be quite impor-
tant to control the acidity of the solution as higher acidity led to
partial decomposition, while at lower acid concentrations in-
complete hydrolysis occurred. Consequently, freeze-drying the
solution, rather than evaporating the solvent was used to isolate
the product. Subsequent silica gel chromatography afforded the
deprotected diols 9 and 10 in high purity and in good yields
(78e91%).
We used a two-prong approach to the regioselective manipu-
lation of the sn-2 and sn-3 hydroxyl groups of compounds 9 and 10
for preparation of the functionalized/substituted sn-3-carbinols
(Scheme 1). One method relied on a sequence of reactions that
included (1) tritylation of the primary hydroxyl group, (2) acylation
of the secondary alcohol function to introduce the sn-2-ester group,
and (3) acid-catalyzed cleavage of the trityl group to obtain the
desired sn-3-alcohol for the subsequent phosphorylation step.
Along these lines, reaction of the dodecyl ester of glyceric acid 9
with triphenylmethyl chloride, in the presence of collidine, in
dichloromethane yielded the corresponding trityl ether 11 (80%),
which was subsequently acylated with coumarin-labeled decanoic
acid using DCC/DMAP (72%), and finally detritylated to produce the
sn-3-carbinol 13 (61%). In a similar series of reactions dodecylamide
of glyceric acid 10 was first converted to 15 by tritylation of the
primary hydroxyl group (72%), followed by acylation at the sn-2-
position (95%) and detritylation to obtain the substituted glycerol
17 in 79% yield.
O
O
O
R
n
O
O
O
O
R'
P
N
m
3
O
HO
HO
O
OH
2
H
N
R
O
n
O
O
R'
O
P
N
m
O
O
4
O
R, R' = reporter groups (i.e., fluorophores, spin labels, ferrocene)
Fig. 2. Structures of the glyceric acid precursor and the phospholipid targets.
This three-step sequence readily produced the desired glyceric
acid derivatives as the precursors needed for phosphorylation,
however, application of the method required development of rig-
orous experimental conditions, specific for each substrate, in the
last step of the sequence, in the course of the detritylation reaction.
Specifically, as it has recently been well demonstrated, the exper-
imental conditions needed to prevent acid-catalyzed acyl migration
to a neighboring hydroxyl group is very much dependent on the
structure of the acyl group involved.15 Along these lines it has been
shown that even minor structural variations (such as the chain-
length of the acyl group) make a significant difference in terms of
the specific experimental conditions that need to be employed to
prevent acyl migration.15b
Consequently, in order to develop a more robust method for
regioselective manipulation of the diol portion of the molecule, we
turned to an alternative strategy relying on orthogonal protection
at the sn-2- and sn-3-positions. To that effect, we used the base-
labile phenoxyacetyl group for protection of the primary alcohol
function,16 and the acid-labile tetrahydropyranyl function to pro-
tect the secondary hydroxyl group. First the phenoxyacetyl group
was introduced in reaction of compounds 9 and 10 with phenox-
yacetyl chloride in chloroform at 0ꢀC, using 2,4,6-collidine as cat-
alyst.16 The sn-3-phenoxyacetyl compounds 12 and 16 were
obtained in 68% yield.17 The regioselectivity of the reaction could
readily be confirmed by the observation that both sn-3-
phenoxyacetyl esters showed base-line 1H NMR absorption in the
Moreover, in designing the PLA2 targeted phospholipid probes
we relied on using chain-terminal reporter groups of small size to
minimize the impact on the physicochemical properties of the fatty
acyl side-chains, including their effect on the packing in phospho-
lipid bilayers and micelles. Specifically, we have shown recently,
that phospholipid probes with coumarin-labeled hydrocarbon
side-chains could readily be incorporated into micellar interfaces of
natural phospholipids, showing near ideal mixing behavior with
the unlabeled phospholipid components in micellar aggregates.14
2. Results and discussion
2.1. Syntheses
Our synthetic approach to the preparation of phospholipid
compounds derived from glyceric acid involved three strategic
components. The first phase of the synthesis, shown in Scheme 1,
started with introduction of the long-chain ester or amide func-
tions at the incipient sn-1-position of the target phospholipids.
Since glyceric acid itself is quite insoluble in organic solvents we
relied on its isopropylidene protected methyl ester 5 as the source
of the required three-carbon scaffold to construct the phospholipid
molecules. Thus, base-catalyzed hydrolysis of the commercially
available methyl ester of 2,3-O-isopropylidene- -glyceric acid 5,
L
followed by treatment with Dowex-Hþ ion exchange resin in
aq dioxane yielded the acetonide protected glyceric acid 6 as a key
intermediate for subsequent structural derivatization. Condensa-
tion of compound 6 with the respective long-chain alcohol or
d 5.00e5.10 range, which is consistent with the presence of the free
sn-2-hydroxyl group in the products.15
In the next step, tetrahydropyranylation of the hydroxyl group at
the sn-2-position was carried out using excess of 3,4-dihydro-2H-

R. Rosseto, J. Hajdu / Tetrahedron 70 (2014) 3155e3165
3157
Scheme 1. Reagents and conditions: (a) 1 M KOH/MeOH; (b) ROH, DCC/DMAP, CH2Cl2; (c) CH3(CH2)11NH2, DCC/DMAP, CH2Cl2; (d) HCl, aq dioxane; (e) (C6H5)3CCl, 2,4,6-
trimethylpyridine, CH2Cl2; (f) C6H5OCH2COCl, 2,4,6-trimethylpyridine, CHCl3; (g) (C6H5)3CCl, pyridine, CHCl3; (h) (i) 10-(70-mercapto-40-methylcoumarin)decanol, DCC/DMAP,
CHCl3, (ii) HCl, aq dioxane); (i) (I) DHP-PPTS, CH2Cl2, (II) tert.-butylamine, (CHCl3/MeOH 1:1).
pyran with PPTS as catalyst in dichloromethane. Subsequent che-
moselective base-hydrolysis of the phenoxyacetyl ester with tert
butylamine in a mixture of methanol/chloroform afforded the sn-3-
carbinols 14 and 18 in 91e97% overall yield. Although introduction
of the tetrahydropyranyl group created a second chiral center in the
molecule, it did not complicate the isolation and purification of the
products.18 It should be pointed out that one significant advantage
of the phenoxyacetyl/tetrahydropyranyl strategy is that it com-
pletely circumvents the problem of acyl migration in the sequence.
The second phase of the synthesis, shown in Scheme 2, focused
on elaboration of the sn-3-phosphocholine headgroup of the tar-
get compounds. To achieve efficient phosphorylation of the
substituted glyceric acid derivatives we used 2-chloro-2-oxo-1,3,2-
dioxaphospholane, as this reagent has been shown to produce
phosphocholine derivatives in good yields and with few byprod-
ucts, even in reactions with substrates that carry nucleophilic
amideecarbonyl groups that are widely regarded as rather difficult
substrates to phosphorylate.19 Thus, reaction between ethylene
chlorophosphate and the substituted glyceric acid series of 13 and/
or 17 in benzene, in the presence of triethylamine as catalyst,
followed by nucleophilic ring-opening of the cyclic phospho-
diester intermediates with trimethylamine in anhydrous acetoni-
trile produced the corresponding phosphocholine compounds
(Scheme 2).
The phospholipid products 19 and 20 were purified by silica
gel chromatography, and isolated in 48e54% overall yield.20
Similarly, phosphorylation of the sn-2-tetrahydropyranyl pro-
tected glyceric acid derivatives 14 and 18 afforded the corre-
sponding phosphorylcholines 21 and 23 in somewhat higher
overall yields (67e74%).
The third and final phase of the synthesis focused on developing
a method to replace the sn-2-tetrahydropyranyl protecting group in
compounds 21 and 23 with the series of sn-2-ester groups of the
target phospholipids. The sequence shown in Scheme 2 proceeded
via formation of lysophospholipid intermediates 22 and 24
that were prepared by acid-catalyzed cleavage of the sn-2-
tetrahydropyranyl group of the phosphorylated compounds. Thus,
phospholipid 21 treated with dilute hydrochloric acid in aq dioxane
yielded lysophospholipid 22 (94%), and deprotection of 23 was
accomplished using a biphasic system comprised of aq HCl and
chloroform that produced the corresponding lysophospholipid 24
isolated by silica gel chromatography in 57% yield.
With compounds 22 and 24 in hand, we proceeded to introduce
a series of sn-2-fatty acyl groups for preparation of mixed-chain
double labeled phospholipid compounds, including those that
would carry chain-terminal reporter groups. We have used two
methods for introduction of the sn-2-substituents: (1) acylation of
the lysophospholipid analogues with fatty acyl groups already

3158
R. Rosseto, J. Hajdu / Tetrahedron 70 (2014) 3155e3165
Scheme 2. Reagents and conditions: (a) (i) ethylene chlorophosphate, Et3N, benzene, (ii) (CH3)3N, MeCN, 65 ꢀC; (b) HCl, aq dioxane; (c) 10-(70-mercapto-40-methylcoumarin)
decanoic acid/DCC/DMAP, CHCl3; (d) 12-(FMOC)-aminododecanoic acid/DCC/DMAP, CHCl3; (e) (i) DBU, CHCl3, (ii) p-nitrophenyl-7-mercapto-4-methylcoumarin-3-carboxylate,
DMAP, CHCl3; (f) (i) as in (e), then (ii) p-nitrophenyl-PROXYL-3-carboxylate, DMAP, CHCl3; (g) (i) as in (e), then ferrocenecarbonyl fluoride, DMAP, CHCl3.
labeled at their chain-end, and (2) incorporating fatty acyl groups
with a chain-terminal protecting group, to elaborate the reporter
group after the acylation step. Thus, reaction between lysophos-
pholipid 22 and 7-mercapto-4-methylcoumarin-labeled decanoic
acid, using DCC/DMAP in chloroform, with added glass beads and
sonication to increase the glass surface in the reaction vessel,21
yielded the phospholipid product 20 (62%), with an sn-2-acyl
group carrying the chain-terminal fluorophore.
In an alternative sequence, lysophospholipid 24 was first acyl-
ated in reaction with FMOC-protected 12-aminododecanoic acid
using DCC/DMAP in chloroform, producing phospholipid 25 that
was isolated by silica gel chromatography in 60% yield. Re-
placement of the FMOC protecting group with the desired chain-
terminal reporter group was carried out in a two-step/one-pot
sequence. Along these lines, base-catalyzed elimination of the flu-
orenylmethoxycarbonyl protecting group with excess of DBU in
chloroform, followed by addition of the p-nitrophenyl active ester
of 7-diethylaminocoumarin-3-carboxylate and DMAP yielded the
desired sn-2-fatty acyl ester with the fluorescent chain-terminal 26.
The product was purified by silica gel chromatography and isolated
in 66% overall yield. The phospholipid compounds carrying the sn-
2-chain-terminal paramagnetic spin label 27 (52%), and the redox-
active ferrocene group 28 (58%), were obtained under similar ex-
perimental conditions.
Specifically, compound 26 with a fluorescent donoreacceptor
pair, and compound 27 with fluorophore-quencher reporter groups
were prepared to detect and measure phospholipase A2 activity
following the changes in fluorescence resonance energy transfer
(FRET), and fluorescence de-quenching on hydrolysis by the en-
zyme. In case of compound 26 the fluorescence emission peak of
the donor, (7-mercapto-4-methylcoumarin), at 390 nm shows
substantial overlap with the excitation spectrum of the acceptor (7-
diethylaminocoumarin) at 405 nm, whose emission is observed at
462 nm. Thus, PLA2 catalyzed cleavage results in increase of the
fluorescence by the donor, and in loss of FRET (i.e., emission of the
acceptor) that can be used to follow the hydrolysis by the enzyme.
Along the same line, hydrolysis of the sn-2-ester bond in 27 causes
an increase in fluorescence at 390 nm via de-quenching due to the
departure of the paramagnetically labeled fatty acid quencher22
from the substrate. Finally, we have applied the synthetic method
to prepare compound 28 incorporating a chain-terminal ferrocene
reporter group that was recently introduced as a redox-active label
of phospholipids.23 Thus, the sequence here described provides
a method that should be widely applicable to the synthesis of
phospholipid compounds including acyl groups with chain-
terminal spectroscopic labels that could be introduced later in the
synthesis such as after the phosphorylation step, either because
they would not survive the phosphorylation conditions or are not

R. Rosseto, J. Hajdu / Tetrahedron 70 (2014) 3155e3165
3159
readily available in the amounts required to carry out multi-step
syntheses.
relative to TMS. IR spectra were measured on a Fourier Transform
infrared spectrometer in chloroform solution. Optical rotations
were measured using sodium light (D line at 589 nm) on a Perkin
Elmer 341 polarimeter. High resolution mass spectra for de-
termination of exact mass of the new compounds were determined
at the Mass Spectrometry Facility at University of California, Riv-
erside. Elemental analyses were determined by Desert Analytics,
Tucson AZ, and Galbraith Laboratories Knoxville, TN. Column
chromatography was carried out using silica gel 60 (230e400
mesh, ASTM, E.M. Science). Reactions were monitored by thin layer
chromatography using MK6F silica gel 60 plates. The compounds
were visualized on the TLC plates by iodine vapor and UV light,
where appropriate. Phospholipids were visualized by molybdenum
spray26 and the primary amines were sprayed with 0.25% ninhydrin
2.2. Enzymatic hydrolysis
Catalytic hydrolysis of the synthetic phospholipid substrates 19,
20, 26e28 was carried with bee-venom phospholipase A2, a widely
used, readily available representative of the low-molecular weight
secretory PLA2 enzymes.24 In an assay mixture containing Triton X-
100/phospholipid mixed micelles,25 in the presence of catalytically
essential Ca2þ, each one of the phospholipid compounds was
completely hydrolyzed by the enzyme, producing the lysophos-
pholipid analogues, and the corresponding fatty acids 29e32 la-
beled with chain-terminal reporter groups, shown in Fig. 3
Fig. 3. Hydrolysis of the synthetic probes by PLA2.
The rates of enzymatic hydrolysis of the coumarin-labeled
compounds were well within the same order of magnitude as the
naturally occurring phospholipids. Specifically, preliminary results
showed that compound 26 was cleaved by bee-venom PLA2 only
2.3 times slower compared to the hydrolysis of dipalmitoyl phos-
phatidylcholine by the same enzyme under similar conditions.
These results indicate that introduction of chain-terminal coumarin
labels maintain good substrate quality, in good agreement
with what we observed with coumarin-labeled fluorescent
glycerophospholipids.14
in acetone solution, followed by heating the plates at 120 ꢀC. Ion
exchange resin AG 50W-X8 (100e200 mesh) was obtained from
Bio-Rad Laboratories. Commercially available reagents used were
reagent grade or better and used as obtained. Dichloromethane and
chloroform were freshly distilled from P2O5; benzene was kept over
sodium wire and distilled from CaH2 before use. Acetonitrile
(spectrograde, Burdick & Jackson) was dried over activated mo-
ꢀ
lecular sieves (3 A). Bee-venom phospholipase A2 was obtained
from Sigma, it was dialyzed against 0.05 M phosphate buffer, pH
8.00, and stored at 4 ꢀC.
4.2. General procedures for preparation of 1,2-disubstituted
glyceric acid derivatives
3. Conclusions
The main significance of the synthesis here reported is in pro-
viding a facile and efficient method for preparation of a new class of
phospholipid compounds, including functionalized phospholipid
analogues with fluorescent, paramagnetic, and redox-active re-
porter groups. The strengths of the method are in its (1) simplicity
and efficiency, (2) flexibility with respect to the substituent groups
that can be introduced, and (3) applicability to the development of
new phospholipid analogues with the desired target structures for
biological and physicochemical studies. Availability of the new
probes opens the way for future in vivo experiments for detection
and measurement of phospholipase A2 activity, and for the design
and development of highly specific activity-based probes for re-
lated studies of phospholipid hydrolyzing enzymes. Work toward
these goals is under way in our laboratory.
4.2.1. Method A: (1) preparation of glyceric esters 9 and amides 10
4.2.1.1. Dodecyl-2,3-dihydroxypropanoate (9a). 4.2.1.1.1. 2,2-
Dimethyl-1,3-dioxolane carboxylic acid (6). To
a
solution of
methyl-2,2-dimethyl-1,3-dioxalane-4-carboxylate
5 (2.057 g,
12.8 mmol) in 5 mL MeOH, kept in an ice-water bath was added
drop-wise 15 mL 1 M potassium hydroxide in methanol. After ad-
dition of KOH, the ice-bath was removed and the mixture was
stirred at room temperature for 45 min. To this solution were added
15 mL of MeOH and 30 mL of Dowex-Hþ ion exchange resin, and
then the mixture was stirred for 3 min. The mixture was then fil-
tered and the resin was washed with MeOH (40 mL). The solvent
was evaporated, the residue was dispersed in benzene and freeze-
dried to give compound 6 (1.695 g, 90.5%) as colorless oil. IR (neat):
3180br m, 1736vs, 1220s, 1104s, 1069s, 837m cmꢁ1; 1H NMR (CDCl3,
4. Experimental section
200 MHz)
d
1.32 (s, 3H), 1.42 (s, 3H), 4.04e4.12 (dd, 1H, J¼8.7,
5.1 Hz), 4.16e4.24 (dd, 1H, J¼7.3, 8.7 Hz), 4.51e4.57 (dd, 1H, J¼5.1,
4.1. General methods
7.3 Hz), 9.45 (s,1H); 13C NMR (CDCl3, 50 MHz)
d 25.2, 25.7, 67.1, 73.5,
20
111.7, 175.8. [
a
]
ꢁ25.2 (c 1.1, CHCl3), known compound.
D
Proton NMR spectra were recorded at 200 MHz, carbon NMR
4.2.1.1.2. Dodecyl-2,2-dimethyl-1,3-dioxolane-4-carboxylate
spectra were recorded at 50 MHz; both are reported in
d
units
(7a). To a solution of compound 6 (1.6213 g, 11.1 mmol) in 35 mL

3160
R. Rosseto, J. Hajdu / Tetrahedron 70 (2014) 3155e3165
of CHCl3 were added 12-dodecanol (2.2920 g, 12.3 mmol), DMAP
(1.5027 g, 12.3 mmol), and DCC (2.5379 g, 12.3 mmol) and the
mixture was stirred for 7 h. The DCCeurea that formed was fil-
tered off, and the solvent was evaporated. The residue was dis-
solved in CH2Cl2 and purified on a silica gel column packed and
eluted with CH2Cl2. The fractions containing the product were
combined, evaporated, dissolved in benzene, and freeze-dried
to give 7a (2.8202 g, 76.4%) as colorless wax. IR (CHCl3):
152.3, 153.7, 160.7, 172.9. Rf (CHCl3/EtOAc 1:1) 0.23. Anal. Calcd for
C
25H36O6S: C, 64.63; H, 7.81. Found: C, 64.84; H, 7.50. MS MHþ
20
C25H36O6SH calcd: 465.2310, found: 465.2307. [
a
]
ꢁ14.2 (c 0.96,
D
CHCl3).
4.2.1.3. N-Dodecyl-2,3-dihydroxypropanamide (10). 4.2.1.3.1. N-
Dodecyl-2,2-dimethyl-1,3-dioxolane-4-carboxamide (8). To a solu-
tion of 6 (1.5009 g, 0.0103 mmol) in 40 mL of CHCl3 were added 12-
dodecylamine (2.2909 g, 12.4 mmol), DMAP (1.3812 g, 11.3 mmol),
and DCC (2.3315 g, 11.3 mmol) and the mixture was stirred at room
temperature for 16 h. The DCCeurea that formed was filtered and
the solvent was evaporated. The residue was re-dissolved in CHCl3
and purified on a silica gel column packed with CHCl3 and eluted
with CHCl3/EtOAc (10:1). The fractions containing the product
were combined, evaporated, re-dissolved in benzene, and freeze-
dried to give 8 (2.9162 g, 90.3%) as colorless oil that solidified in
1740br cmꢁ1 1H NMR (CDCl3, 200 MHz)
; d 0.86 (br t, 3H), 1.24 (br
s, 18H), 1.39 (s, 3H), 1.48 (s, 3H), 1.64 (m, 2H), 4.18 (m, 4H), 4.56 (t,
1H, J¼6.2 Hz). 13C NMR (CDCl3, 50 MHz)
d 14.2, 22.8, 25.7, 25.9,
26.0, 28.7, 29.3, 29.5, 29.6, 29.7, 29.7, 32.0, 65.6, 67.5, 74.3, 111.4,
20
171.4. Rf (CH2Cl2) 0.69. [
a
]
ꢁ10.4 (c 1.02, CHCl3/MeOH 4:1),
D
known compound.
4.2.1.1.3. 12-[(40-Methyl-20-oxo-2H0-chromen-70-yl)thio]dodecyl-
2,2-dimethyl-1,3-dioxolane-4-carboxylate (7b). To a solution of 6
(0.400 g, 2.7 mmol) in 25 mL CHCl3 were added 7-[(12-
hydroxydodecyl)thio]-4-methyl-2H-chromen-2-one (0.8010 g,
2.1 mmol), DMAP (0.2598 g, 2.1 mmol), and DCC (0.5614 g,
2.1 mmol). The mixture was stirred for 6 h, then the DCCeurea
that formed was filtered and the solvent was evaporated. The
residue was re-dissolved in CHCl3 and purified on a silica gel
column packed with CHCl3 and eluted with CHCl3/EtOAc (20:1).
The fractions containing the product were combined, evaporated,
dissolved in benzene, and freeze-dried to give 7b (0.9342 g,
the freezer. IR (CHCl3): 3345, 1680br cmꢁ1
200 MHz) 0.83 (br t, 3H), 1.21 (br s, 18H), 1.35 (s, 3H), 1.42 (s, 3H),
1.69 (m, 2H), 3.24 (m, 2H), 4.04 (m, 1H), 4.23 (br t, 1H), 4.43 (br t,
1H), 6.58 (m, 1H). 13C NMR (CDCl3, 50 MHz)
14.0, 22.5, 24.5, 24.9,
;
1H NMR (CDCl3,
d
d
26.0, 26.7, 29.1, 29.2, 29.4, 29.5, 30.6, 31.8, 38.8, 67.6, 74.9, 110.9,
170.9. Rf (CHCl3/EtOAc 5:1) 0.80. [
a
]
20 ꢁ12.4 (c 1.03, CHCl3), known
D
compound.11
4.2.2. Compound (10). To a solution of 8 (2.8241 g, 9 mmol) in
25 mL of 1,4-dioxane was added a solution of HCl (1.6 mL of 12 M
aq HCl diluted in 23.5 mL of 1,4-dioxane) at room temperature.
After 2.5 h, the mixture became cloudy and formed a white pre-
cipitate. To this mixture was added 40 mL benzene, the precipitate
was filtered, and washed with benzene. The solid was then dis-
persed in benzene and freeze-dried to give 8 (1.5584 g, 5.7 mmol,
63%) as white powder. The filtrate was collected, freeze-dried,
dissolved in CHCl3/EtOAc (1:1), and purified on a silica gel col-
umn loaded and eluted with CHCl3/EtOAc (1:1). The fractions cor-
responding to the product were combined, evaporated, and
dispersed in benzene, and freeze-dried to give an additional crop of
10 (0.6398 g, 26%) to a combined yield of 89%. IR (CHCl3): 3380br,
88.2%) as a white solid. IR (CHCl3): 3257, 1732br, 1621, 1246 cmꢁ1
1H NMR (CDCl3, 200 MHz)
1.17 (br s, 16H), 1.30 (s, 3H), 1.39
;
d
(s, 3H), 1.56 (m, 4H), 2.28 (s, 3H), 2.87 (t, 2H, J¼7.2 Hz),
4.05 (m, 4H), 4.48 (t, 1H, J¼5.4 Hz), 6.06 (s, 1H), 7.03 (m, 2H),
7.33 (d, 1H, J¼8.1 Hz). 13C NMR (CDCl3, 50 MHz)
d 18.2, 25.2,
25.4, 25.5, 28.2, 28.3, 28.6, 28.8, 29.1, 31.7, 65.1, 67.0, 73.8,
110.9, 113.2, 116.5, 122.4, 124.2, 143.5, 151.9, 153.5, 160.1, 171.0. Rf
20
(CHCl3/EtOAc 20:1) 0.67. [
compound.
a
]
ꢁ9.7 (c 0.99, CHCl3), known
D
4.2.1.1.4. Compound (9a). To
a solution of 7a (1.7502 g,
5.26 mmol) in 25 mL of 1,4-dioxane was added a solution of HCl
(1.6 mL of 12 M aq HCl diluted with 23.5 mL 1,4-dioxane) at room
temperature. The reaction was stopped after 2 h. To this mixture
was added 30 mL benzene and then freeze-dried to give a white
residue. The solid was re-dissolved in CHCl3 and purified on a silica
gel column loaded with CHCl3 and eluted with CHCl3/EtOAc (2:1).
The fractions corresponding to the product were combined and
evaporated, dissolved in benzene, and freeze-dried to give 9a
1683br cmꢁ1
;
1H NMR (CDCl3þCD3OD, 200 MHz)
d 0.82 (br t, 3H),
1.20 (br s, 18H), 1.45 (m, 2H), 3.19 (t, 2H, J¼7.32 Hz), 3.37 (br s, 2H)
3.70 (m, 2H), 4.01 (br t, 1H). 13C NMR (CDCl3, 50 MHz)
d 14.0, 22.5,
26.2, 27.0, 29.1, 29.2, 29.4, 29.5, 30.6, 31.8, 38.8, 67.9, 75.0, 170.7. Rf
(CHCl3/EtOAc 1:1) 0.21. Anal. Calcd for C15H31NO3$1/3CHCl3: C,
58.80; H, 10.08; N, 0.47, Found: C, 58.83; H, 9.77; N, 4.96. MS MHþ
20
(1.3134 g, 91%) as white solid. IR (CHCl3): 3340, 1742br cmꢁ1 1H
;
C15H31NO3H calcd: 274.2382, found: 274.2378. [
CHCl3/MeOH 4:1).
a
]
ꢁ9.4 (c 1.01,
D
NMR (CDCl3, 200 MHz) 0.86 (br t, 3H), 1.24 (br s, 18H), 1.57 (m, 2H),
3.84 (m, 2H), 4.16 (t, 2H, J¼6.8 Hz), 4.25 (m, 1H). 13C NMR (CDCl3,
4.2.3. Method A: (2) protection of the primary hydroxyl group of
glyceric acid derivatives via tritylation
50 MHz) d 14.2, 22.8, 25.8, 29.1, 29.3, 29.5, 29.7, 29.7, 29.7, 32.1, 65.8,
67.6, 74.5, 173.0. Rf (CHCl3/EtOAc 1:1) 0.36. [
MeOH 4:1), known compound.
a]
20 ꢁ8.4 (c 1.02, CHCl3/
D
4.2.3.1. Dodecyl-2-hydroxy-3-(trityloxy)propanoate (11). To a so-
lution of 9a (1.3502 g, 4.6 mmol) in 30 mL of CH2Cl2 were added
collidine (0.64 mL, 4.8 mmol) and trityl chloride (1.4003 g,
4.8 mmol). The reaction mixture was kept at room temperature for
26 h. The volume of the solution was reduced to one-third by
evaporation and it was then loaded onto a silica gel column and
eluted with CHCl3. The fractions corresponding to the product were
combined and evaporated, dissolved in benzene, and freeze-dried
to give 11 (1.9008g, 80.4%) as a white waxy solid. IR (CHCl3):
4.2.1.2. 12-[(4-Methyl-2-oxo-2H-chromen-7-yl)thio]dodecyl-2,3-
dihydroxypropanoate (9b). To a solution of 7b (0.9304g, 1.84 mmol)
in 25 mL 1,4-dioxane was added a solution of HCl (1.6 mL of 12 M
aq HCl diluted with 23.5 mL 1,4-dioxane) at room temperature. The
reaction was stopped after 2 h. To this mixture 30 mL of benzene
were added and then freeze-dried to give a white residue. The solid
was dissolved in CHCl3 and purified on a silica gel column loaded
with CHCl3 and eluted with CHCl3/EtOAc (5:1). The fractions cor-
responding to the product were combined and evaporated, dis-
solved in benzene and freeze-dried to give 9b (0.6612 g, 78%) as
white solid. IR (CHCl3): 3330 br, 1740 br, 1614, 1215 cmꢁ1; 1H NMR
3370, 1736 cmꢁ1 1H NMR (CDCl3, 200 MHz)
; d 0.88 (br t, 3H), 1.25
(br s, 18H), 1.58 (m, 2H), 3.25 (d, 1H, J¼9.4 Hz), 3.33 (dd, 1H, J¼3,
9.4 Hz), 3.49 (dd, 1H, J¼3, 9.4 Hz), 4.17 (t, 2H, 6.7 Hz), 4.26 (br m,
1H), 7.18e7.44 (m, 15H). 13C NMR (CDCl3, 50 MHz)
d 14.1, 22.6, 25.7,
28.5, 29.2, 29.3, 29.4, 29.6, 31.9, 65.3, 65.9, 70.7, 86.3, 127.0, 127.8,
(CDCl3, 200 MHz) d 1.24 (br s,16H),1.62 (m, 4H), 2.36 (s, 3H), 2.94 (t,
128.3, 143.6, 173.2. Rf (CHCl3) 0.55. Anal. Calcd for C34H44O4$1/
2H, J¼7.2 Hz), 3.20 (br s, 2H), 3.84 (m, 2H), 4.16 (t, 2H, J¼6.8 Hz),
3CHCl3: C, 77.83; H, 8.45. Found: C, 77.85; H, 8.40. MS MNaþ
4.24 (m, 1H), 6.16 (s, 1H), 7.08e7.12 (m, 2H), 7.41 (d, 1H, J¼8.8 Hz).
20
13C NMR (CDCl3, 50 MHz)
d 18.4, 25.6, 28.3, 28.5, 28.7, 29.0, 30.0,
C34H44O4Na calcd: 539.3137, found: 539.3125. [
a
]
ꢁ5.7 (c 1.13,
D
CHCl3/MeOH 4:1).
29.2, 29.3, 32.0, 64.0, 65.9, 71.6,113.4,113.6,116.7,122.7,124.4,143.7,

R. Rosseto, J. Hajdu / Tetrahedron 70 (2014) 3155e3165
3161
4.2.3.2. N-Dodecyl-2-hydroxy-3-(trityloxy)propanamide (15). To
a solution of 10 (0.5547 g, 2 mmol) in 35 mL of CHCl3 were added
trityl chloride (1.0201 g, 3.66 mmol) and pyridine (0.5 mL), and
the reaction mixture was kept under reflux for 24 h. The mixture
was then directly loaded on a silica gel column packed with CHCl3
and eluted with CHCl3/EtOAc (9:1). The fractions containing the
product were combined, evaporated, and the residue re-dissolved
in benzene and freeze-dried to give the product 15 (0.7427 g, 72%)
Found: C, 68.57; H, 8.50. MS MNaþ C35H54O7S calcd: 618.3590,
20
found: 618.3583. [
a
]
D
ꢁ8.9 (c 1.23, CHCl3/MeOH 4:1).
4.2.4.2. 1-(Dodecylamino)-3-hydroxy-1-oxopropan-2-yl-10-((4-
methyl-2-oxo-2H-chromen-7-yl)thio)decanoate (17). 4.2.4.2.1. 1-
(Dodecylamino)-1-oxo-3-(trityloxy)propan-2-yl-10-[(4-methyl-2-
oxo-2H-chromen-7-yl)thio]decanoate. To a solution of 15 (0.5402 g,
1.05 mmol) in 30 mL of CHCl3 were added (40-methyl-70-mercap-
tocoumarin)-10-decanoic acid (0.4567 g, 1.26 mmol), DCC
(0.3362 g,1.26 mmol), and DMAP (32 mg, 0.26 mmol, 20 mol %). The
reaction was over after 1 h stirring at room temperature. The
DCCeurea that formed was filtered and the solvent was evaporated.
The residue was re-dissolved in CHCl3 and purified on a silica gel
column packed with CHCl3 and eluted with CHCl3/EtOAc (9:1). The
fractions containing the product were combined, evaporated, re-
dissolved in benzene, and freeze-dried to give the analytically
pure product (0.8602 g, 95%) as pale yellow wax. IR (CHCl3): 3330,
as a white waxy solid. IR (CHCl3): 3340br m, 1657, 1236 cmꢁ1
NMR (CDCl3, 200 MHz) 0.90 (br t, 3H), 1.27 (br s, 18H), 1.48 (m,
2H), 3.30 (m, 4H), 4.14 (dd, 1H, J¼5.4, 9.4 Hz), 4.16 (m, 1H), 6.81
(m, 1H), 7.25e7.45 (m, 15H). 13C NMR (CDCl3, 50 MHz)
14.1, 22.7,
;
1H
d
d
26.9, 29.3, 29.2, 29.4, 29.5, 29.6, 29.7, 29.6, 31.9, 39.3, 65.0, 70.4,
87.3, 127.3, 127.9, 128.9, 129.0, 128.5, 143.3, 171.5. Rf (CHCl3/EtOAc
4:1) 0.78. Anal. Calcd for C34H45NO3$1/3H2O: C, 77.38; H, 8.85; N,
2.65. Found: C, 77.06; H, 8.82; N, 2.78. MS MNaþ C34H45NO3Na
20
calcd: 538.3297, found: 538.3317. [
4:1).
a
]
ꢁ2.4 (c 0.91, CHCl3/MeOH
D
1731br, 1672 cmꢁ1; 1H NMR (CDCl3, 200 MHz)
d 0.88 (br t, 3H), 1.25
(br s, 28H), 1.66 (m, 6H), 2.39 (m, 5H), 2.97 (t, 2H, J¼7.2 Hz), 3.30 (m,
2H), 3.42 (dd, 1H, J1¼3.3 Hz, J2¼9.9 Hz), 3.54 (dd, 1H, J1¼4.8 Hz,
J2¼9.9 Hz), 5.34 (br t, 1H, J1¼3.3 Hz), 6.20 (s, 1H), 6.29 (m, 1H),
4.2.4. Method A: (3) acylation at the secondary hydroxyl group and
detritylation to produce compounds 13 and 17
7.14e7.47 (m, 18H). 13C NMR (CDCl3, 50 MHz)
d 14.1, 18.5, 22.6, 24.8,
4.2.4.1. 1-(Dodecyloxy)-3-hydroxy-1-oxopropan-2-yl(10-(4-
26.8, 26.9, 28.6, 28.8, 28.9, 29.0, 29.1, 29.2, 29.2, 29.3, 29.5, 29.6,
31.8, 32.1, 34.2, 39.4, 63.3, 73.0, 86.6, 113.6, 116.9, 122.9, 124.5, 127.1,
127.8, 128.6, 143.4, 152.1, 152.2, 153.9, 160.6, 167.7, 172.0. Anal. Calcd
for C54H69NO6S$1/2H2O: C, 74.62; H, 8.12; N, 1.61. Found: C, 74.22;
H, 8.23; N, 1.74. MS MNaþ C54H69NO6SNa calcd: 882.4738, found:
methyl-2-oxo-2H-chromen-7-yl)thio)decanoate
(13). 4.2.4.1.1. 1-
(Dodecyloxy)-1-oxo-3-(trityloxy)propan-2-yl-10-[(4-methyl-2-oxo-
2H-chromen-7-yl)thio]decanoate. To a solution of 11 (1.0012 g,
1.93 mmol) in 30 mL of CHCl3 were added (40-methyl-70-mercap-
tocoumarin-70-yl)-10-decanoic acid (0.7002 g, 1.93 mmol), DCC
(0.4008 g, 1.93 mmol), and DMAP (50 mg, 0.41 mmol). The reaction
was stopped after 4 h stirring at room temperature. The mixture
was filtered and the solvent was evaporated. The residue was re-
dissolved in CHCl3 and purified on silica gel column packed with
CHCl3/hexane (1:1) and eluted with a stepwise gradient of CHCl3/
hexane (1:1, 7:5, 2:1 and 5:1). The fractions corresponding to the
product were isolated, evaporated, dissolved in benzene, and
freeze-dried to give the analytically pure product (1.1903 g, 72%) as
a colorless wax. IR (CHCl3): 3340, 1735br, 1625, 1210 cmꢁ1; 1H NMR
882.4774. Rf (CHCl3/EtOAc 9:1) 0.78. [
a
]
D
20 ꢁ7.4 (c 1.19, CHCl3/MeOH
4:1).
4.2.4.2.2. Compound (17). To a solution of 1-(dodecylamino)-1-
oxo-3-(trityloxy)propan-2-yl-10-[(4-methyl-2-oxo-2H-chromen-
7-yl)thio]decanoate (0.6502 g, 0.76 mmol) in 30 mL 1,4-dioxane
was added 0.2 mL of 12 M HCl. The reaction was stopped after
5 h stirring at room temperature. To this mixture was added 30 mL
benzene and then freeze-dried. The white residue obtained was
dissolved in CHCl3 and purified on a silica gel column packed with
CHCl3 and eluted with CHCl3/EtOAc (5:2). The fractions containing
the product were combined, evaporated, re-dissolved in benzene,
and freeze-dried to give 17 (0.3662 g, 79%) as white solid. IR
(CHCl3): 3335 m, 1729, 1666, 1209 cmꢁ1; 1H NMR (CDCl3, 200 MHz)
(CDCl3, 200 MHz) d 0.91 (br t, 3H), 1.28 (br s, 28H),1.65 (m, 6H), 2.32
(s, 3H), 2.50 (t, 2H, J¼7.4 Hz), 2.94 (t, 2H, J¼6.8 Hz), 3.52 (br s, 2H),
4.16 (m, 2H), 5.25 (m, 1H), 6.16 (s, 1H), 7.10e7.49 (m, 18H). 13C NMR
(CDCl3, 50 MHz)
d 13.9, 18.2, 22.5, 24.6, 25.5, 28.3, 28.4, 28.6, 28.9,
d
0.87 (br t, 3H), 1.26 (br s, 30H), 1.66 (m, 4H), 2.35 (m, 5H), 2.98 (t,
29.0, 29.1, 29.2, 29.4, 31.7, 31.8, 33.8, 62.9, 65.3, 71.63, 86.4, 113.4,
116.6, 122.5, 124.3, 126.9, 127.6, 128.0, 128.4, 143.3, 143.6, 151.9,
153.6, 160.2, 167.93, 172.70. Rf (CHCl3) 0.27. Anal. Calcd for
2H, J¼6.8 Hz), 3.22 (m, 2H), 3.93 (m, 2H), 5.26 (t, 1H, J¼4 Hz), 6.28
(s,1H), 6.33 (m,1H), 7.12e7.20 (m, 2H), 7.43 (d,1H, J¼8 Hz). 13C NMR
C
54H68O7S: C, 75.31; H, 7.96. Found: C, 75.71; H, 7.66. MS MNaþ
(CDCl3, 50 MHz)
d 14.1, 18.5, 22.6, 24.8, 26.8, 28.6, 28.7, 29.0, 29.1,
20
29.1, 29.2, 29.3, 29.37, 29.58, 31.9, 32.1, 34.1, 39.3, 62.5, 67.1, 71.5,
73.6, 113.6, 117.2, 123.0, 124.5, 143.1, 152.3, 153.5, 160.2, 168.4, 172.4.
Rf (CHCl3/EtOAc 5:2) 0.38. Anal. Calcd for C35H55NO6S$1/2H2O: C,
67.06; H, 9.00; N, 2.23. Found: C, 67.04; H, 8.47; N, 2.22. MS MHþ
C54H68O7SNa calcd: 883.4583, found: 883.4575. [
CHCl3/MeOH 4:1).
a
]
ꢁ7.2 (c 1.21,
D
4.2.4.1.2. Compound (13). To a solution of the product 1-
(dodecyloxy)-1-oxo-3-(trityloxy)propan-2-yl-10-((4-methyl-2-
oxo-2H-chromen-7-yl)thio)decanoate (0.8012 g, 0.93 mmol) in
25 mL of 1,4-dioxane was added a solution of HCl (0.25 mL 1 M
aq HCl diluted with 5 mL of 1,4-dioxane). The reaction mixture was
kept at room temperature overnight. To this mixture was added
30 mL benzene and then freeze-dried. The white residue obtained
was dissolved in CHCl3 and purified on silica gel column packed
with CHCl3 and eluted with CHCl3/EtOAc (9:1). The fractions con-
taining the product were isolated, evaporated, re-dissolved in
benzene, and freeze-dried to give 13 (0.3402 g, 61%) as white solid.
C35H55NO6SNa calcd: 618.3828, found: 618.3795. [
CHCl3/MeOH 4:1).
a
]
D
20 ꢁ6.1 (c 1.21,
4.2.5. Method B: preparation of 2-tetrahydropyranyl glyceric acid
esters and amides (14 and 18)
4.2.5.1. 12-[(4-Methyl-2-oxo-2H-chromen-7-yl)thio]dodecyl-3-
hydroxy-2-[(tetrahydro-2H-pyran-2-yl)oxy]propanoate
(14). 4.2.5.1.1. 12-[(4-Methyl-2-oxo-2H-chromen-7-yl)thio]dodecyl-
2-hydroxy-3-(2-phenoxyacetoxy)propanoate (12). To a solution of
9b (2.1021 g, 4.5 mmol) and phenoxyacetyl chloride (0.82 mL,
5.85 mmol) in 40 mL of CHCl3 kept at 0 ꢀC was slowly (0.25 mL/min)
added a solution of collidine (1.1021 g, 9 mmol) in 10 mL of CHCl3.
After 90 min stirring, more phenoxyacetyl chloride was added
(0.1 mL, 0.72 mmol) and the reaction mixture was stirred for an
additional 90 min. This solution was then loaded directly on a silica
gel column packed with CHCl3 and eluted with CHCl3/EtOAc (9:1).
Two products were isolated: one corresponding to the monoacyl
IR (Nujol): 3350, 1738br, 1630 cmꢁ1
;
1H NMR (CDCl3, 200 MHz)
d
0.91 (br t, 3H), 1.26 (br s, 30H), 1.65 (m, 4H), 2.35 (m, 5H), 2.93 (t,
2H, J¼6.8 Hz), 3.94 (br m, 2H), 4.11 (t, 3H, J¼6.8 Hz), 5.12 (t, 1H,
J¼4 Hz), 6.15 (s, 1H), 7.07e7.43 (m, 3H). 13C NMR (CDCl3, 50 MHz)
d
14.0, 18.4, 22.5, 24.6, 25.6, 28.3, 28.5, 28.7, 28.9, 29.0, 29.1, 29.2,
29.3, 29.4, 29.5, 31.8, 32.0, 33.7, 62.0, 65,6, 73.1, 113.5, 113.6, 116.7,
122.7, 124.4, 143.7, 152.2, 153.7, 160.6, 168.2, 172.8. Rf (CHCl3/EtOAc
9:1) 0.49. Anal. Calcd for C35H54O7S$1/4C6H6: C, 68.67; H, 8.76.

3162
R. Rosseto, J. Hajdu / Tetrahedron 70 (2014) 3155e3165
and one to the diacyl compound with Rf (CHCl3/EtOAc 5:1) 0.81 and
0.33, respectively, as identified by 1H NMR. The fractions containing
the monoacyl compound were combined, evaporated re-dissolved
in benzene, and freeze-dried to give 12 (1.8376 g, 68.2%) as a col-
orless oil that turned into a white waxy solid. IR (CHCl3): 3300,
then loaded directly on a silica gel column packed with CH2Cl2 and
eluted with CH2Cl2/EtOAc (5:1). The fractions containing the
product were combined, evaporated, the residue dissolved in
benzene and freeze-dried to give 16 (1.1221 g, 68%) as a white solid.
IR (CHCl3): 3350, 1728, 1679 cmꢁ1; 1H NMR (CDCl3, 200 MHz)
d
0.88
(br t, 3H), 1.25 (br s, 18H), 1.47 (m, 2H), 3.24 (m, 2H), 4.32 (m, 1H),
4.40e4.60 (m, 2H), 4.66 (s, 2H), 6.87 (m, 1H), 6.90e7.33 (m, 5H). 13
NMR (CDCl3, 50 MHz) 14.0, 22.6, 26.8, 29.2, 29.3, 29.4, 29.5, 29.6,
1738br, 1710br, 1614 cmꢁ1 1H NMR (CDCl3, 200 MHz)
; d 1.26 (br s,
16H), 1.66 (m, 4H), 2.39 (s, 3H), 2.97 (t, 2H, J¼7.2 Hz), 4.16 (t, 2H,
C
J¼6.8 Hz), 4.48 (m, 3H), 4.65 (s, 2H), 6.19 (s, 1H), 6.87e7.47 (m, 8H).
d
13C NMR (CDCl3, 50 MHz)
d
18.5, 25.6, 28.4, 28.6, 28.8, 29.1, 29.4,
31.9, 39.3, 65.1, 67.2, 70.7, 114.5, 122.0, 129.6, 157.6, 169.7. Rf (CHCl3/
EtOAc 1:1) 0.48. Anal. Calcd for C23H37NO5: C, 67.78; H, 9.15; N, 3.44.
Found: C, 67.66; H, 9.20; N, 3.17. MS MHþ C23H37NO5H calcd:
29.4, 32.1, 61.9, 65.0, 65.9, 66.5, 69.0, 113.6, 113.7, 114.6, 116.8, 121.8,
122.9, 124.4, 129.5, 143.7, 152.2, 153.8, 157.6, 160.7, 168.6, 171.7. Rf
(CHCl3/EtOAc 5:1) 0.33. Anal. Calcd for C33H42O8S: C, 66.20; H, 7.07.
Found: C, 66.49; H, 7.25. MS MNaþ C33H42O8SNa calcd: 621.2498,
408.2749, found: 408.2739. [
a
]
20 ꢁ2.4 (c 0.81, CHCl3/MeOH 4:1).
D
4.2.5.2.2. 3-(Dodecylamino)-3-oxo-2-[(tetrahydro-20H-pyran-20-
found: 621.2480. [
a]
20 ꢁ7.3 (c 0.95, CHCl3).
yl)oxy]propyl-200-phenoxyacetate. To
a cloudy solution of 16
D
4.2.5.1.2. 12-[(4-methyl-2-oxo-2H-chromen-7-yl)thio]dodecyl-3-
(2-phenoxyacetoxy)-2-[(tetrahydro-2H-pyran-2-yl)oxy]prop-
anoate. To a solution of 12 (2.4 g, 4 mmol) in 20 mL of CH2Cl2 were
added 3,4-dihydro-2H-pyran (1.7 g, 20 mmol) and PPTS (0.3 g,
1.2 mmol). The reaction mixture was over after 3 h. The mixture
was then loaded directly on a silica gel column packed with CHCl3
and eluted with CHCl3/EtOAc (8:1). The fractions corresponding to
the product were combined and evaporated, re-dissolved in ben-
zene, and freeze-dried to give the tetrahydropyranyl product
(2.6313 g, 96%) as white solid. IR (CHCl3): 2928, 2850, 1738br, 1719,
(0.6551 g, 1.6 mmol) in 5 mL of CH2Cl2 was added 3,4-dihydro-2H-
pyran (0.6762 g, 8 mmol) and PPTS (80 mg, 0.32 mmol). The re-
action mixture was stirred at room temperature for 2 h. The mix-
ture was loaded directly on a silica gel column packed with CHCl3
and eluted with CHCl3/EtOAc (4:1). The fractions containing the
product were combined, evaporated, the residue dissolved in
benzene and freeze-dried to give the product (0.7701 g, 98%) as
colorless oil. IR (CHCl3): 3340, 2926, 2850, 1729, 1682 cmꢁ1
;
1H
NMR (CDCl3, 200 MHz) 0.84 (br t, 3H), 1.22 (br s, 18H), 1.60 (m,
d
8H), 3.22 (m, 2H), 3.47 (m, 1H), 3.80 (m, 1H), 4.32 (m, 2H),
1609, 1206 cmꢁ1; 1H NMR (CDCl3, 200 MHz)
d
1.25 (br s, 16H), 1.68
4.35e4.60 (m, 4H), 6.53 (m, 1H), 6.70e7.28 (m, 5H). 13C NMR
(m, 10H), 2.38 (s, 3H), 2.96 (t, 2H, J¼7.2 Hz), 3.48 (m, 1H), 3.85 (m,
1H), 4.13 (t, 2H, J¼6.8 Hz), 4.41 (m, 1H), 4.56 (m, 2H), 4.62 (br s,
2H),4.70e4.81 (m, 1H), 6.18 (s, 1H), 6.86e7.46 (m, 8H). 13C NMR
(CDCl3, 50 MHz) d 14.0, 18.9, 20.1, 22.5, 24.8, 24.9, 25.2, 25.3, 26.7,
29.0, 29.1, 29.3, 29.4, 29.5, 30.3, 30.5, 30.8, 31.7, 39.0, 62.4, 63.9,
65.0, 65.5, 74.8, 75.5, 98.8, 99.3, 114.4, 121.6, 129.35, 157.70, 168.75.
Rf (CHCl3/EtOAc 4:1) 0.49. Anal. Calcd for C28H45NO6: C, 68.40; H,
9.23; N, 2.85. Found: C, 68.51; H, 9.35; N, 2.75. MS MNaþ
C28H45NO6Na calcd: 514.3145, found: 514.3144.
(CDCl3, 50 MHz)
d 18.5, 25.1, 25.2, 25.7, 28.4, 28.6, 28.8, 29.0, 29.1,
29.4, 29.9, 32.04, 61.85, 62.0, 64.1, 64.9, 65.0, 65.4, 65.5, 71.8, 73.5,
97.3, 98.4, 113.6, 113.7, 114.5, 114.6, 116.8, 121.7, 122.8, 124.4, 129.4,
143.7, 152.2, 153.8, 157.5, 157.6, 160.5, 168.5, 169.6. Rf (CHCl3/EtOAc
5:1) 0.88. Anal. Calcd for C38H50O9S$1/12C6H6: C, 67.08; H, 7.38.
Found: C, 67.07; H, 7.42. MS MNaþ C38H50O9SNa calcd: 705.3073,
found: 705.3100.
4.2.5.2.3. Compound (18). To a solution of 3-(dodecylamino)-3-
oxo-2-[(tetrahydro-20H-pyran-20-yl)oxy]propyl-200-phenox-
yacetate (0.7502 g, 1.53 mmol) in 8 mL of CHCl3/MeOH (5:3) cooled
at 0 ꢀC, was added tert-butylamine (0.8 mL, 7.6 mmol). The reaction
mixture was stirred at 0 ꢀC for 28 h and then the mixture was
loaded directly on a silica gel column packed with CHCl3 and eluted
with a gradient of CHCl3/EtOAc (4:1, followed by 2:1). The fractions
containing the product were combined, evaporated, dissolved in
benzene and freeze-dried to give 18 (0.4902 g, 92%) as white solid.
4.2.5.1.3. Compound (14). To a solution of 12-[(4-methyl-2-oxo-
2H-chromen-7-yl)thio]dodecyl-3-(2-phenoxyacetoxy)-2-[(tetra-
hydro-2H-pyran-2-yl)oxy]propanoate (2.5532 g, 3.73 mmol) in
8 mL mixture of CHCl3/MeOH (1:1) kept at 0 ꢀC was added tert-
butylamine (2 mL, 19 mmol). The reaction mixture was kept at 0 ꢀC
for 14 h. The mixture was purified directly by loading it on a silica
gel column packed with CHCl3 and eluted with a stepwise gradient
of CHCl3/EtOAc (9:1 and 6:1). The fractions containing the product
were combined, evaporated, re-dissolved in benzene, and freeze-
dried to give 14 as a white solid (1.9821 g, 96.7%). IR (CHCl3):
IR (CHCl3): 3350 br, 2854, 1677 cmꢁ1
0.84 (br t, 3H), 1.22 (br s, 18H), 1.54 (m, 6H), 1.82 (m, 2H), 3.22 (m,
2H), 3.49 (m, 1H), 3.70e3.80 (m, 2H), 4.17 (m, 1H), 4.52e4.63 (m,
1H), 6.57 (m, 1H), 6.92 (m, 1H). 13C NMR (CDCl3, 50 MHz)
14.0,
;
1H NMR (CDCl3, 200 MHz)
d
d
20.2, 20.6, 22.6, 24.8, 24.9, 26.7, 29.2, 29.2, 29.3, 29.4, 29.5, 30.8, 31.1,
31.8, 38.9, 62.6, 64.0, 64.2, 64.5, 77.3, 80.7, 100.2, 100.4, 169.9, 171.4.
Rf (CHCl3/EtOAc 5:2) 0.22. Anal. Calcd for C20H39NO4: C, 67.19; H,
10.99; N, 3.92. Found: C, 67.39; H, 10.92; N, 3.79. MS MHþ
C20H39NO4H calcd: 358.2964, found: 358.2946.
3300, 2855, 1735br, 1617, 1206 cmꢁ1 1H NMR (CDCl3, 200 MHz)
;
d
1.26 (br s, 16H), 1.65 (m, 10H), 2.38 (s, 3H), 2.96 (t, 2H, J¼7.2 Hz),
3.51 (m, 1H), 3.85 (m, 3H), 4.16 (t, 2H, J¼6.8 Hz), 4.20e4.40 (m, 1H),
4.65e4.81 (m, 1H), 6.18 (s, 1H), 7.10e7.46 (m, 3H). 13C NMR (CDCl3,
50 MHz) d 18.5, 18.7, 25.1, 25.7, 28.4, 28.6, 28.8, 29.0, 29.1, 29.4, 30.2,
30.3, 32.0, 62.6, 62.7, 62.9, 65.1, 65.2, 75.9, 77.2, 98.5, 98.8, 113.6,
116.8, 122.8, 124.4, 143.7, 152.2, 153.8, 160.6, 170.4, 170.8. Rf (CHCl3/
EtOAc 5:1) 0.25. Anal. Calcd for C30H44O7S: C, 65.66; H, 8.08. Found:
C, 65.89; H, 8.13. MS MNaþ C30H44O7SNa calcd: 571.2705, found:
571.2687.
4.3. General procedures for phosphorylation of the
1,2-disubstituted glyceric acid derivatives
4.3.1. 3-(Dodecyloxy)-2-[(100-(700-mercapto-400methylcoumarin-7yl)
decanoyl)oxy]-3-(oxopropyl)-phosphocholine (19). To a solution of
13 (0.2802 g, 0.46 mmol) in 40 mL freshly distilled benzene par-
tially submerged in an ice-bath was added 2-chloro-2-oxo-1,2,3-
4.2.5.2. N-Dodecyl-3-hydroxy-2-[(tetrahydro-2H-pyran-2-yl)oxy]
propanamide (18). 4.2.5.2.1. 3-(Dodecylamino)-2-hydroxy-3-
oxopropyl-20-phenoxyacetate (16). To a stirred suspension of 10
(1.1061 g, 4.05 mmol) in 60 mL CHCl3, kept in ice-water bath was
added phenoxyacetyl chloride (0.85 mL, 6.07 mmol), followed by
slow drop-wise addition of a solution of 2,4,6-collidine (0.7355 g,
6.07 mmol) in 10 mL of CHCl3, over 30 min. After 2 h of reaction
more collidine (0.2879 g, 2.4 mmol) was added in 5 mL of CHCl3.
One hour later the mixture became clear. The reaction mixture was
then left stirring at room temperature overnight. The solution was
dioxaphospholane (130
triethylamine (80 L, 0.57 mmol) in 10 mL benzene, added drop-
wise. Eight hours later more phosphorylating agent was added
(150 L, 1.6 mmol) and the reaction mixture was stirred at room
mL, 1.4 mmol) followed by a solution of
m
m
temperature overnight. The precipitate that formed was filtered,
and the filtrate was evaporated to give a white waxy residue. This
residue was dispersed in 25 mL of anhydrous acetonitrile, and the
dispersion was transferred to pressure bottle and cooled to ꢁ10 ꢀC.
To this mixture was added excess trimethylamine (4 mL), the

R. Rosseto, J. Hajdu / Tetrahedron 70 (2014) 3155e3165
3163
pressure bottle was sealed, heated to 65 ꢀC, and kept for 24 h.
Cooling to room temperature and later in an ice-bath led to for-
mation of a white precipitate. This precipitate was filtered, washed
with 3ꢂ20 mL cold acetonitrile, and chromatographed on silica gel
with a solution of CHCl3/MeOH/H2O (65:25:4). The combined
CH3CN phase was evaporated to give an oily residue, which was
dissolved in the mixture of CHCl3/MeOH/H2O (65:25:4) and puri-
fied on a separate silica gel column packed with CHCl3/MeOH (4:1)
and eluted with CHCl3/MeOH/H2O (65:25:4). The fractions con-
taining the product were collected, evaporated, dispersed in ben-
zene, and freeze-dried to give 19 as a white solid (in a combined
obtained was dissolved in 25 mL of CH3CN, the solution was
transferred to pressure bottle and frozen in a dry ice-bath at ꢁ10 ꢀC.
To this solid was added excess trimethylamine (5 mL), and then the
pressure bottle was sealed, heated to 65 ꢀC, and kept for 60 h. After
60 h, the pressure bottle was cooled to room temperature, and then
in an ice-bath. A white precipitate formed on the walls of pressure
bottle. The solvent was evaporated, and the residue in combination
with the precipitate was dissolved in CHCl3/MeOH/H2O (65:25:4)
and loaded on a silica gel column packed with the same solvent,
and eluted with CHCl3/MeOH/aq NH3 (1:9:1). The fractions con-
taining the product were collected, evaporated, dissolved in ben-
zene, and freeze-dried to give 21 (0.4372 g, 67%) as white solid. IR
yield of 0.1695 g, 48%). IR (CHCl3): 3330, 1731br, 1632, 1206 cmꢁ1
;
1H NMR (CDCl3þCD3OD, 200 MHz)
d
0.79 (br t, 3H), 1.18 (br s, 26H),
(CHCl3): 3312, 2852, 1672 cmꢁ1 1H NMR (CDCl3, 200 MHz)
; d 0.86
1.58 (m, 8H), 2.21 (t, 2H, J¼7.2 Hz), 2.35 (s, 3H), 2.91 (t, 2H,
(br t, 3H), 1.24 (br s, 18H), 1.52 (m, 6H), 1.77 (m, 2H), 3.10 (m, 2H),
J¼6.8 Hz), 3.15 (br s, 9H), 4.07 (m, 4H), 4.25e4.38 (m, 4H), 4.87 (m,
3.36 (br s, 10H), 3.79 (m, 2H), 3.95e4.15 (m, 2H), 4.28 (m, 4H), 4.81
1H), 6.15 (s, 1H), 7.08e7.27 (m, 3H). 13C NMR (CDCl3, 50 MHz)
d
8.6,
(m, 1H), 7.00 (m, 1H). 13C NMR (CDCl3, 50 MHz)
d 14.9, 17.3, 20.1,
14.1, 18.5, 22.6, 24.7, 25.8, 28.5, 28.7, 28.8, 29.0, 29.2, 29.3, 29.5, 29.6,
31.8, 32.1, 33.9, 45.7, 54.4, 59.8, 64.5, 65.6, 66.0, 72.2, 113.6, 116.8,
122.8, 124.5, 143.7, 152.3, 153.8, 160.6, 169.2, 173.1. 31P NMR (CDCl3,
22.6, 25.1, 27.4, 29.3, 29.6, 30.6, 31.8, 39.2, 52.9, 53.4, 55.8, 59.7, 63.5,
67.2, 74.9, 100.0, 170.1. 31P NMR (CDCl3, 160 MHz, pyrophosphate
ref. ext.)
d
ꢁ0.62 Rf (CHCl3/MeOH/H2O 65:25:4) 0.25. Anal. Calcd for
160 MHz, pyrophosphate ref. ext.)
d
ꢁ2.91. Rf (CHCl3/MeOH/H2O
C25H51N2O7P$H2O: C, 55.54; H, 9.88; N, 5.18. Found: C, 55.18; H,
65:25:4) 0.45. Anal. Calcd for C40H66NO10PS$2H2O: C, 58.59; H,
9.83; N, 5.14. MS MHþ C25H51N2O7PHþ calcd: 523.3512, found:
523.3503.
8.60; N, 1.71. Found: C, 58.91; H, 8.47; N, 1.94. MS MHþ
20
C40H66NO10PSH calcd: 784.4223, found: 784.4206. [
1.09, CHCl3/MeOH 4:1).
a]
ꢁ6.4 (c
D
4.3.4. 3-[(120-(700-Mercapto-400-methylcoumarin-700yl))dodecyloxy]-
3-oxo-2-[(tetrahydro-2H-pyran-2-yl)oxy]propyl
phosphocholine
4.3.2. 3-(Dodecylamino)-2-[(100-(700-mercapto-400-methylcoumarin-
700yl)decanoyl)oxy]-3-(oxopropyl)-phosphocholine (20) (route I). To
a suspension of 17 in 25 mL freshly distilled benzene, partially
submerged in an ice-water bath was added 2-chloro-2-oxo-1,2,3-
(23). To a solution of 14 (0.9651 g, 1.76 mmol) in 30 mL benzene in
an ice-bath were added 2-chloro-2-oxo-1,2,3-dioxaphospholane
(0.32 mL, 3.5 mmol) followed by triethylamine (0.5 mL,
3.5 mmol) drop-wise. After the addition of NEt3, the ice-bath was
removed and the reaction mixture was left stirring at room tem-
perature for 4 h. The mixture was filtered and the crystalline pre-
cipitate of triethylamine hydrochloride was removed. The solvent
was evaporated and the oily residue was dissolved in 45 mL of
anhydrous acetonitrile, the resulting solution was transferred to
a pressure bottle and frozen to ꢁ10 ꢀC. To this frozen solution was
added excess trimethylamine (5 mL), the pressure bottle was then
sealed and heated to 65 ꢀC for 24 h. After that the mixture was
cooled to room temperature, and then kept at 7 ꢀC overnight, when
a white precipitate formed. The precipitate was separated from the
acetonitrile solution, which was then evaporated to give an oily
residue. Both samples were purified by silica gel chromatography
using separate columns, packed with CHCl3/MeOH (4:1) and eluted
with In both cases, with CHCl3/MeOH/H2O (65:25:4) the fractions
corresponding to the product were isolated, evaporated, dispersed
in benzene, and freeze-dried to give 23 as a white solid (overall
yield: 0.9217 g 74%, from precipitate 0.7201 g, and from the MeCN
phase 0.2016 g). IR (CHCl3): 3350, 2852, 1732, 1621, 1207 cmꢁ1; 1H
dioxaphospholane (70
triethylamine (82 L, 0.60 mmol) in 5 mL benzene drop-wise. Six
hours later, more phosphorylating agent was added (30 L,
mL, 0.57 mmol) followed by a solution of
m
m
0.32 mmol) to this mixture. The reaction mixture was stirred at
room temperature for 22 h, then the mixture was filtered and the
solvent was evaporated to give a white residue. This residue was
dispersed in 25 mL MeCN, the dispersion was transferred to pres-
sure bottle and cooled to ꢁ10 ꢀC, followed by addition of trime-
thylamine (2 mL). The pressure bottle was sealed and heated to
65 ꢀC for 48 h. Cooling to room temperature and then with an ice-
bath yielded a white precipitate, and the acetonitrile phase was
evaporated to give an oily residue. The precipitate and the oily
residue were purified on separate silica gel columns, packed with
CHCl3/MeOH (4:1), and eluted with CHCl3/MeOH/H2O (65:25:4).
Both columns yielded identical product 20 as a white solid (overall
0.211 g, 54%). IR (CHCl3): 3340br, 1728, 1656, 1210 cmꢁ1
;
1H NMR
(CDCl3, 200 MHz) 0.83 (br t, 3H),1.20 (br s, 32H),1.52 (m, 2H), 2.38
d
(s, 3H), 2.94 (t, 2H, J¼7.4 Hz), 3.10 (br s, 9H), 3.20e3.35 (m, 6H), 3.72
(m, 2H), 4.22 (m, 2H), 5.25 (m, 1H), 6.17 (s, 1H), 7.08e7.12 (m, 2H),
NMR (CDCl3, 200 MHz) d 1.27 (br s, 16H), 1.66 (m, 10H), 2.39 (s, 3H),
7.41 (d, 1H, J¼8 Hz). 13C NMR (CDCl3, 50 MHz)
d
8.8, 14.3, 19.8, 22.8,
2.97 (t, 2H, J¼7.2 Hz), 3.36 (br s, 9H), 3.80 (m, 6H), 4.10 (t, 2H,
24.9, 27.1, 28.8, 29.0, 29.3, 29.5, 29.6, 29.7, 29.8, 29.9, 32.1, 34.1, 39.6,
46.0, 54.5, 59.8, 64.7, 65.1, 73.2, 113.8, 117.0, 123.0, 124.7, 144.0,
152.6, 154.0, 160.9, 167.7, 173.9, 173.7. 31P NMR (CDCl3, 160 MHz,
J¼6.8 Hz), 4.31 (m, 3H), 4.80 (m, 1H), 6.19 (s, 1H), 7.12e7.43 (m, 3H).
13C NMR (CDCl3, 50 MHz)
d 18.5, 19.1, 25.2, 25.8, 25.9, 28.5, 28.6,
28.9, 29.1, 29.3, 29.5, 32.1, 54.3, 59.4, 62.3, 65.2, 66.2, 98.9, 113.7,
pyrophosphate ref. ext.)
d
ꢁ1.4 br. Rf (CHCl3/MeOH/H2O 65:25:4)
116.9, 122.9, 124.5, 143.8, 152.2, 153.8, 160.6, 170.7. 31P NMR (CDCl3,
0.45. Anal. Calcd for C40H67N2O9PS$CHCl3: C, 54.57; H, 7.60; N, 3.10.
160 MHz, pyrophosphate external reference)
d
ꢁ1.71 and ꢁ1.07. Rf
Calcd for
Found: C, 54.84; H, 7.41; N, 3.93. MS MHþ C40H67N2O9PSH calcd:
(CHCl3/MeOH/H2O 65:25:4) 0.31. Anal.
20
783.4378, found: 783.4331. [
a
]
ꢁ4.7ꢀ (c 1.11, CHCl3/MeOH 4:1).
C35H56NO10PS$2H2O$CHCl3: C, 49.74; H, 7.07; N, 1.61. Found: C,
D
49.77; H, 7.07; N, 1.52. MS MNaþ C35H56NO10PSNa calcd: 736.3260,
found: 736.3239.
4.3.3. 3-(Dodecylamino)-3-oxo-2-[(tetrahydro-2H-pyran-2-yl)oxy]
propyl phosphocholine (21). To solution of 18 (0.4462 g,
a
1.25 mmol) in 15 mL freshly distilled benzene, partially submerged
in an ice-bath, was added 2-chloro-2-oxo-1,2,3-dioxaphospholane
(0.17 mL, 1.86 mmol) followed by a solution of triethylamine
(0.26 mL, 1.86 mmol). After addition of NEt3, the ice-bath was re-
moved and the mixture was stirred for 8 h at room temperature.
The mixture was filtered and the residue collected was washed
with benzene. The solvent was evaporated and the oily residue
4.4. General procedure for hydrolytic cleavage of the tetra-
hydropyranyl protecting group to produce the lysophospho-
lipid analogues
4.4.1. 3-(Dodecylamino)-2-hydroxy-3-oxopropyl
phosphocholine
(22). To a cloudy solution of 21 (0.3521 g, 0.67 mmol) in 25 mL 1,4-
dioxane was added 0.15 mL 12 M aq HCl. The reaction mixture was

3164
R. Rosseto, J. Hajdu / Tetrahedron 70 (2014) 3155e3165
stirred at room temperature for 2 h. At the end of the reaction
30 mL dioxane was added, and then the reaction mixture was
freeze-dried. The white residue obtained was dissolved in CHCl3/
MeOH/H2O (65:25:4) and purified on a short silica gel column
packed with CHCl3 and eluted with CHCl3/MeOH/H2O (65:25:4).
The fractions containing the product were collected, evaporated,
dispersed in benzene, and freeze-dried to give 22 (0.2762 g, 94%) as
white solid. IR (CHCl3): 3352, 1675 cmꢁ1; 1H NMR (CDCl3, 200 MHz)
and purified on silica gel column packed with CHCl3 and eluted first
with CHCl3/MeOH (4:1) to remove the impurities, and then with
CHCl3/MeOH/H2O (65:25:4) to elute the product. The fractions
containing the product were combined, evaporated, dispersed in
benzene, and freeze-dried to give 25 (0.5020 g, 60%) as white solid.
IR (Nujol): 3335, 1730 br, 1690, 1608, 1207 cmꢁ1
;
1H NMR (CDCl3,
1.23 (br s, 30H), 1.62 (m, 8H), 2.34 (br s, 5H), 2.93 (t, 2H,
200 MHz)
d
J¼7.2 Hz), 3.16 (br m, 2H), 3.35 (br s, 9H), 3.80 (m, 2H), 4.06e4.12
(m, 2H), 4.25e4.40 (m, 5H), 4.35 (m, 2H), 5.09 (m, 1H), 5.20 (m, 1H),
6.15 (s, 1H), 7.07e7.42 (m, 7H), 7.58 (d, 2H, J¼7.3 Hz), 7.75 (d, 2H,
d
0.86 (br t, 3H), 1.24 (br s, 16H), 1.52 (m, 2H), 3.33 (br s, 13H), 3.72
(m, 3H), 4.21 (m, 4H). 13C NMR (CDCl3, 50 MHz)
14.0, 22.6, 27.1,
29.3, 29.5, 29.7, 31.9, 39.2, 54.2, 59.7, 65.9, 68.2, 71.8, 171.6. 31P NMR
(CDCl3, 160 MHz, pyrophosphate ref. ext.)
d
J¼7.3 Hz). 13C NMR (CDCl3, 50 MHz)
d 18.4, 24.6, 25.6, 26.7, 28.4,
d
ꢁ1.01. Rf (CHCl3/MeOH/
28.5, 28.8, 29.0, 29.2, 29.4, 29.9, 32.0, 33.8, 41.0, 47.2, 54.3, 59.3, 64.1,
65.7, 66.4, 72.2, 113.5, 116.8, 119.8, 122.8, 124.4, 124.9, 126.9, 127.5,
141.1, 143.9, 152.2, 153.7, 156.3, 160.6, 168.1, 173.0. 31P NMR (CDCl3,
H2O 65:25:4) 0.25. Anal. Calcd for C20H43N2O6P$1.5H2O: C, 51.60; H,
9.96; N, 6.02. Found: C, 51.49; H, 9.99; N, 6.06. MS MHþ
C20H43N2O6PH calcd: 439.2937, found: 439.2927. [
CHCl3/MeOH 4:1).
a
]
D
20 ꢁ5.4 (c 1.12,
160 MHz, pyrophosphate reference)
d
ꢁ1.07. Rf (CHCl3/MeOH/H2O
65:25:4) 0.44. Anal. Calcd for C57H81N2O12PS$0.5H2O: C, 64.69; H,
7.81; N, 2.65. Found: C, 64.75; H, 7.62; N, 3.16. MS MHþ
4.4.2. 2-Hydroxy-3-[(120-(700-mercapto-400-methylcoumarin))dode-
cyloxy]-3-oxopropyl phosphocholine (24). To solution of 23
(0.1202 g, 0.17 mmol) in 2 mL of CHCl3 was added 50 L of 12 M
C57H81N2O12PSH calcd: 1049.5326, found: 1049.5314. [
0.78, CHCl3/MeOH 4:1).
a
]
20 ꢁ2.4 (c
D
a
m
aq HCl. The reaction mixture was stirred at room temperature for
2 h. At the end of the reaction to this solution was added 100 mL
benzene, and the mixture was freeze-dried to give a white residue.
This residue was dissolved in CHCl3/MeOH/H2O (65:25:4) and pu-
rified on a silica gel column packed with CHCl3/MeOH (4:1) and
eluted with CHCl3/MeOH/H2O (65:25:4). The fractions containing
the product were isolated, evaporated, dispersed in benzene, and
freeze-dried to give 24 (62 mg, 57%) as white solid. IR (CHCl3): 3330,
4.5.3. 2-((12-(7-(Diethylamino)-2-oxo-2H-chromene-3-
carboxamido)dodecanoyl)oxy)-3-((12-((4-methyl-2-oxo-2H-chro-
men-7-yl)thio)dodecyl)oxy)-3-oxopropyl phosphocholine (26). To
a solution of 25 (0.1309 g, 0.125 mmol) in 5 mL CHCl3 was added
DBU (0.1021 g, 0.66 mmol), and the reaction mixture was stirred
at room temperature for 1 h, when cleavage of the FMOC protecting
group was completed. To this solution was then added p-nitro-
phenyl ester of 7-N,N-diethylaminocoumarin-3-carboxylate (0.095
g, 0.25 mmol) and DMAP (30 mg, 0.25 mmol). The reaction was
stopped after 4 h, when the negative ninhydrin test indicated that
acylation of the chain-terminal amino group reached completion.
The mixture was loaded directly on a silica gel column packed with
CHCl3 and eluted with CHCl3/MeOH (4:1) to remove the impurities,
and then with CHCl3/MeOH/H2O (65:25:4). The fractions contain-
ing the product were combined, evaporated, dispersed in benzene,
and freeze-dried to give 26 (87.8 mg, 66%) as yellow solid. IR
(CHCl3): 3331, 1732 br, 1598, 1208 cmꢁ1; 1H NMR (CDCl3, 200 MHz)
1737 br, 1615, 1205 cmꢁ1; 1H NMR (CDCl3þCD3OD, 200 MHz)
d 1.28
(br s,16H),1.69 (m, 6H), 2.64 (s, 3H), 2.98 (t, 2H, J¼7.2 Hz), 3.24 (br s,
9H), 3.36 (s, 1H), 3.66 (br s, 2H), 3.68 (m, 2H), 4.35 (m, 1H), 6.23 (s,
1H), 7.18e7.56 (m, 3H). 13C NMR (CDCl3, 50 MHz)
d 18.4, 25.6, 28.5,
28.7, 29.0, 29.3, 29.5, 30.5, 32.0, 54.2, 59.2, 66.3, 113.3, 113.6, 116.1,
122.9, 124.5, 143.9, 152.8, 153.6, 161.2. 31P NMR (CDCl3, 160 MHz,
pyrophosphate external reference)
d
ꢁ1.16. Rf (CHCl3/MeOH/H2O
65:25:4) 0.31. Anal. Calcd for C30H48NO9PS$5/2H22O0: C, 53.40; H,
7.92; N, 2.08. Found: C, 53.32; H, 7.56; N, 1.71. [
CHCl3/MeOH 4:1).
a
]
D
ꢁ3.7 (c 0.94,
d
1.25 (br s, 36H), 1.61 (m, 8H), 2.39 (br s, 5H), 2.97 (t, 2H, J¼7.2 Hz),
3.36 (br m, 16H), 3.80 (m, 2H), 4.10 (m, 2H), 4.20e4.35 (m, 4H), 5.22
(m, 1H), 6.18 (s, 1H), 6.46 (s, 1H), 6.65 (d, 1H, J¼6.8 Hz), 7.09e7.47
4.5. General procedure for acylation of the 2-hydroxy group
of the lysophospholipid analogues
(m, 5H), 8.76 (s, 1H), 8.78 (m, 1H). 13C NMR (CDCl3, 50 MHz)
d 12.4,
18.5, 24.7, 25.7, 27.0, 28.4, 28.6, 28.8, 29.1, 29.2, 29.3, 29.5, 32.1, 33.8,
39.6, 45.0, 54.5, 59.3, 64.2, 65.7, 66.5, 72.1, 96.5, 108.3, 109.9, 110.4,
113.6,116.8, 122.8,124.5, 131.0, 143.8,147.9, 152.3,153.8,157.5, 160.7,
162.7, 168.2, 173.0. 31P NMR (CDCl3, 160 MHz, pyrophosphate ref.
4.5.1. .Compound (20) (route II). To a solution of (22) (0.2497 g,
0.57 mmol) in 15 mL of CHCl3 were added 10-(70-mercapto-40-
methyl-70-yl)decanoic acid (0.4129 g, 1.14 mmol), DCC (0.2352 g,
1.14 mmol), DMAP (0.1393 g, 1.14 mmol), and 0.5 g of glass beads.21
The mixture was sonicated for 5 h at 25 ꢀC. The glass beads and the
DCCeurea were filtered off, and the filtrate was evaporated. The
residue was dissolved in CHCl3/MeOH (4:1) and purified on silica
gel column packed with CHCl3, and eluted first with CHCl3/MeOH
(4:1), to remove the impurities, and then with CHCl3/MeOH/H2O
(65:25:4) to elute the product. The fractions containing the product
were combined, evaporated, dispersed in benzene, and freeze-
dried to give 20 (0.2767 g, 0.35 mmol, 62%) as white solid (ana-
lytical data are described above, at the synthesis of compound 20 in
route I).
ext.)
d
ꢁ1.11. Rf (CHCl3/MeOH/H2O 65:25:4) 0.45. Anal. Calcd for
C
56H86N3O13PS$2H2O: C, 60.68; H, 8.18; N, 3.79. Found: C, 60.83; H,
7.83; N, 3.88. MS [MꢁH]þ C56H86N3O13PSꢁHþ calcd: 1070.5540,
found: 1070.5573. [
a
]
20 ꢁ3.7 (c 0.81, CHCl3/MeOH 4:1).
D
4.5.4. 3-((12-((4-Methyl-2-oxo-2H-chromen-7-yl)thio)dodecyl)oxy)-
2-(120-((100-pyrrolidinyloxy-300-carboxy)dodecyl)oxy)-3-oxopropyl
phosphocholine (27). Yield: 52%. IR (CHCl3): 3349, 1739br,
1210m cmꢁ1; Rf (CHCl3/MeOH/H2O 65:25:4) 0.50. Anal. Calcd for
C
51H85N3O12PS$3/2H2O: C, 59.92; H, 8.68; N, 4.11. Found: C, 60.09;
H, 8.69; N, 3.82. MS MHþ C51H85N3O12PSH calcd: 995.5664, found:
995.5715. [
a
]
20 ꢁ3.9 (c 0.92, CHCl3/MeOH 4:1).
D
4.5.2. (2-[(120-[(900H-Fluoren-9-yl)methoxycarbonyl]amino]dodeca-
noyl)oxy)-3-(12-((7-mercapto-4-methylcoumarin-7-yl)dodecyl)oxy)-
3-oxopropyl phosphocholine (25). To a solution of 24 (0.4995 g,
0.8 mmol) in 25 mL CHCl3 were added 12-(9-fluorenylmethoxy
carbonyl)-N-aminododecanoic acid (0.9987 g, 2.3 mmol), DCC
(0.6621 g, 3.2 mmol), DMAP (0.4012 g, 3.2 mmol), and 1 g of glass
beads,21 and the mixture was sonicated for 5 h at 25 ꢀC. The glass
beads and the DCC/urea were filtered off, the filtrate was evapo-
rated, and the residue obtained was dissolved in CHCl3/MeOH (4:1)
4.5.5. 2-((12-(Ferrocenyl)carboxamidododecyl)oxy)-2-oxo-3-((12-
((4-methyl-2-oxo-2H-chromen-7-yl)thio)dodecyl)oxy)-3-oxopropyl
phosphocholine (28). Yield: 58%. IR (CHCl3): 3350, 1735br, 1650,
1540m, 1208 cmꢁ1 1H NMR (CDCl3, 200 MHz)
; d 1.24 (br s, 30H),
1.58 (m, 8H), 2.37 (br s, 5H), 2.94 (t, 2H, J¼7.2 Hz), 3.34 (br m, 10H),
3.80 (m, 2H), 4.05e4.16 (br s, 12H), 4.28 (br s, 4H), 4.69 (br s, 2H),
5.22 (m, 1H), 6.17 (br s, 1H), 7.04e7.55 (m, 3H). 13C NMR (CDCl3,
50 MHz) d 15.0, 18.5, 24.7, 25.8 26.9, 28.4, 28.6, 28.8, 29.0, 29.4, 29.9,

R. Rosseto, J. Hajdu / Tetrahedron 70 (2014) 3155e3165
3165
4. (a) Murakami, M.; Taketomi, Y.; Sato, H.; Yamamoto, K. J. Biochem. 2011, 150,
233e255; (b) Murakami, M.; Taketomi, Y.; Miki, Y.; Sato, H.; Hirabayashi, T.;
Yamamoto, K. Prog. Lipid Res. 2011, 50, 152e192.
5. Lambeau, G.; Gelb, M. H. Annu. Rev. Biochem. 2008, 77, 495e520.
6. Funk, C. D. Science 2001, 294, 1871e1875.
7. Morris, A. J.; Smyth, S. S. J. Lipid Res. 2013, 54, 1153e1157.
8. Prescott, S. A.; Zimmerman, G. A.; Stafforini, D. M.; McIntire, T. M. Annu. Rev.
Biochem. 2000, 69, 419e445.
9. Murakami, M.; Lambeau, G. Biochimie 2013, 95, 43e50.
10. Schaloske, R. H.; Dennis, E. A. Biochim. Biophys. Acta 2006, 1761, 1246e1259.
11. Rosseto, R.; Tcacenco, C. M.; Ranganathan, R.; Hajdu, J. Tetrahedron Lett. 2008,
49, 3500e3503.
32.0, 33.7, 38.4, 54.4, 59.4, 65.8, 68.0, 69.6, 70.2, 72.0, 113.5, 116.8,
122.8, 124.5, 143.8, 152.3, 153.8, 160.6, 168.1, 168.7, 170.0, 173.0. 31P
NMR (CDCl3, 160 MHz, pyrophosphate ref. ext.): no signal was ob-
served, a broad area around zero. Rf (CHCl3/MeOH/H2O 65:25:4)
0.57. Anal. Calcd for C53H79FeN2O11PS$2H2O: C, 59.21; H, 7.78; N,
2.61. Found: C, 59.28; H, 7.70; N, 2.25. MS MHþ C53H79FeN2O11PSH
20
calcd: 1039.4569, found: 1039.4593. [
MeOH 4:1).
a]
ꢁ4.4ꢀ (c 0.95, CHCl3/
D
4.6. Enzymatic hydrolysis of the phospholipids 19, 20, 26e28
12. Linderoth, L.; Andresen, T. L.; Jorgensen, K.; Madsen, R.; Peters, G. H. Biophys. J.
2008, 94, 14e26.
13. (a) Satoh, T.; Hosokawa, M. Annu. Rev. Pharmacol. Toxicol. 1998, 38, 257e288; (b)
Chahinian, H.; Sarda, L. Protein Pept. Lett. 2009, 16, 1149e1161; (c) Yang, Y.;
Babiak, P.; Reymond, J.-L. Org. Biomol. Chem. 2006, 4, 1746e1754.
14. Wang, M.; Pinnamaraju, S.; Ranganathan, R.; Hajdu, J. Chem. Phys. Lipids 2013,
172, 78e85.
15. (a) Chen, H.-J.; Chen, C.-Y.; Gao, P.; Wu, Y. Tetrahedron 2013, 69, 9848e9851; (b)
Chen, C.-Y.; Han, W.-B.; Chen, H.-J.; Wu, Y.-K.; Gao, P. Eur. J. Org. Chem. 2013,
4311e4318.
In a typical experiment to a sample of phosphocholine (2.5 mg,
2.5 mmol) was added to a solution of 4.1 mL Tris buffer (0.05 M, pH
8.50), containing 0.1 mL Triton X-100 and 50 mM CaCl2. The mix-
ture was vortexed, followed by incubation of the resulting disper-
sion at 40 ꢀC for 10 min in a constant temperature water bath. To
the optically clear dispersion that resulted was added bee-venom
16. Ishihara, K.; Kurihara, H.; Yamamoto, H. J. Org. Chem. 1993, 58, 3791e3793.
17. Under the experimental conditions, keeping the temperature at 0ꢀ C, and with
the slow rate of addition of the catalyst, the reaction proceeded with high re-
gioselectivity, producing 30% of the 2,3-(bis)phenoxyacetyl ester as the only
byproduct that could easily be separated by silica gel chromatography
18. The tetrahydropyranyl compounds were isolated as diastereomeric mixtures
and they were used as such in the subsequent step for phosphorylation of the
primary hydroxyl group
19. (a) Kim, U. T.; Hajdu, J. J. Chem. Soc., Chem. Commun. 1993, 70e72; (b) Marx, M.
H.; Piantadosi, C.; Noseda, A.; Daniel, L. W.; Modest, E. J. J. Med. Chem. 1988, 31,
858e863.
20. The phospholipids were obtained in high yield, however, isolation of the
products purified by silica gel chromatography frequently results in substantial
losses in recovery, because of irreversible adsorption of phospholipids on the
silica gel columns, and yields of 50e70% are usually realized; see: Blank, M. L.;
Robinson, M.; Snyder, F. In Platelet Activating Factor and Related Lipid Mediators;
Snyder, F., Ed.; Plenum: New York, NY, 1987; pp 33e52.
phospholipase A2 (8 mg in 45 mL buffer) to initiate the reaction.
The reaction mixture was kept at 40 ꢀC, and formation of the
products was analyzed by thin layer chromatography, (CHCl3/
MeOH/H2O, 65:25:4). The compounds were visualized by UV-
absorption, fluorescence, iodine adsorption, and molybdic acid
spray. TLC analysis showed complete hydrolysis for each substrate
in the series of the synthetic phospholipids. Identity of the products
was confirmed with authentic samples of the lysophospholipids
and the labeled fatty acids.
Acknowledgements
We are grateful to the National Institutes of Health, grant
1SC3GM096878 for financial support.
21. Rosseto, R.; Hajdu, J. Tetrahedron Lett. 2005, 46, 2941e2944.
22. Ranganathan, R.; Tcacenco, C. M.; Rosseto, R.; Hajdu, J. Biophys. Chem. 2006, 122,
79e89.
23. Correia-Ledo, D.; Arnold, A. A.; Mauzeroll, J. J. Am. Chem. Soc. 2010, 132,
15120e15123.
References and notes
24. Singh, J.; Ranganathan, R.; Hajdu, J. Anal. Biochem. 2010, 407, 253e260.
25. Balet, C.; Clingman, K. A.; Hajdu, J. Biochem. Biophys. Res. Commun. 1988, 150,
561e567.
1. Bhattacharya, S.; Biswas, J. Langmuir 2010, 26, 4642e4654.
2. Skaug, M. J.; Longo, M. L.; Faller, R. J. Phys. Chem. B 2011, 115, 8500e8505.
3. Dennis, E. A.; Cao, J.; Hsu, H. Y.; Magrioti, V.; Kotokos, G. Chem. Rev. 2011, 111,
6130e6185.
26. Dittmer, J. C.; Lester, R. L. J. Lipid Res. 1964, 5, 126e127.