Stereospecific synthesis of phosphatidylglycerol using a cyanoethyl phosphoramidite precursor

Article abstract of DOI:10.1016/j.chemphyslip.2020.104933

Phosphatidylglycerols (PG) are a family of naturally occurring phospholipids that are responsible for critical operations within cells. PG are characterized by an (R) configuration in the diacyl glycerol backbone and an (S) configuration in the phosphoglycerol head group. Herein, we report a synthetic route to provide control over the PG stereocenters as well as control of the acyl chain identity.

Full text of DOI:10.1016/j.chemphyslip.2020.104933

ChemistryandPhysicsofLipids231(2020)104933
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
Short communication
Stereospecific synthesis of phosphatidylglycerol using a cyanoethyl
phosphoramidite precursor
Zachary J. Struzika, Ashley N. Weertsa, Judith Storchb, David H. Thompsona,*
a Department of Chemistry, Purdue University, Multi-disciplinary Cancer Research Facility, Bindley Bioscience Center, 1203 W. State Street, West Lafayette, IN 47907,
United States
b Department of Nutritional Sciences and Rutgers Center for Lipid Research, Rutgers University, New Brunswick, NJ 08901, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
Phosphatidylglycerols (PG) are a family of naturally occurring phospholipids that are responsible for critical
operations within cells. PG are characterized by an (R) configuration in the diacyl glycerol backbone and an (S)
configuration in the phosphoglycerol head group. Herein, we report a synthetic route to provide control over the
PG stereocenters as well as control of the acyl chain identity.
Stereospecific synthesis
Phosphatidylglycerol
Phosphoramidite methodology
Dioleoyl glycerophospholipid
1. Introduction
utilized in other cellular activities such as electron transport in photo-
synthesis, attenuation of inflammatory responses, lipoprotein matura-
Glycerophospholipids represent a diverse class of biological mole-
cules that play vital roles in all living systems including cell membrane
structure, vesicular transport, and intracellular signaling (Dowhan
et al., 2008). PL are generally characterized by a glycerol moiety
phosphorylated at the sn-3 position and fatty acid ester linkages in the
sn-1 and sn-2 positions of the DAG. Deficiencies in PL biogenesis and
metabolism have been attributed to a host of inherited and acquired
diseases including mitochondrial disorders, breast cancer, neurode-
generation, and obesity (Anjani et al., 2015; Lu and Claypool, 2015;
Mesa-Herrera et al., 2019; Yang et al., 2015). Interestingly, PL have also
been used successfully as drug delivery vehicles over the past few
decades for numerous anticancer drugs, several of which are FDA ap-
proved (Savla et al., 2017; Yingchoncharoen et al., 2016).
PG are a subset of glycerophospholipids that are distinguished by
the presence of an (R)-glycerylphosphoester linkage in the lipid head-
group region (Fig. 1), originally discovered in Scenedesmus (Benson and
Maruo, 1958). PG are suspected as biosynthetic precursors to a range of
PL including cardiolipin, bis(monoacylglycerol)phosphate (BMP; also
known as lysobis phosphatidic acid or LBPA), bacterial proteolipids,
lipoteichoic lipids, and other glycophospholipids (Emdur and Chiu,
1975; Poorthuis and Hostetler, 1975; Stillwell, 2016). They are also
tion, and extracellular stress sensing (Dugail et al., 2017). This wide use
of PG by cells makes it an important PL whose roles in fundamental
mechanisms of cellular processes and disease pathology need better
elucidation. Naturally occurring PG is phosphorylated at the sn-3 and
sn-1’positions of the two glycerol motifs making enantiomerically pure
PG essential for biological studies. Syntheses of PG has previously been
demonstrated (Burugupalli et al., 2017; Murakami et al., 1999; Sato
et al., 2004). Herein, we report an improved synthetic route designed to
afford diastereochemically pure PG using two chiral starting materials,
(R)-solketal and (S)-solketal while maintaining control over acyl chain
identity.
Our strategy for the synthesis of sn-3:sn-1’ PG 1 was based on a
convergent synthesis employing DAG and phosphoglycerol headgroup
intermediates, each with stereochemistries that were fixed via the
corresponding solketal precursors (Scheme 1). Global deprotection of
protected PG intermediate 2 using a naked fluorine source can be uti-
lized to synthesize 1. The stereospecific phosphorylation of the DAG 3
and glycerol headgroup 4 can be realized through coupling of the re-
spective glycerol fragments and a PIII phosphoramidite reagent com-
monly employed in DNA oligonucleotide synthesis and other phos-
pholipid syntheses (Hughes and Ellington, 2017; Keith and Townsend,
Abbreviations: PG, Phosphatidyl glycerols; BMP, Bis(monoacylglycerolphosphate); PMB, p-methoxybenzylether; DAG, Diacylglycerol; DMF, Dimethylformamide;
DCM, Dichloromethane; THF, Tetrahydrofuran; MeOH, Methanol; t-BuOOH, tert-Butylhydroperoxide; EtOAc, Ethyl acetate; AcOH, Acetic acid; TBAF, Tetra-n-
butylammonium fluoride; DCC, N,N'-Dicyclohexylcarbodiimide; TBS, tert-Butyldimethylsilylether; DDQ, 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone; MeCN,
Acetonitrile; DMAP, 4-Dimethylaminopyridine; TMS, Tetramethylsilylether; PNN, 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite; PNCl,
N,Ndiisopropylchlorophosphoramidite
⁎ Corresponding author.
E-mail address: davethom@purdue.edu (D.H. Thompson).
https://doi.org/10.1016/j.chemphyslip.2020.104933
Received 26 January 2020; Received in revised form 22 May 2020; Accepted 3 June 2020
Availableonline11June2020
0009-3084/©2020ElsevierB.V.Allrightsreserved.

Z.J. Struzik, et al.
ChemistryandPhysicsofLipids231(2020)104933
adversities have historically been reported with lysophospholipid
synthesis (D’Arrigo and Servi, 2010; Plueckthun and Dennis, 1982), but
are also relevant in synthesizing the DAG in other PL syntheses such as
phosphatidylserine (Mallik et al., 2018) and glycosylpho-
sphatidylinositols (Guo, 2013). Installation of the acyl chains via Ste-
glich esterification of 7 can be employed to afford 5. Acetonide de-
protection of 9 would yield 7. Protection of (S)-solketal precursor 11
with PMB can be used to synthesize 9. Following PMB deprotection of
the glycerol headgroup, intermediate 6 would correctly expose the
primary sn-1′ alcohol of 4. TBS protection would allow for conservation
of the sn-2′ stereocenter in 4 during the latter steps of the synthesis.
Prior methods for glycerol backbone synthesis can be used to make the
PMB protected, and acetonide deprotected species 8 and 10, respec-
tively, from the (R)-solketal precursor 12 (Jiang et al., 2006).
Fig. 1. General structure of PG with known acyl chain identities as reported in
Lipidomics Gateway (2020).
2. Materials and methods
2.1. General information
Commercial reagents were used as purchased from TCI Chemicals
and MilliporeSigma. Organic solvents used were reagent grade pur-
chased from Fisher Scientific. Dry solvents were purified using a Glass
Contour Solvent System from Pure Process Technology, LLC, with
Fisher HPLC grade DCM, Aldrich anhydrous DMF, and Fisher HPLC/
ACS grade THF. Reactions were monitored by thin-layer chromato-
graphy carried out on silica gel 60 F254 plates (Merck). UV light (254
nm) and staining with aqueous KMnO4 was used to visualize the de-
veloped chromatograms. Flash chromatography was performed using a
Biotage instrument (SP4 A2A0) with RediSep Rf silica flash columns (12
g, 60 mg-1.2 g sample size) and collected in 9 mL fraction volumes or
performed via manual silica column chromatography using silica gel 60
(MilliporeSigma, 0.040−0.063 MM). Compounds purified via auto-
matic flash chromatography are accompanied with a gradient table in
the Supplementary Material. All 1H, 13C and 31P NMR spectra were
recorded on a Bruker-AV-III-500-HD instrument. Chemical shifts (δ) are
reported in parts per million relative to CDCl3 (1H NMR residual peak at
δ =7.26 ppm, 13C NMR residual peaks at δ = 77.3, 77.0, 76.8 ppm)
and coupling constants (J) are given in Hz. Unless otherwise stated, all
reactions occurred at 24 °C. Reported yields in schemes are given as
averages over the three most successful trials.
2.2. Experimental procedures and data
2.2.1. HPLC/MS-MS analysis
The method used was based on a previously reported protocol
(Vosse et al., 2018). Separation was performed on an Agilent Rapid Res
1200 HPLC system using a HILICON iHILIC-Fusion (2.1x150 mm, 3.5
μm) column. Mobile phase A was water with 35 mM NH4OH, pH 5.75
and mobile phase B was ACN. Initial conditions were 3:97 A:B, held for
0.5 min, followed by a linear gradient to 25:75 at 26.5 min, 40:60 at 27
min, and held until 33 min. Column re-equilibration was performed by
returning to 3:97 A:B at 35 min and held until 45 min. Column flow rate
was 0.3 mL/min. The retention time for PG was 7.5 min. Analytes were
detected by MS/MS, based on multiple reaction monitoring, utilizing an
Agilent 6460 triple quadrupole mass spectrometer. Electrospray ioni-
zation in negative mode was used with a transition of 773.4–281.2,
with a collision energy of 45 V, for both BMP and PG. A fragmentor
energy of 100 V and a dwell time of 200 ms was used. Source para-
meters were as follows: nitrogen gas temperature =325 °C and flow
rate = 9 L/min, nebulizer pressure =40 psi, sheath gas temperature
=250 °C, sheath gas flow rate = 7 L/min, and capillary potential =3.8
kV. All data were collected and analyzed with Agilent MassHunter B.03
software.
Scheme 1. Retrosynthetic Analysis of 1,2-Dioleyl-sn-glycero-3-phospho-(1’-sn-
glycerol).
2019). Most recently, the phosphoramidite method has been utilized
during the installation of a phosphocholine headgroup (Xu, et al. 2020).
Because this reagent has been used successfully in the synthesis of BMP,
a structural isomer of PG, we decided to model our synthetic route
closely to this strategy (Chevallier et al., 2000; Jiang et al., 2005, 2006).
The crucial deprotection of the PMB group on 3 would expose the
stereochemically correct primary alcohol for phosphorylation. One of
the challenges commonly encountered in PL synthesis is acyl chain
migration that occurs within the glycerol backbone from the sn-2 po-
sition to the sn-1 position under mildly acidic or basic conditions. These
2

Z.J. Struzik, et al.
ChemistryandPhysicsofLipids231(2020)104933
2.2.2. Procedure for the synthesis of 1,2-dioleyl-sn-glycero-3-phospho-(1′-
sn-glycerol) (1)
2.2.4. Procedure for the synthesis of (S)-3-hydroxypropane-1,2-diyl
dioleate (3)
In an oven dried 2-neck flask equipped with a magnetic stir bar was
placed 2 (0.200 g, 0.19 mmol). The flask was dried via Schlenk tech-
niques and equipped with an Ar balloon. Dry THF (3 mL) was then
placed in the flask to dissolve 2, followed by the addition of AcOH
(0.227 g, 3.79 mmol). TBAF (1.0 M in dry THF) (0.990 g, 3.79 mmol)
was subsequently added to the flask, and the reaction was allowed to
stir for 24 h at 23 °C before removal of the solvent in vacuo. The reaction
mixture was then dissolved in 30 mL of CHCl3 and placed directly into a
separatory funnel followed by the addition of 20 mL of MeOH, and then
10 mL of water. After shaking, the organic layer separated, dried with
anhydrous Na2SO4, and was concentrated in vacuo. The crude product
was purified on a Biotage flash purification system (DCM:MeOH). After
two rounds of silica chromatography (both followed the same gradient),
the tetrabutyl ammonium salt of the product was removed by passage
through 10 g of SP-Sephadex C-25 ion exchange medium that was
swelled in 0.22 M NaCl over 2 d. After elution (MeOH:H2O, 1:1), the
pure product was removed from water by using a similar extraction to
that of the crude product. Here, the fractions containing 1 were added
to 30 mL of CHCl3 placed in a separatory funnel followed by the ad-
dition of 20 mL of MeOH, and then 10 mL of water. Occasionally,
centrifugation (7000 rpm for 5 min) was required to separate the layers.
After shaking and/or centrifugation, the organic layer was collected
and concentrated under reduced pressure to afford 1 as a clear colorless
oil in 56 % yield (0.082 g); [α]D27 +0.975 (c 0.003, CHCl3); Rf = 0.46
(DCM:MeOH:H2O, 80:18:2); 1H NMR (500 MHz, CDCl3) δ 5.33 (q, J =
5.0, 4.5 Hz, 4 H), 5.23 (dq, J = 8.8, 5.1 Hz, 1 H), 4.43 – 4.34 (m, 1 H),
4.15 (dd, J = 12.1, 6.8 Hz, 1 H), 4.00 – 3.83 (m, 5 H), 3.71 – 3.56 (m, 2
H), 2.29 (dt, J = 14.7, 7.5 Hz, 4 H), 2.00 (q, J =6.4 Hz, 8 H), 1.57 (t, J
=7.1 Hz, 4 H), 1.38 – 1.17 (m, 42 H), 0.88 (t, J =6.7 Hz, 7 H); 13C
NMR (126 MHz, CDCl3) δ 130.0, 129.6, 77.3, 77.0, 76.8, 34.3, 34.1,
31.9, 29.8, 29.6, 29.3, 27.2, 24.9, 24.9, 22.7, 14.1; 31P NMR (203 MHz,
CDCl3) δ 1.45; QTOF-HRMS (ESI) for C42H79O10P [M + Na+]: found
797.5303, calcd 797.5303.
This protocol was based on a previously reported method (Jiang
et al., 2006). In a 100 mL round bottom flask was placed 5 (1.0 eq., 2
mmol) and DCM/H2O (15 mL, 5% H2O). Following the addition of DDQ
(1.5 eq., 3 mmol) the reaction flask was completely wrapped in alu-
minum foil and stirred at 20 °C for 4 h. The reaction mixture turned
from dark green to a dark shade of red. The mixture was diluted with
DCM and vacuum filtered through a coarse glass frit with celite. The
collected filtrate was washed with saturated NaHCO3 solution (40 mL),
swirling gently to mix. The organic layer was washed again with
NaHCO3 (80 mL) and swirled vigorously to mix. Again the organic layer
was washed with NaHCO3 (2 × 100 mL), this time shaken to combine.
The resulting organic solution was dried with anhydrous Na2SO4 before
concentration under reduced pressure. The crude product was purified
on a Biotage flash purification system (hexane:EtOAc) to yield the de-
26
sired product as a clear colorless oil in an 84 % yield (1.1 g); [α]D
+3.0 (c 0.016, EtOAc); Rf = 0.08 (Hexane:EtOAc, 9:1); 1H NMR (500
MHz, CDCl3) δ 5.38 – 5.30 (m, 4 H), 5.08 (p, J =5.0 Hz, 1 H), 4.34 –
4.21 (m, 2 H), 3.73 (ddd, J = 6.8, 4.9, 1.7 Hz, 2 H), 2.33 (dt, J = 11.0,
7.5 Hz, 4 H), 2.05 – 1.98 (m, 9 H), 1.61 (p, J =7.3 Hz, 4 H), 1.35 – 1.23
(m, 41 H), 0.88 (t, J =6.9 Hz, 6 H); 13C NMR (126 MHz, CDCl3) δ
173.8, 173.4, 130.0, 129.7, 77.3, 77.0, 76.8, 72.1, 62.0, 61.5, 34.3,
34.1, 31.9, 29.8, 29.7, 29.5, 29.3, 29.2, 29.1, 27.21, 27.16, 24.9, 22.7,
14.1; HRMS (ESI) C39H72O5 [M + Na+]: found 643.5262, calcd
643.5272.
2.2.5. Procedure for the synthesis of (R)-2,3-bis((tert-butyldimethylsilyl)
oxy)propan-1-ol (4)
This protocol was based on a previously reported method (Abe
et al., 2010). In a 100 mL round bottom flask was placed 6 (1.0 eq., 3
mmol) and DCM:H2O (15 mL, 5% H2O). Following the addition of DDQ
(1.5 eq., 5 mmol) the reaction flask was completely wrapped in alu-
minum foil and stirred at 20 °C for 4 h. The reaction mixture turned
from dark green to a dark shade of red. The mixture was diluted with
DCM and vacuum filtered through a coarse glass frit with celite. The
collected filtrate was washed with saturated NaHCO3 solution (40 mL),
swirling gently to mix. The organic layer was washed again with
NaHCO3 (80 mL), swirled vigorously to mix. Again, the organic layer
was washed with NaHCO3 (2 × 120 mL), this time shaken to combine.
The resulting organic solution was dried with anhydrous Na2SO4 and
concentrated under reduced pressure. The crude product was purified
using a Biotage flash purification system (hexane:EtOAc) to give the
2.2.3. Procedure for the one-pot synthesis of (2R)-3-((((S)-2,3-bis((tert-
butyldimethylsilyl)oxy)propoxy)(2-cyanoethoxy)phosphoryl)oxy)propane-
1,2-diyl dioleate (2)
In an oven dried three-neck 25 mL round bottom flask was placed 3
(1.2 eq., 0.5 mmol). Schlenk techniques were used before before the
flask was equipped with an Ar balloon. 1-H tetrazole (3.0 eq., 1 mmol)
and dry DCM (1.5 mL) was added and stirred until dissolved. A solution
of 14 (1.0 eq, 0.4 mmol) in dry DCM (1.5 mL) was added dropwise to
the reaction flask and stirred for 16 h. Then, under an open atmosphere,
t-BuOOH (4.0 eq., 2 mmol, 70 % in H2O) was added. After mixing for 1
h, the reaction mixture was diluted with ethyl acetate (30 mL) and
washed with saturated NaHCO3 solution (2 × 20 mL). The combined
aqueous washes were rinsed with ethyl acetate (2 × 25 mL) and all
organic fractions were combined, dried with anhydrous Na2SO4 and
concentrated under reduced pressure. The resulting crude product was
purified on a Biotage flash purification system (hexane:EtOAc) to give
the purified product as a clear colorless oil in 71 % yield (0.29 g);
25
desired product as a clear colorless oil in 78 % yield (0.85 g); [α]D
+9.45 (c 0.005, EtOAc); Rf = 0.35 (hexane:EtOAc, 9:1); 1H NMR (500
MHz, CDCl3) δ 3.78 – 3.73 (m, 1 H), 3.66 – 3.54 (m, 4 H), 2.17 (dd, J =
7.5, 4.9 Hz, 1 H), 0.88 (d, J =1.6 Hz, 18 H), 0.08 (d, J =2.4 Hz, 6 H),
0.05 (d, J =1.9 Hz, 6 H); 13C NMR (126 MHz, CDCl3) δ 72.6, 64.9, 64.8,
25.9, 25.8, 18.3, 18.1, -4.6, -4.9, -5.5, -5.5; HRMS (ESI) for C15H36O3Si2
[M + Na+]: found 321.2280, calcd 321.2276.
2.2.6. Procedure for the synthesis of (S)-3-((4-methoxybenzyl)oxy)
propane-1,2-diyl dioleate (5)
[α]D +2.73 (c 0.01, CHCl3); Rf = 0.51 (DCM:MeOH, 96:4); 1H NMR
This protocol was based on a previously reported method (Abe
et al., 2011). In an oven dried 100 mL three-neck round bottom flask
was placed 7 (1.0 eq., 5 mmol) and oleic acid (2.1 eq., 10 mmol). The
contents of the flask were dried using Schlenk techniques, the flask was
equipped with an Ar balloon and dry DCM (15 mL) was added. A so-
lution of DCC (2.3 eq., 11 mmol) and DMAP (2.3 eq., 11 mmol) in dry
DCM (15 mL) was prepared in a separate 100 mL round bottom flask
and equipped with an Ar balloon. This solution was transferred to the
three-neck reaction flask via a cannula and stirred at 20 °C for 16 h. The
formed salt was removed by vacuum filtration through a coarse glass
frit containing celite and the resulting filtrate was collected and con-
centrated under reduced pressure. To the crude product was added a
minimal amount of hexane and the mixture was sonicated to dissolve
27
(500 MHz, CDCl3) δ 5.37 – 5.30 (m, 4 H), 5.24 (h, J =4.8 Hz, 1 H), 4.33
(ddd, J = 11.9, 9.4, 4.4 Hz, 1 H), 4.28 – 4.11 (m, 6 H), 3.99 (dq, J =
10.1, 5.7 Hz, 1 H), 3.89 – 3.83 (m, 1 H), 3.59 – 3.51 (m, 2 H), 2.76 (td, J
= 6.4, 2.8 Hz, 2 H), 2.32 (dt, J = 11.4, 7.6 Hz, 4 H), 2.01 (q, J =6.4
Hz, 8 H), 1.60 (p, J =6.8 Hz, 4 H), 1.29 (dd, J = 19.1, 5.5 Hz, 42 H),
0.88 (d, J =4.6 Hz, 24 H), 0.09 (d, J =3.8 Hz, 6 H), 0.06 (s, 6 H); 13C
NMR (126 MHz, CDCl3) δ 173.1, 172.7, 116.1, 77.3, 77.0, 76.8, 71.8,
71.8, 69.6, 69.3, 65.8, 63.9, 63.8, 91.9, 61.5, 34.1, 34.0, 31.9, 29.7,
29.5, 29.3, 29.2, 29.1, 27.2, 27.2, 25.9, 25.7,24.8, 22.7, 19.6, 19.6,
18.3, 18.1, 14.1, -4.7, -4.76; 31P NMR (203 MHz, CDCl3) δ -1.37, -1.50;
QTOF-HRMS (ESI) for
C
57H110NO10
P
[M
+
Na+]: found
1078.7299.5303, calcd 1078.7297.
3

Z.J. Struzik, et al.
ChemistryandPhysicsofLipids231(2020)104933
the solid. The resulting solution was purified on a 4 cm diameter, 32 cm
long silica gel column (hexane:EtOAc) to yield the desired product clear
colorless oil in 82 % yield (2.9 g). The use of a shorter column resulted
concentrated under reduced pressure. The crude product was purified
on a Biotage flash purification system (DCM:MeOH) to yield the pro-
25
duct as a white solid in 93 % yield (1.6 g); [α]D −3.24 (c 0.010,
EtOAc); Rf = 0.13 (DCM:MeOH, 98:2); 1H NMR (500 MHz, CDCl3) δ
7.25 – 7.22 (m, 2 H), 6.89 – 6.86 (m, 2 H), 4.47 (s, 2 H), 3.86 (q, J =5.6
Hz, 1 H), 3.80 (s, 3 H), 3.69 – 3.56 (m, 2 H), 3.50 (qd, J = 9.7, 5.2 Hz, 2
H), 2.96 (d, J =4.8 Hz, 1 H), 2.54 (t, J =6.0 Hz, 1 H); 13C NMR (126
MHz, CDCl3) δ 159.4, 129.8, 129.5, 113.9, 73.2, 71.5, 70.7, 64.1, 55.3;
26
in co-elution of oleic acid; [α]D +7.08 (c 0.029, EtOAc); Rf = 0.32
(hexane:EtOAc, 9:1); 1H NMR (500 MHz, CDCl3) δ 7.25 – 7.20 (m, 2 H),
6.90 – 6.84 (m, 2 H), 5.38 – 5.30 (m, 4 H), 5.24 – 5.20 (m, 1 H), 4.46 (q,
J =11.7 Hz, 2 H), 4.33 (dd, J = 11.9, 3.8 Hz, 1 H), 4.17 (dd, J = 11.9,
6.4 Hz, 1 H), 3.80 (s, 3 H), 3.55 (dd, J = 5.2, 1.9 Hz, 2 H), 2.29 (dt, J =
20.5, 7.5 Hz, 4 H), 2.01 (q, J =6.6 Hz, 8 H), 1.64 – 1.55 (m, 4 H), 1.29
(td, J = 13.2, 5.2 Hz, 40 H), 0.88 (t, J =6.9 Hz, 6 H); 13C NMR (126
MHz, CDCl3) δ 173.4, 173.1, 159.3, 130.0, 129.8, 129.7, 129.3, 113.8,
72.9, 70.0, 67.9, 62.7, 55.3, 34.3, 34.1, 31.9, 29.8, 29.7, 29.5, 29.3,
29.2, 29.1, 29.1, 29.1, 27.2, 27.2, 24.9, 24.9, 22.7, 14.1; HRMS (ESI)
for C47H80O6 [M + Na+]: found 763.5837, calcd 763.5847.
HRMS (ESI) for
235.0941.
C11H16O4 [M +
Na+]: found 235.0945, calcd
2.2.10. Procedure for the synthesis of (S)-4-(((4-methoxybenzyl)oxy)
methyl)-2,2-dimethyl-1,3-dioxolane (9)
This protocol was based on a previously reported method (Jiang
et al., 2006). To an oven dried 250 mL multineck round bottom flask
was added NaH (3.0 eq., 113 mmol). Schlenk techniques were utilized
to evacuate the flask and dry DMF (50 mL) was added. The flask was
maintained under an Ar atmosphere for the duration of the reaction.
The reaction flask was lowered into an ice bath and (S)-solketal (1.0 eq.,
38 mmol) was added dropwise and allowed to react with vigorous
stirring at 5 °C for 45 min. PMBCl (1.1 eq., 42 mmol) was added and
stirred at 20 °C for 4 h. The completed reaction was quenched slowly
with saturated NH4Cl solution and extracted with EtOAc (3 × 150 mL).
The combined organic extracts were washed with water (2 × 150 mL),
washed with brine (2 × 150 mL), dried over anhydrous Na2SO4, and
concentrated under reduced pressure. The crude product was purified
on a 5 cm diameter silica gel column (DMC:MeOH 98:2) to yield the
desired product as a yellow oil in 96 % yield (9.2 g); [α]D25 +39.85 (c
0.001, EtOAc); Rf = 0.50 (DMC:MeOH, 98:2); 1H NMR (500 MHz,
CDCl3) δ 7.28 – 7.23 (m, 2 H), 6.90 – 6.85 (m, 2 H), 4.50 (q, J =11.7
Hz, 2 H), 4.28 (p, J =6.0 Hz, 1 H), 4.04 (dd, J = 8.3, 6.4 Hz, 1 H), 3.80
(s, 3 H), 3.72 (dd, J = 8.3, 6.3 Hz, 1 H), 3.48 (ddd, J = 43.4, 9.8, 5.7
Hz, 2 H), 1.42 (s, 3 H), 1.36 (s, 3 H); 13C NMR (126 MHz, CDCl3) δ
159.3, 130.0, 129.4, 113.8, 109.4, 74.8, 73.2, 70.8, 66.9, 55.3, 26.8,
25.4. HRMS (ESI) for C14H20O4 [M + Na+]: found 275.1252, calcd
275.1254.
2.2.7. Procedure for the synthesis of (R)-5-(((4-methoxybenzyl)oxy)
methyl)-2,2,3,3,8,8,9,9-octamethyl-4,7-dioxa-3,8-disiladecane (6)
In an oven dried 100 mL three-neck round bottom flask was placed
8 (4 eq., 19 mmol), and TBSCl (4 eq., 19 mmol). The contents were
dried using Schlenk techniques and the closed flask was equipped with
an Ar balloon. The contents were dissolved in dry DMF (15 mL) and the
reaction was heated to 50 °C in an oil bath. TEA (2.4 eq., 11 mmol) was
added and stirred at 50 °C for 4 h. After completion the reaction was
quenched with water (60 mL) and extracted with EtOAc (3 × 80 mL).
The combined organic layers were washed with water (3 × 50 mL) and
brine (2 × 25 mL) before being dried with anhydrous Na2SO4 and
concentrated under reduced pressure. The crude product was purified
using a Biotage flash purification system (hexane:EtOAc) to give the
26
desired product as a clear colorless oil in 96 % yield (2.0 g); [α]D
+4.39 (c 0.006, EtOAc); Rf = 0.49 (hexane:EtOAc, 9:1); 1H NMR (500
MHz, CDCl3) δ 7.25 (d, J =7.9 Hz, 2 H), 6.89 – 6.85 (m, 2 H), 4.46 (s, 2
H), 3.84 (qd, J = 5.6, 4.7 Hz, 1 H), 3.80 (s, 3 H), 3.60 (dd, J = 10.2, 5.8
Hz, 1 H), 3.56 – 3.48 (m, 2 H), 3.39 (dd, J = 9.9, 5.5 Hz, 1 H), 0.88 (s,
18 H), 0.06 (d, J =1.2 Hz, 6 H); 13C NMR (126 MHz, CDCl3) δ 159.1,
130.7, 129.2, 113.7, 73.0, 72.8, 71.9, 65.1, 55.3, 26.0, 25.9, 18.4, 18.2,
1.0, -4.6, -4.7, -5.3, -5.4; HRMS (ESI) for C23H44O4Si2 [M + Na+]:
found 463.2670, calcd 463.2657.
2.2.11. Procedure for the synthesis of (R)-4-(((4-methoxybenzyl)oxy)
methyl)-2,2-dimethyl-1,3-dioxolane (10)
2.2.8. Procedure for the synthesis of (R)-3-((4-methoxybenzyl)oxy)
propane-1,2-diol (7)
This protocol was based on a previously reported method (Jiang
et al., 2006). To an oven dried 250 mL multineck round bottom flask
was added NaH (3.0 eq., 113 mmol). Schlenk techniques were utilized
to evacuate the flask and dry DMF (50 mL) was added. The flask was
maintained under a continuous Ar atmosphere for the duration of the
reaction. The reaction flask was lowered into an ice bath and (R)-
solketal (1.0 eq., 38 mmol) was added dropwise and allowed to stir
vigorously at 0 °C for 45 min. PMBCl (1.1 eq., 42 mmol) was added and
stirred at 20 °C for 4 h. The completed reaction was quenched slowly
with saturated NH4Cl solution and extracted with ethyl acetate (3 ×
150 mL). Combined organic extracts were washed with water (2 × 150
mL), washed with brine (2 × 150 mL), dried over anhydrous Na2SO4,
and concentrated under reduced pressure. The crude product was
purified on a 5 cm silica gel column (DMC:MeOH, 98:2) to yield the
desired product as a yellow oil in 99 % yield (9.5 g); [α]D25 −14.22 (c
0.025, EtOAc); Rf = 0.50 (DMC:MeOH, 98:2); 1H NMR (500 MHz,
CDCl3) δ 7.28 – 7.22 (m, 2 H), 6.89 – 6.84 (m, 2 H), 4.50 (q, J =11.7
Hz, 2 H), 4.31 – 4.24 (m, 1 H), 4.04 (dd, J = 8.3, 6.4 Hz, 1 H), 3.79 (s, 3
H), 3.72 (dd, J = 8.3, 6.3 Hz, 1 H), 3.48 (ddd, J = 43.3, 9.8, 5.7 Hz, 2
H), 1.38 (d, J =29.6 Hz, 6 H); 13C NMR (126 MHz, CDCl3) δ 159.3,
130.1, 129.4, 113.8, 109.4, 74.8, 73.2, 70.8, 66.9, 55.3, 26.8, 25.4;
This protocol was based on a previously reported method (Jiang
et al., 2006). To a 100 mL round bottom flask containing a solution of 9
(1.0 eq., 8 mmol) in 21 mL THF was added aqueous HCl (1 M, 2.8 eq.,
22 mmol). The reaction mixture was stirred at 20 °C for 2 h. Saturated
NaHCO3 solution was then added to quench the reaction and the re-
action mixture was extracted with ethyl acetate (3 × 100 mL). The
combined organic extracts were dried with anhydrous Na2SO4 and
concentrated under reduced pressure. The crude product was purified
on a Biotage flash purification system (DCM:MeOH) to give a white
25
solid in 90 % yield (1.5 g); [α]D +3.77 (c 0.009, EtOAc); Rf = 0.13
(DCM:MeOH, 98:2); 1H NMR (500 MHz, CDCl3) δ 7.26 – 7.22 (m, 2 H),
6.89 – 6.85 (m, 2 H), 4.46 (s, 2 H), 3.85 (td, J = 6.0, 3.0 Hz, 1 H), 3.79
(s, 3 H), 3.67 – 3.55 (m, 2 H), 3.49 (qd, J = 9.7, 5.2 Hz, 2 H), 3.08 (s, 1
H), 2.69 (s, 1 H); 13C NMR (126 MHz, CDCl3) δ 159.4, 129.8, 129.5,
113.9, 73.2, 71.5, 70.7, 64.1, 55.3; HRMS (ESI) for C11H16O4 [M +
Na+]: found 235.0940, calcd 235.0941.
2.2.9. Procedure for the synthesis of (S)-3-((4-methoxybenzyl)oxy)
propane-1,2-diol (8)
This protocol was based on a previously reported method (Jiang
et al., 2006). To a 100 mL round bottom flask containing a solution of
10 (1.0 eq., 8 mmol) in 21 mL THF was added aqueous HCl (1 M, 2.77
eq., 22 mmol). The reaction mixture was allowed to react at 20 °C for 2
h. Saturated NaHCO3 solution was added to quench the reaction and
the reaction mixture was extracted with EtOAc (3 × 100 mL). The
combined organic extracts were dried with anhydrous Na2SO4 and
HRMS (ESI) for
275.1254.
C14H20O4 [M +
Na+]: found 275.1257, calcd
2.2.12. Procedure for the synthesis of (S)-2,3-bis((tert-butyldimethylsilyl)
oxy)propyl(2-cyanoethyl) diisopropylphosphoramidite (14)
This method was based on previously reported protocol (Ching
4

Z.J. Struzik, et al.
ChemistryandPhysicsofLipids231(2020)104933
et al., 2010). In dry 25 mL round bottom flask was placed 4 (1.0 eq., 0.6
mmol). The flask was dried with Schlenk techniques and fitted with an
Ar balloon. To the flask was added 1-H tetrazole (1.5 eq., 0.8 mmol) and
dry ACN (2 mL). Phosphoramidite PNN (2.0 eq., 1 mmol) was added to
the reaction flask dropwise at a rapid rate and stirred at 20 °C for 18 h.
Then, the reaction mixture was diluted with EtOAc (10 mL) and filtered
through a coarse glass frit. The collected filtrate was washed with sa-
turated NaHCO3 solution (15 mL), after which it was dried with an-
hydrous Na2SO4 and the solvent removed under reduced pressure. A
Biotage flash purification system was used to purify the crude product
to give the desired product in 79 % yield (0.23 g); clear colorless liquid;
[α]D27 +6.26 (c 0.009, CHCl3) Rf = 0.55 (Hexane:EtOAc, 9:1); 1H NMR
(500 MHz, CDCl3) δ 3.88 – 3.77 (m, 3 H), 3.69 – 3.47 (m, 6 H), 2.63
(tdd, J = 6.3, 3.3, 2.0 Hz, 2 H), 1.20 – 1.16 (m, 12 H), 0.89 (d, J =2.7
Hz, 18 H), 0.09 – 0.04 (m, 12 H); 13C NMR (126 MHz, CDCl3) δ 127.3,
77.3, 77.0, 76.8, 65.0, 64.8, 58.5, 43.04, 26.0, 25.9, 25.7, 24.7, 24.6,
20.4, 18.4, 18.2, -4.6, -4.7, -5.4, -5.4 ; 31P NMR (203 MHz, CDCl3) δ
148.48, 147.88; QTOF-HRMS (ESI) for C24H53N2O4PSi2 [M + Na+]:
found 521.3339, calcd 521.3354.
3. Results and discussion
3.1. Synthesis of glycerol head group
Protection with PMBCl and NaH of the primary sn-1’ alcohol of 12
gave 10 in 97 % yield. (Scheme 2). Acetonide deprotection to expose
the sn-2’ and sn-3’ alcohols of 10 was performed using 1.0 M HCl in THF
to afford 8 in 88 % yield. We chose to use the bulkier TBS groups in-
stead of TMS groups to protect the 1,2-diol of 8 during the synthesis of
6 to provide increased stability of the glycerol headgroup intermediate
throughout the synthetic pathway. Several reported protocols used
DCM as the solvent during the silyl protection of intermediate 8 and
related compounds (Abe et al., 2010, 2015). Unfortunately, we typi-
cally observed yields that were significantly lower than the reported
values (20 % or less) that most often resulted in partial conversion to
the monosilated product. In turn, we set out to identify a more efficient
method to protect the sn-2’ and sn-3’ alcohols. Patschinski et al. ex-
amined the effects of solvent and auxiliary base in the Corey silyl ether
protection of alcohols (Patschinski et al., 2014). It was found that DMF
acted as a catalyst based on the observed turnover of starting material
in the absence of imidazole, while the use of DCM resulted in little to no
reaction under the same conditions. Furthermore, the addition of non-
nucleophilic bases such as TEA further enhanced the product yield. This
observation was attributed to the regeneration of the imidazole catalyst
by the auxiliary base. We, therefore, applied these conditions to our
synthesis by reacting the 1,2 - diol with 3.5 equivalencies of TBSCl and
imidazole, and 2 equivalents of TEA in DMF with heating over 4 h from
24 °C to 50 °C to afford 6 in 93 % yield. PMB deprotection of 6 was then
inducted with DDQ in a DCM/H2O mixture to produce 4 in 77 % yield.
Scheme 3. Synthesis of the 1,2-Dioleyl-sn-glycerol Intermediate 3.
3.2. Synthesis of glycerol backbone
Next, we synthesized DAG intermediate 3 in a similar manner to
that for glycerol intermediate 4 (Scheme 3). When 11 was mixed with
NaH and PMBCl in DMF, 9 was observed in 96 % yield. Subsequent
acetonide deprotection in 1.0 M HCl exposed the sn-2 and sn-1 alcohols
to give 7 in 90 % yield. Steglich esterification was then implemented to
install the oleyl acyl chains onto the protected glycerol backbone to
give 5. Formation of DAG 3 was affected via PMB deprotection by DDQ
in 82 % yield. To minimize the amount of acyl chain migration from the
sn-2 position to the more thermodynamically stable sn-3 position, an
optimum reaction time of 4 h is required to produce greater than 99 %
of the observed esterified product at the sn-2 and sn-1 positions (Fig. 2).
We base this conclusion on the characteristic 1H NMR peaks observed at
5.08 ppm and 3.73 ppm, corresponding to the methine proton at the
esterfied sn-2 position and methylene protons at the unfunctionalized
sn-3 position, respectively (Fig. 2A). Conversely, 38 % of the diacyl
glycerol product had rearranged after a 24 h reaction time. This product
is characterized by the disappearance of the formerly mentioned peaks
and the appearance of the unesterified methine proton peak that was
shifted up field to 4.09 ppm in 13 (Fig. 2B).
3.3. Phosphorylation and global deprotection
With the glycerol backbone and headgroup intermediates in hand,
efforts to phosphorylate and ultimately globally deprotect to yield PG
were initiated (Scheme 4). Our synthesis began with phosphorylation of
4 via coupling with a chloro-substituted phosphoramidite PNCl in the
presence of 1-H tetrazole. The attractiveness of PNCl was the ability to
selectively phosphorylate both the glycerol head group and backbone in
separate steps due to the asymmetric nature of the phosphoramidite.
However, PNCl proved to be difficult to handle because the product
streaked excessively on silica and gave low reaction yields (30 % or
lower). Multiple byproducts formed due to degradation on the column
and hydrolysis of the excess monochlorophosphoramidite during the
Scheme 2. Synthesis of the 1’-sn-Glycerol Intermediate 4.
5

Z.J. Struzik, et al.
ChemistryandPhysicsofLipids231(2020)104933
Fig. 2. 1H NMR Comparison of the 1,2-Dioleyl-sn-glycerol Product 3 (A) Versus
the Acyl Chain Migration Product 13 (B).
Fig. 3. HILIC-MS/MS of Racemic (A) and Stereospecific PG (B).31P NMR of 1 as
a Tetrabutylammonium salt (C).
the reaction that is easily removed by filtration after work-up, thereby
simplifying purification. By combining 4 with PNN and 1-H tetrazole in
MeCN, we were able to obtain 14 in 77 % yield. A one-pot procedure
was used to phosphorylate 3 with phosphoramidite 14 in the presence
of 1-H tetrazole followed by PIII→PV oxidation with 70 % tBuOOH to
give 2 in 74 % yield. Our global deprotection strategy was adopted from
Chevallier et al. who also utilized a naked fluorine source to remove
both the cyanoethyl and silyl ether protecting groups in a single step
(Chevallier et al., 2000). Phospholipid precursor 2 was mixed with
TBAF and an equivalent amount of acetic acid to neutralize the mildly
basic fluorine in THF, giving 1 in 42 % yield after silica chromato-
graphy and ion exchange chromatography using Sephadex C-25.
3.4. HILIC-MS/MS analysis
Scheme 4. Synthesis of 1,2-Dioleyl-sn-glycero-3-phospho-(1’-sn-glycerol) (1).
To validate our synthetic protocol, we compared our diaster-
eomerically pure PG 1 to the racemic PG that is commercially available
from MilliporeSigma using hydrophilic interaction liquid chromato-
graphy coupled to tandem mass spectrometry (HILIC-MS/MS) (Fig. 3).
The racemic compound had a retention time of 7.5 min (Fig. 3A), si-
milar to that observed for 1 at 7.6 min (Fig. 3B) in HILIC-MS/MS. The
1H NMR spectra share similar chemical shifts positions and integrations
at key points such as the methine peak at 5.22 ppm that integrates to
workup, consistent with the observations of Ching et al.(Ching et al.,
2010), leading us to pursue a different approach with this reagent. We
then explored the use of a symmetric phosphoramidite containing two
amine subsituents, PNN, that is less air and moisture sensitive. Ad-
ditionally, a diisopropylammonium tetrazolide salt was formed during
6

Z.J. Struzik, et al.
ChemistryandPhysicsofLipids231(2020)104933
1.00 indicating that acyl chain migration did not occur (Fig. S1). Ad-
ditionally, the peak pattern in the glycerol region is consistent between
the two samples for peaks appearing at 3.38, 3.61, 3.65, 4.16, and 4.39
ppm, further indicating esterifcations at the sn-2 and sn-1 positions.
While the peaks belonging to PG are similar between the commercial
standard and synthesized sample, a significant difference between the
two spectra is a broad singlet appearing at 2.41 ppm that integrates to
5.83 protons in the commercial source that does not appear to belong to
PG. 31P NMR (Fig. 3C) data collected for 1 as a tetrabutyl ammonium
salt further validates the structure as the PG since a single peak at 1.77
ppm was observed in CDCl3, whereas a single signal at 1.68 ppm was
observed for commercial PG (Fig. S2), thus confirming that the syn-
thesized PG belongs to the same lipid class as the commercial standard.
While the goal of the ion exchange step was to obtain the sodium salt of
PG, the CHCl3/MeOH/H2O extraction required after chromatography
prevented salt formation entirely and thus resulted in a broad 31P NMR
spectrum most likely due to self-assembly of the lipid into higher or-
dered structures (Filippov et al., 2015).
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In summary, we have successfully developed a synthetic protocol to
make stereochemically pure PG from two chiral solketal precursors. The
DAG was synthesized without acyl chain migration by quenching the
reaction at 4 h; significant acyl chain migration was observed for re-
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Declaration of Competing Interest
The authors have no conflicts of interest to declare.
Murakami, K., Molitor, E.J., Liu, H.-w., 1999. An efficient synthesis of unsymmetrical
optically active phosphatidyl glycerol. J. Org. Chem. 64, 648–651.
Patschinski, P., Zhang, C., Zipse, H., 2014. The Lewis Base-Catalyzed Silylation of alco-
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Acknowledgments
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phosphatidylation. Lipids 39, 1025–1030.
Support of NIH grant [GM115866] is gratefully acknowledged. We
would like to thank Bruce Cooper, director of the Purdue University
Metabolomics facility, for the assistance in the HILIC MS/MS method.
Appendix A. Supplementary data
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