Surface properties of sulfur- and ether-linked phosphonolipids with and without purified hydrophobic lung surfactant proteins

Article abstract of DOI:10.1016/j.chemphyslip.2005.07.002

Two novel C16:0 sulfur-linked phosphonolipids (S-lipid and SO ₂-lipid) and two ether-linked phosphonolipids (C16:0 DEPN-8 and C16:1 UnDEPN-8) were studied for surface behavior alone and in mixtures with purified bovine lung surfactant proteins

Full text of DOI:10.1016/j.chemphyslip.2005.07.002

Chemistry and Physics of Lipids 137 (2005) 77–93
Surface properties of sulfur- and ether-linked phosphonolipids with
and without purified hydrophobic lung surfactant proteins
Yusuo Changa,c, Zhengdong Wanga, Adrian L. Schwand, Zhongyi Wangd,
Bruce A. Holme, John E. Baatzf, Robert H. Nottera,b,∗
a
Department of Pediatrics, University of Rochester, 601 Elmwood Avenue, Rochester, NY 14642, USA
b
Department of Environmental Medicine, University of Rochester, Rochester, NY 14642, USA
c
Department of Chemical Engineering, University of Rochester, Rochester, NY 14642, USA
d
Department of Chemistry, University of Guelph, Guelph, Ont., Canada N1G 2W1
e
Department of Pediatrics, State University of New York (SUNY) at Buffalo, Buffalo, NY 14214, USA
f
Department of Pediatrics, Medical University of South Carolina, Charleston, SC 29425, USA
Received 12 January 2005; accepted 5 July 2005
Available online 25 July 2005
Abstract
Two novel C16:0 sulfur-linked phosphonolipids (S-lipid and SO2-lipid) and two ether-linked phosphonolipids (C16:0 DEPN-8
and C16:1 UnDEPN-8) were studied for surface behavior alone and in mixtures with purified bovine lung surfactant proteins
(SP)-B and/or SP-C. Synthetic C16:0 phosphonolipids all had improved adsorption and film respreading compared to dipalmitoyl
phosphatidylcholine, and SO2-lipid and DEPN-8 reached maximum surface pressures of 72 mN/m (minimum surface tensions of
<1 mN/m) in compressed films on the Wilhelmy balance (23 ◦C). Dispersions of DEPN-8 (0.5 mg/ml) and SO2-lipid (2.5 mg/ml)
also reached minimum surface tensions of <1 mN/m on a pulsating bubble surfactometer (37 ◦C, 20 cycles/min, 50% area
compression). Synthetic lung surfactants containing DEPN-8 or SO2-lipid + 0.75% SP-B + 0.75% SP-C had dynamic surface
activity on the bubble equal to that of calf lung surfactant extract (CLSE). Surfactants containing DEPN-8 or SO2-lipid plus
1.5% SP-B also had very high surface activity, but less than when both apoproteins were present together. Adding 10 wt.%
of UnDEPN-8 to synthetic lung surfactants did not improve dynamic surface activity. Surfactants containing DEPN-8 or SO2-
lipid plus 0.75% SP-B/0.75% SP-C were chemically and biophysically resistant to phospholipase A2 (PLA2), while CLSE was
severely inhibited by PLA2. The high activity and inhibition resistance of synthetic surfactants containing DEPN-8 or SO2-lipid
plus SP-B/SP-C are promising for future applications in treating surfactant dysfunction in inflammatory lung injury.
© 2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Lung surfactants; Surfactant therapy; Phosphonolipids; Lipid analogs; DEPN-8
∗
Corresponding author. Tel.: +1 585 275 5948; fax: +1 585 756 7780.
E-mailaddress:Robert Notter@urmc.Rochester.edu (R.H. Notter).
0009-3084/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2005.07.002

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Y. Chang et al. / Chemistry and Physics of Lipids 137 (2005) 77–93
1. Introduction
in CLSE were susceptible to cleavage by this enzyme
(Wang et al., 2003).
Lung surfactant is a complex mixture of surface-
active phospholipids and proteins that promotes normal
respiration by lowering and varying surface tension at
the liquid–air alveolar interface (Notter, 2000). A defi-
ciency in lung surfactant in premature infants causes
the neonatal respiratory distress syndrome (RDS), and
surfactant dysfunction is an important contributor to
clinical acute lung injury (ALI) and the acute respira-
tory distress syndrome (ARDS) (Notter, 2000; Wang et
al., 2005). When endogenous surfactant is deficient or
dysfunctional, it can be replaced by active exogenous
substitutes. Therapy with active exogenous surfactants
is life-saving in premature infants, and is currently
being extended to pediatric and adult patients with
ALI/ARDS (Chess et al., 2005; Notter, 2000; Will-
son et al., 2005). Optimizing surfactant therapy for
ALI/ARDS is challenging, and requires exogenous sur-
factants with maximal surface activity plus the abil-
ity to resist inhibition from endogenous substances in
injured, inflamed lungs. The most widely used cur-
rent clinical exogenous surfactants are animal-derived
rather than synthetic in nature (Chess et al., 2005; Not-
ter, 2000). However, synthetic exogenous surfactants
containing novel chemical components with specific
structural and activity benefits are of significant inter-
est.
In addition to carrying out further studies with
DEPN-8, the current paper investigates the surface-
active properties of new synthetic phosphonolipids
alone and in combination with purified bovine
hydrophobic SP-B and/or SP-C. New compounds
include two disaturated C16:0 sulfur-linked phospho-
nolipids (S-lipid and SO2-lipid), as well as a C16:1
unsaturated diether phosphonolipid (UnDEPN-8), all
having the same choline N-headgroup as DEPN-8
and DPPC. Synthetic surfactants containing phospho-
nolipids plus pure SP-B, pure SP-C, or an equal
weight of SP-B + SP-C are examined to assess syn-
ergy between the hydrophobic apoproteins. Biophysi-
cal measurements include surface pressure–area (π–A)
isotherms and respreading in interfacial films on the
Wilhelmy balance, adsorption from a stirred subphase,
and overall dynamic surface activity on the physiolog-
ically relevant pulsating bubble surfactometer (37 ◦C,
20 cycles/min, 50% area compression). The ability of
synthetic lung surfactants containing DEPN-8 or SO2-
lipid + 0.75% SP-B + 0.75% SP-C to resist inhibition
from PLA2 is also specifically examined.
2. Materials and methods
In prior work, we have reported the synthesis of
several phospholipase-resistant phosphonolipids that
are structurally analogous to dipalmitoyl phosphatidyl-
choline (DPPC), the most prevalent glycerophospho-
lipid in native lung surfactant (Liu et al., 1994a, 1994b,
1995; Turcotte et al., 1991, 1977; Wang et al., 2003).
These studies have shown that DEPN-8, a synthetic
diether analog, has better adsorption and spreading
properties than DPPC while maintaining an ability
to reduce surface tension to <1 mN/m in dynamically
compressed interfacial films (Liu et al., 1994a; Tur-
cotte et al., 1991; Wang et al., 2003). In addition,
a synthetic exogenous surfactant containing DEPN-
8 plus 1.5 wt.% of mixed (unseparated) bovine lung
surfactant proteins (SP)-B/C was shown to have over-
all surface activity equivalent to calf lung surfactant
extract (CLSE, the substance of the highly active clini-
cal exogenous surfactant Infasurf) (Wang et al., 2003).
This synthetic surfactant was resistant to inhibition by
phospholipaseA2 (PLA2), whileglycerophospholipids
2.1. Synthetic lipids
DPPC
(1,2-dipalmitoyl-sn-3-phosphocholine,
>99% pure) was purchased from Avanti Polar Lipids
Inc. (Alabaster, AL). The four synthetic phospho-
nolipids studied (DEPN-8, S-lipid, SO2-lipid, and
UnDEPN-8) are shown schematically in Fig. 1. DEPN-
8 [( )-trimethyl(3-phosphonopropyl)ammonium, mo-
no(2,3-bis(hexadecyloxy)propyl) ester] was made and
purified as detailed previously (Wang et al., 2003).
Details on the methods used in preparing S-lipid, SO2-
lipid, andUnDEPN-8aregivenin AppendixA. Inbrief,
1-thioglycerolforthesynthesisofS-lipidandSO2-lipid
was converted to the 1-S-hexadecyl-rac-thioglycerol
by alkylation with hexadecyl bromide in alcoholic
KOH (95%) (Chang et al., 2004). The primary
hydroxyl group was selectively tritylated to produce 1-
S-hexadecyl-3-O-trityl-rac-thioglycerol (93% yield),
and the remaining hydroxyl group was alkylated
with hexadecyl bromide to give 1-S-hexadecyl-2-O-

Y. Chang et al. / Chemistry and Physics of Lipids 137 (2005) 77–93
79
Fig. 1. Schematic diagram of the synthetic phosphonolipid compounds studied. Shown schematically are DEPN-8, S-lipid, SO2-lipid, and
UnDEPN-8. Arrows (⇒) indicate the position of functional group changes in relation to DPPC. The molecular changes at these locations impart
structural resistance to phospholipases A1, A2, and D (Lin et al., 1997; Wang et al., 2003). In addition, DEPN-8 has been shown to be partially
resistant to cleavage by phospholipase C (Lin et al., 1997).
hexadecyl-3-O-trityl-rac-thioglycerol (94% yield).
The trityl group was cleaved in 95% yield with aqueous
methanol to produce 1-S-hexadecyl-2-O-hexadecyl-
rac-thioglycerol (Nali et al., 1986), which was oxidized
to the corresponding sulfone 1-SO2-hexadecyl-2-O-
hexadecyl-rac-sulfonylglycerol prior to installation of
the phosphono headgroup (Chang et al., 2004). Final
isolation and recrystallization afforded S-lipid [( )-
trimethyl(3-phosphonopropyl)ammonium, mono(2-
hexadecyloxy-3-hexadecylsulfanylpropyl)ester, 48%
yield] and SO2-lipid [( )-trimethyl(3-phosphonopro-
pyl)ammonium, mono(2-hexadecyloxy-3-hexadecyls-
ulfonylpropyl)ester, 59% yield]. UnDEPN-8 [( )-
trimethyl(3-phosphonopropyl) ammonium, mono(2-
hexadec-9-enyloxy-3-hexadecyloxypropyl)ester] was
prepared by alkylation of 1-O-hexadecyl-3-O-
trityl-rac-glycerol with commercially available
Z-hexadec-9-enyl mesylate under KOH/DMSO condi-
tions (Johnstone and Rose, 1979) as used in preparing
DEPN-8 (Wang et al., 2003). Double bond geometry
remained intact over all manipulations based on 1H and
13C NMR spectroscopy, and the final waxy UnDEPN-
8 compound was obtained at a yield of 51%. All
compounds had single spots on thin layer chromatog-
raphy with a solvent system of 30:9:25:7:25 (v/v)
chloroform:methanol:2-propanol:water:triethylamine
(Touchstone et al., 1980).
aggregate surfactant lavaged from intact calf lungs as
reported previously by Notter and co-workers (e.g.,
Hall et al., 1992b, 1994; Wang et al., 1995). SP-
B and SP-C were purified from CLSE by isocratic
normal-phase liquid chromatography (LC) on Silica
C8 (60 ␮m mesh, J.T. Baker, Phillipsburg, NJ) (Baatz
et al., 2001). All columns, tubing, and fittings were con-
structed of organic-resistant materials to avoid degra-
dation of plastics (Spectrum Medical Industries Inc.,
Los Angeles, CA). Approximately 700 mg of CLSE
in 7:1 MeOH:chloroform + 5% 0.1N HCl was concen-
trated by evaporation under nitrogen to 4 ml volume
and applied to a 450 ml LC column pre-equilibrated
with the same solvent. Added CLSE was allowed to
completely absorb to the column prior to elution with
additional 7:1 MeOH:chloroform + 5% 0.1N HCl at
a flow rate of 0.4 ml/min. Eluted components were
assessed by a UV detector (Pharmacia Biotech, Upp-
sala, Sweden) at 254 nm. The phospholipid content
of eluted fractions was determined by the method-
ology of Shin et al. (Shin, 1962) with the aid of a
Fiske-Subbarow phosphate analysis kit (Sigma Chem-
ical Co., St. Louis, MO). Chloroform and methanol
(HPCL grade) and HCl (ACS grade) were from Baxter
Scientific (Chicago, IL). Final isolates of pure bovine
SP-B and SP-C had no detectable contaminating pro-
teins based on SDS polyacrylamide gel electrophore-
sis (SDS-PAGE) and N-terminal amino acid analysis
(Baatz et al., 2001).
2.2. CLSE and hydrophobic lung surfactant
proteins (SP-B, SP-C)
2.3. Phospholipase A2 (PLA2) studies
CLSE (a gift from ONY Inc., Amherst, NY) was
prepared by chloroform:methanol extraction of large
PLA2 (Sigma Chemical, St. Louis, MO) was sus-
pended in 5 mM Tris(hydroxymethylaminomethane)

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Y. Chang et al. / Chemistry and Physics of Lipids 137 (2005) 77–93
buffer containing 5 mM CaCl2 (pH 7.4), and was
incubated with surfactants dispersed in the same sol-
vent for 30 min at 37 ◦C (final enzyme concentration
was 0.1 units/ml) (Enhorning et al., 1992; Holm et
al., 1991). After incubation, surfactant–PLA2 mixtures
were examined for surface activity on the pulsating
bubble (see below). In addition, aliquots of PLA2-
incubated surfactants containing S-lipid and SO2-lipid
were also examined for chemical degradation by thin
layerchromatography(Touchstoneetal., 1980). Chem-
ical degradation was assessed by scraping and analyz-
ing chromatographic bands using the phosphate assay
of Ames (1966).
area of zero indicates complete respreading between
the designated cycles, and larger areas indicate poorer
respreading) (Notter, 2000; Wang et al., 1995).
2.5. Adsorption apparatus
Adsorption studies were done at 37 0.5 ◦C in a
Teflon dish containing a 35 ml subphase of 150 mM
NaCl + 1.5 mM CaCl2 (Notter et al., 1982, 1983). A
Teflon-coated magnetic bar was used to gently stir the
subphase to minimize diffusion resistance. Adsorption
experiments were initiated at time zero by injecting
2.5 mg of phosphonolipid dispersed in 5 ml of subphase
by probe sonication on ice into the 35 ml stirred sub-
phase (final surfactant concentration was 0.0625 mg/ml
in40 mloftotalsubphase). Adsorptionsurfacepressure
was followed as a function of time from the force on
a partially submerged sandblasted platinum Wilhelmy
slide (Notter et al., 1982, 1983).
2.4. Wilhelmy balance studies on spread
interfacial films
Surface pressure–area (π–A) isotherms were mea-
sured on a custom-designed Wilhelmy surface balance
with a Teflon trough and continuous ribbon barrier to
minimize film leakage as described previously (Tabak
and Notter, 1977). Surfactant mixtures were dissolved
in 9:1 (v/v) hexane–ethanol and spread dropwise from
a syringe at the air–water interface of a saline subphase
(1.5 mM CaCl2, 150 mM NaCl). Surface pressure (the
magnitude by which surface tension was lowered rel-
ative to the pure subphase) was determined from the
force on a hanging sandblasted platinum slide dipped
into the interface, and film compression was initiated
after a 10 min pause for solvent evaporation. Two types
of films were investigated: (1) monolayers (“lift-off”
2.6. Pulsating bubble surfactometer
The dynamic surface tension lowering ability of
surfactant dispersions was measured at a cycling rate
of 20 cycles/min at 37 0.5 ◦C on a pulsating bub-
ble surfactometer (General Transco, Largo, FL). This
instrument, based on the original design of Enhorning
(1977), gives a physiologically relevant assessment of
overall surface activity that includes a combination of
dynamic film behavior and adsorption at physical con-
ditions similar to those in the pulmonary alveoli. An air
bubble, communicating with ambient air, was formed
in 40 ␮l of dispersed surfactant held in a sample cham-
ber mounted on a precision pulsator unit. The bubble
was pulsated between maximum and minimum radii of
0.55 and 0.4 mm (50% area compression), and surface
tension at minimum bubble radius (minimum surface
tension) was calculated as a function of time of pulsa-
tion from the pressure drop across the bubble interface
using the Laplace equation (Enhorning, 1977; Hall et
al., 1993; Notter, 2000). Surfactants were dispersed in
1.5 mM CaCl2 + 150 mM NaCl by probe sonication on
ice (three 15 s bursts at 40 W power) at concentrations
of 0.5 or 2.5 mg phosphonolipid/ml.
films) spread to a dilute initial surface concentration
2
˚
of 120–150 A /molecule and cycled at 10 min/cycle;
2
˚
(2) “surface excess” films spread to 15 A /molecule
and cycled at a rate of 5 min/cycle. Studies of surface
excess films are of particular interest for lung sur-
factants because they emphasize respreading behavior
during continuous cycling in the collapse regime where
the highest surface pressures (lowest surface tensions)
are reached. The compression ratio of the balance
was 4.35:1, and temperature was 23 1 ◦C. Dynamic
respreading in surface excess films was defined from
calculated π–A isotherm areas between compressions
2/1 and 7/1 as reported previously (Notter, 2000; Wang
et al., 1995). These isotherm areas (arbitrary units)
provide a measure of surfactant material ejected from
the interface during compression that re-incorporates
effectively back into the film during expansion (an
3. Results
Representative π–A isotherms for solvent-spread
films of S-lipid, SO2-lipid, DEPN-8, UnDEPN-8, and

Y. Chang et al. / Chemistry and Physics of Lipids 137 (2005) 77–93
81
Fig. 2. Surface pressure-area isotherms for spread monolayers of phosphonolipids in a Wilhelmy balance. (Panel A) S-lipid; (Panel B) SO2-
2
˚
lipid; (Panel C) DEPN-8; (Panel D) UnDEPN-8. Monolayers were spread in hexane–ethanol (9:1, v/v) to 120–150 A /molecule and compressed
at 10 min/cycle at 23 ◦C. Isotherms shown are representative first compressions from four to five closely reproduced experiments for each
compound. The isotherm for S-lipid is dotted above a surface pressure of 51 mN/m, where a change to lower compressibility occurred. Lift-off
areas (mean S.E.M.) for the complete isotherm groups for each compound are in Table 1.
DPPC are shown in Fig. 2 (monolayers) and Fig. 3
(surface excess films). Average monolayer lift-off areas
(the point where non-zero surface pressures were ini-
tially apparent during compression), as well as max-
imum surface pressures and respreading ratios from
surface excess films, are given in Table 1. Mono-
layer lift-off areas were similar for all phosphonolipids
(Table 1), and monolayers and surface excess films of
SO2-lipid, DEPN-8, and DPPC reached maximum sur-
face pressures of 72 mN/m (minimum surface tensions
<1 mN/m) during compression (Figs. 2 and 3). Mono-
layers of S-lipid had lower maximum pressures that
continued to increase after compression past collapse
lipid reached maximum surface pressures of 72 mN/m
on cycles 4–7, but had lower maximum pressures of
51–56 mN/m on cycles 1–3 (Fig. 3, Table 1). Mono-
layer and surface excess films of UnDEPN-8 had the
lowest maximum surface pressures (∼50 mN/m), con-
sistent with the fluid unsaturated C16:1 chain present in
this compound. S-lipid had the best respreading of any
compound studied, and all phosphonolipids had greatly
improved respreading compared to DPPC (Fig. 3,
Table 1). Both sulfur-containing phosphonolipids also
had greater respreading than DEPN-8 (Table 1).
In addition to studying solvent-spread interfacial
films in the Wilhelmy balance, the adsorption and
dynamic surface activity of phosphonolipids were also
investigated following dispersal in the aqueous phase.
based on the limiting area of the hydrocarbon chains
2
˚
(36 A /molecule) (Fig. 2A). Surface excess films of S-

82
Y. Chang et al. / Chemistry and Physics of Lipids 137 (2005) 77–93
As pure compounds, all phosphonolipids had improved
adsorption compared to DPPC, with overall adsorption
facility ordered as S-lipid ∼ SO2-lipid > UnDEPN-
8 > DEPN-8 > DPPC (Fig. 4). In binary mixtures, 9:1
DEPN-8:UnDEPN-8 had increased adsorption com-
pared to DEPN-8 alone, while 9:1 SO2-lipid:UnDEPN-
8 had reduced adsorption compared to SO2-lipid alone
(Fig. 5). In pulsating bubble experiments, DEPN-8
Fig. 3. Surface excess films of phosphonolipids compared to DPPC. (Panel A) S-lipid; (Panel B) SO2-lipid; (Panel C) DEPN-8; (Panel D)
2
◦
˚
UnDEPN-8; (Panel E) DPPC. Surface excess films were spread to 15 A /molecule and compressed at 5 min/cycle at 23 C. Isotherms shown
are representative of three to five closely reproduced experiments per compound, and include compression curves 1, 2, and 7 plus expansion
curve 1. In addition, compressions 3 and 4 are shown for S-lipid, which did not reach 72 mN/m until cycle 4. Table 1 gives maximum surface
pressures and respreading areas (mean S.E.M.) for the complete sets of isotherms obtained for each compound.

Y. Chang et al. / Chemistry and Physics of Lipids 137 (2005) 77–93
83
Table 1
Respreading areas and maximum surface pressures for spread interfacial films of synthetic phosphonolipids compared to DPPC on a Wilhelmy
balance
Compound
Dynamic respreading based on
isotherm areas between compressions
Maximum surface
pressure (mN/m)
Lift-off area
2
˚
(A /molecule)
Cycle 2/1
Cycle 7/1
DPPC
28.3 0.3
19.5 1.0
14.3 0.7
48 0.3
33.1 1.5
21.6 0.4
–
72
72
72
104
105
102
101
103
1
1
1
1
2
DEPN-8
SO2-lipid
S-lipid
0.2
0
72 (≥cycle 4)
UnDEPN-8
3.1 0.3
26.6 0.4
50
2
˚
Data are mean S.E.M. for n = 4–6. Lift-off areas are for monolayers initially spread to 120–150 A /molecule and compressed at 10 min/cycle
◦
2
˚
at 23 C. Respreading areas and maximum surface pressures are for surface excess films spread to 15 A /molecule (5 min/cycle, 4:35 to 1
compression, 23 ◦C). Largerrespreadingareas(arbitraryunits)indicatelessrespreading. MaximumsurfacepressuresforS-lipidweresignificantly
less than 72 mN/m for cycles 1–3 (51–56 mN/m). Data for DPPC are from Wang et al. (2002, 2003).
had the greatest overall dynamic surface activity of
the phosphonolipids studied, reaching minimum sur-
face tensions <1 mN/m at both 0.5 and 2.5 mg/ml
(Tables 2 and 3). Dispersions of SO2-lipid also reached
minimum surface tensions of <1 mN/m on the bubble at
2.5 mg/ml (Table 3), but had minimum surface tension
values of only 14.2 0.3 mN/m after 20 min of pulsa-
tion at 0.5 mg/ml (Table 2). S-lipid reached minimum
surface tensions of only 25.4 0.2 mN/m (0.5 mg/ml)
and 13.3 0.3 mN/m (2.5 mg/ml) after 20 min of pul-
sation on the bubble apparatus, and UnDEPN-8 had
the worst dynamic surface tension lowering ability as
a pure component (Tables 2 and 3).
In added studies related to synthetic exogenous sur-
factant development, phosphonolipids (DEPN-8, SO2-
lipid, and S-lipid) were examined for activity on the
bubble apparatus when combined with purified bovine
SP-B and/or SP-C. Synthetic surfactants containing
DEPN-8 or SO2-lipid plus either 1.5% SP-B or 0.75%
SP-B + 0.75% SP-C had very high activity, rapidly
reducing surface tension to <1 mN/m on the pulsat-
ing bubble (Tables 2 and 3). SP-C had significantly
less activity than SP-B when present individually with
Fig. 4. Surface pressure-time adsorption isotherms for aqueous dis-
persions of synthetic phosphonolipids. Phosphonolipids in 150 mM
NaCl + 1.5 mM CaCl2 were injected at time 0 into a stirred sub-
phase of the same solvent (final phosphonolipid concentration was
0.0625 mg/ml), and surface pressure was measured as a function of
time with a hanging Wilhelmy slide at 37 ◦C (Section 2). Adsorp-
tion curve for DPPC is from Wang et al. (2002, 2003). Data are
mean S.E.M. for n = 3–5.
Fig. 5. Adsorption of binary mixtures of phosphonolipids to the
air–water interface. Surfactants were injected at time 0 into a
stirred subphase as in the legend to Fig. 4 (final lipid concentra-
tion was 0.0625 mg/ml). Binary phosphonolipid mixtures contained
90 wt.% DEPN-8 or SO2-lipid plus 10 wt.% UnDEPN-8. Data are
mean S.E.M. for n = 3–5.

Table 2
Dynamic surface tension lowering of synthetic phosphonolipids with and without hydrophobic surfactant proteins at low concentration (0.5 mg lipid/ml)
Surfactant mixture
Dynamic surface tension (mN/m) at minimum bubble radius at
0.25 min
0.5 min
1 min
2 min
5 min
10 min
15 min
20 min
UnDEPN-8
S-lipid
SO2-lipid
DEPN-8
CLSE
57.8 2.8
48.2 1.1
20.0 0.3
38.0 1.2
17.6 0.4
50.9 1.9
44.5 1.2
19.7 0.4
26.7 3.4
14.8 1.4
27.5 0.4
41.7 1.1
19.4 0.5
22.2 2.5
10.9 2.2
25.8 0.4
35.8 0.6
18.7 0.6
17.7 1.8
9.2 2.1
25.3 0.1
30.6 0.5
16.6 0.5
12.0 1.8
6.8 2.0
25.5 0.2
28.1 0.3
15.3 0.3
8.6 1.0
< 1
25.4 0.4
25.7 0.3
14.2 0.3
4.7 1.0
25.1 0.1
25.4 0.2
14.2 0.3
< 1
S-lipid + 1.5% SP-B
S-lipid + 1.5% SP-C
S-lipid + 0.75% SP-B + 0.75% SP-C
36 1.1
43.8 1.6
22.8 1.5
33.6 1.2
40.5 2.0
20.7 1.3
30.1 1.1
36.6 2.1
18.9 0.9
25.8 2.1
31.8 1.7
16.7 0.9
22.9 1.0
26.9 0.7
15.1 0.5
20.7 0.4
24.1 0.2
13.6 0.4
18.6 0.4
23.5 0.1
11.4 0.3
17.6 0.2
23.3 0.0
10.3 0.4
SO2-lipid + 1.5% SP-B
SO2-lipid + 1.5% SP-C
SO2-lipid + 0.75% SP-B + 0.75% SP-C
19.1 1.0
18.5 0.7
7.8 2.6
18.4 1.2
17 0.7
4.1 2.1
16 0.3
15.7 0.5
1.8 1.5
11.6 1.6
14.8 0.2
< 1
9.0 1.0
14.1 0.3
6.9 0.7
13.1 0.3
< 1
10.6 0.4
8.8 0.2
DEPN-8 + 1.5% SP-B
DEPN-8 + 1.5% SP-C
DEPN-8 + 0.75% SP-B + 0.75% SP-C
19.6 1.0
25.6 1.5
10.9 0.8
17.9 0.9
23.9 1.1
8.3 1.2
14.7 0.3
22.4 1.2
5.7 2.0
10.7 0.8
17.3 0.6
3.1 1.6
4.9 0.2
11.5 0.3
1.2 1.2
< 1
6.1 0.6
< 1
1.3 0.5
< 1
Data are mean S.E.M. for n = 4–6. Surface tension at minimum radius (minimum surface tension) is on a bubble surfactometer (37 ◦C, 20 cycles/min, 50% area compression,
0.5 mg phospholipid/ml in 150 mM NaCl + 1.5 mM CaCl2).

Table 3
Dynamic surface tension lowering of synthetic phosphonolipids with and without hydrophobic surfactant proteins at high concentration (2.5 mg lipid/ml)
Surfactant mixture
Dynamic surface tension (mN/m) at minimum bubble radius at
0.25 min
0.5 min
1 min
2 min
5 min
10 min
15 min
20 min
UnDEPN-8
S-lipid
SO2-lipid
DEPN-8
CLSE
35.2 5.1
41.7 1.2
21.0 0.4
31.7 1.6
4.5 2.0
25.5 0.4
37.8 1.0
20.0 0.6
27.4 1.9
< 1
24.9 0.5
32.8 1.4
17.6 0.9
21.2 1.6
24.7 0.5
24.6 1.6
15.3 1.3
14.0 1.5
24.1 0.3
19.6 0.9
11.3 1.4
5.6 1.3
23.8 0.1
16.9 0.5
6.3 1.4
1.9 1.1
24.0 0.4
14.5 0.2
2.8 1.4
< 1
23.8 0.2
13.3 0.3
< 1
S-lipid + 1.5% SP-B
S-lipid + 1.5% SP-C
S-lipid + 0.75% SP-B + 0.75% SP-C
22.6 0.7
36.2 1.2
21.8 1.2
19.3 0.1
29.2 0.6
19.1 0.8
17.0 0.8
22.7 0.9
17.5 0.8
13.4 1.0
18.8 0.9
14.2 0.9
12.4 1.0
16.1 0.5
11.7 0.8
11.1 0.8
12.3 0.4
10.7 0.2
10.9 0.7
11.8 0.2
10.2 0.3
9.7 0.2
11.6 0.2
9.2 0.1
SO2-lipid + 1.5% SP-B
SO2-lipid + 1.5% SP-C
SO2-lipid + 0.75% SP-B + 0.75% SP-C
17.7
19.6 0.4
4.4 2.1
1
13.4 1.0
17.7 0.2
3.0 1.4
7.8 0.5
14.2 0.5
1.9 1.0
4.2 0.6
7.2 1.1
< 1
< 1
1.9 0.8
< 1
DEPN-8 + 1.5% SP-B
DEPN-8 + 1.5% SP-C
DEPN-8 + 0.75% SP-B + 0.75% SP-C
18.5 0.3
29.3 4.0
10.8 1.5
12.4 1.5
23.0 2.4
7.8 1.2
6.8 2.0
18.5 1.6
5.6 1.0
1.6 1.0
13.4 1.0
1.8 0.6
< 1
5.1 0.5
< 1
< 1
Data are mean S.E.M. for n = 4–6. Surface tension at minimum radius (minimum surface tension) is on a bubble surfactometer (37 ◦C, 20 cycles/min, 50% area compression,
2.5 mg phospholipid/ml in 150 mM NaCl + 1.5 mM CaCl2).

86
Y. Chang et al. / Chemistry and Physics of Lipids 137 (2005) 77–93
DEPN-8 or SO2-lipid in synthetic surfactants. How-
ever, overall dynamic surface activity was greatest for
surfactants that contained 0.75% SP-B + 0.75% SP-C
(Tables 2 and 3), indicating possible synergy between
these hydrophobic apoproteins that was most pro-
nounced at a low surfactant concentration of 0.5 mg/ml.
Addition of UnDEPN-8 at 10 wt.% did not improve
the surface activity of synthetic surfactants contain-
ing DEPN-8 or SO2-lipid + 0.75% SP-B + 0.75% SP-C
(Table 4 compared to Tables 2 and 3). In fact, addition
of 10% UnDEPN-8 had a negative rather than posi-
tive effect on overall dynamic surface activity for the
mixtures studied.
A final set of experiments examined the abil-
ity of synthetic surfactants to resist inhibition by
PLA2. When incubated with 0.1 units/ml of PLA2 for
30 min, syntheticsurfactantscontainingS-lipidorSO2-
lipid + 0.75% SP-B + 0.75% SP-C exhibited no chem-
ical degradation based on phosphate analysis of thin
layer chromatographic bands (Table 5). Surfactants
containing DEPN-8 + 1.5% of mixed bovine SP-B/C
have previously been shown to be resistant chemi-
cally to PLA2 (Wang et al., 2003), while glycerophos-
pholipids in CLSE are highly susceptible to PLA2-
induced degradation (Table 5). Consistent with the
structural resistance of phosphonolipids to degrada-
tionbyPLA2, syntheticsurfactantscontainingDEPN-8
or SO2-lipid + 0.75% SP-B + 0.75% SP-C maintained
high dynamic surface activity in the presence of this
lytic enzyme (Fig. 6). However, the surface activity of
CLSE on the bubble apparatus was severely impaired
by the presence of PLA2 (Fig. 6).
4. Discussion
This study has examined the surface-active bio-
physics of several novel phosphonolipids as pure com-
pounds and in synthetic lung surfactants containing
purified bovine hydrophobic SP-B and/or SP-C. All of
the disaturated C16:0 phosphonolipids studied (SO2-
lipid, DEPN-8, and S-lipid) were synthesized to have
structural analogy to DPPC, the most prevalent glyc-
erophospholipidinmammalianlungsurfactant(Fig. 1).
However, modified structural linkages and groups
(ether, sulfur, and phosphono) in analog compounds
were by design resistant to cleavage by biological phos-
pholipases (Lin et al., 1997; Wang et al., 2003). These

Y. Chang et al. / Chemistry and Physics of Lipids 137 (2005) 77–93
87
structural modifications also influence molecular pack-
ing and interactions relative to glycerophospholipids,
and thus affect surface tension lowering, adsorption,
and film spreading behavior. All of the C16:0 phos-
phonolipid analogs studied here had surface properties
that differed significantly from those of DPPC. In addi-
tion, SO2-lipid and S-lipid had substantial differences
in adsorption, spreading, and dynamic surface ten-
sion lowering compared to the diether phosphonolipid
DEPN-8 (Figs. 2–4, Tables 1–3). As a pure compound,
DEPN-8 had the greatest overall dynamic surface activ-
ity of any phosphonolipid investigated, followed by
SO2-lipid (Tables 2 and 3). DEPN-8 and SO2-lipid
had even greater activity when combined with purified
bovine SP-B or SP-B/SP-C in synthetic lung surfac-
tants (Tables 2 and 3, Fig. 6).
Our prior research has investigated the behavior of
DEPN-8 in surface films and aqueous dispersions (Liu
et al., 1994a, 1994b, 1995; Skita et al., 1995; Turcotte
et al., 1991, 1977; Wang et al., 2003). DEPN-8 has
a gel to liquid–crystal transition temperature (Tc) of
45 ◦C compared to 41 ◦C for DPPC (Liu et al., 1995;
Skita et al., 1995), and forms tightly packed interfacial
films able to reduce surface tension to <1 mN/m dur-
Fig. 6. Effects of PLA2 on the dynamic surface activity of synthetic
surfactants containing SO2-lipid or DEPN-8 + 0.75% SP-B + 0.75%
SP-C. Surface tension at minimum bubble radius was measured as
a function of time of pulsation on a bubble surfactometer (37 ◦C,
20 cycles/min, 50% area compression, 0.5 mg phosphonolipid/ml)
for dispersions of SO2-lipid or DEPN-8 combined with 1.5 wt.% of
purified bovine hydrophobic apoproteins (0.75% SP-B + 0.75% SP-
C) with and without PLA2 (0.1 units/ml). Also shown for comparison
is the activity of calf lung surfactant extract (CLSE) in the presence
and absence of PLA2. Data are mean S.E.M. for n = 6–8.

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Y. Chang et al. / Chemistry and Physics of Lipids 137 (2005) 77–93
ing dynamic compression (Liu et al., 1994a, 1994b;
Turcotte et al., 1991). DEPN-8 has also been shown to
have significantly greater adsorption and film respread-
ing than DPPC (Liu et al., 1994a, 1994b; Turcotte
et al., 1991). Ether linkages between the fatty chains
and glycerol backbone in DEPN-8 have greater flex-
ibility than ester linkages, and contribute to the abil-
ity of this compound to form interdigitated bilayers
that may enhance respreading and adsorption (Skita
et al., 1995). The relative hydrophobicity and size
of chain linkage groups in phosphonolipid molecules
also potentially affect surface behavior. The thioether
linkage in S-lipid was the most hydrophobic of those
studied, while the chain linkage of SO2-lipid was the
largest and most hydrophilic. Sulfur linkages in S-
lipid and SO2-lipid were associated with improved
adsorptionandfilmrespreadingcomparedtoetherlink-
ages in DEPN-8 (Table 1, Fig. 4). S-lipid had the
best respreading and adsorption of the C16:0 com-
pounds studied, suggesting that the thioether linkage
was particularly beneficial for these surface proper-
ties. However, the SO2-linkage was more beneficial to
dynamic surface tension lowering in SO2-lipid com-
pared to the thioether linkage in S-lipid (Tables 1–3).
Moreover, despite having reduced adsorption com-
paredtosulfur-linkedphosphonolipids, thedietherana-
log DEPN-8 had the greatest overall dynamic surface
activity on the pulsating bubble (Tables 2 and 3). Spe-
cific molecular biophysical mechanisms for the surface
property differences found between sulfur-linked and
ether-linked analogs were not addressed in our experi-
ments, and need to be elucidated in more detail in future
research.
An important emphasis in the present study was
on the overall dynamic surface activity of synthetic
lung surfactants containing phosphonolipids combined
with hydrophobic apoproteins purified from lavaged
bovine lung surfactant. Surface activity assessments
were done on surfactants that contained pure SP-B,
pure SP-C, and equal weights of the two together to
address potential apoprotein synergy. We have pre-
viously reported that a surfactant containing DEPN-
8 + 1.5 wt.% of mixed (unpurified) bovine SP-B/C had
very high surface activity that equaled or exceeded
that of CLSE (Wang et al., 2003). However, this prior
work did not examine DEPN-8 combined with pure
isolates of SP-B and SP-C as done here. Synthetic sur-
factants containing DEPN-8 or SO2-lipid plus 1.5%
SP-B were highly active, but dynamic surface ten-
sion lowering was found to be maximal for prepa-
rations that contained equal amounts by weight of
both hydrophobic proteins (0.75% SP-B + 0.75% SP-
C) (Tables 2 and 3). Evidence of apoprotein synergy
was particularly apparent in the activity of surfac-
tants containing SO2-lipid + 1.5% SP-B versus 0.75%
SP-B + 0.75% SP-C at a concentration of 0.5 mg/ml
(Table 2). Previous studies by Wang et al. (1996) did
not report appreciable synergy between bovine SP-B
and SP-C in mixtures with biological glycerophos-
pholipids. The small but noticeable synergy observed
here may reflect differences in interactions of these
apoproteins with synthetic phosphonolipids as opposed
to glycerophospholipids, as well as newer preparative
methodology used in purifying bovine SP-B and SP-C
(Baatz et al., 2001).
In addition to studying surfactant apoproteins com-
bined with C16:0 disaturated phosphonolipids in syn-
thetic surfactants, the C16:1 compound UnDEPN-8
was examined to assess the effects of a fluid sec-
ondary lipid on surface activity. Endogenous lung sur-
factant is known to contain a mix of rigid disaturated
phospholipids together with fluid unsaturated phospho-
lipids that have increased respreading ability (Notter,
2000). Addition of 10 wt.% of UnDEPN-8 improved
the adsorption of DEPN-8, but not that of SO2-lipid
(Fig. 5). Moreover, addition of 10% UnDEPN-8 did
not improve (and in fact was detrimental to) dynamic
surface tension lowering in synthetic surfactants con-
tainingDEPN-8orSO2-lipidplushydrophobicapopro-
teins (Table 4). Unsaturated lipids like UnDEPN-8
have decreased molecular packing ability relative to
disaturated lipids, and reach lower maximum surface
pressures (higher minimum surface tensions) during
dynamic compression. Although the addition of fluid
lipids can potentially increase adsorption and film
spreading in synthetic lung surfactants, phosphono-
lipids like DEPN-8 and SO2-lipid already have sig-
nificant improvements in these surface behaviors com-
pared to glycerophospholipids like DPPC. This may
lessen the need for secondary unsaturated zwitterionic
components in synthetic surfactants that contain these
active disaturated phosphonolipids.
The results of the present study are positive for
the general approach of using phospholipase-resistant
phosphonolipids like DEPN-8 or SO2-lipid in syn-
thetic surfactants for treating surfactant dysfunction

Y. Chang et al. / Chemistry and Physics of Lipids 137 (2005) 77–93
89
during inflammatory lung injury. Phospholipases are
known to be released in the lungs during states of
inflammatory injury in vivo (Holm et al., 1991; Touqui
and Arbibe, 1999; Vadas and Pruzanski, 1986). Lytic
enzymes including PLA2 are also found in meconium
(Schrama et al., 2001), which can be aspirated to cause
lung injury in newborns. A number of studies have
shown that phospholipases are directly inhibitory to
surfactant activity (Duncan et al., 1996; Enhorning et
al., 1992; Holm et al., 1991; Schrama et al., 2001;
Wang et al., 2003). Phospholipases not only degrade
active surfactant glycerophospholipids, but also pro-
duce reaction products (lysophosphatidylcholine, free
fatty acids) that can further inhibit surfactant function
(Hall et al., 1992a; Holm et al., 1999; Wang and Notter,
1998) and injure the alveolocapillary membrane (Hall
et al., 1990; Niewoehner et al., 1987). Ether-, sulfur-,
and phosphono-linkages in phosphonolipids are, on a
structural basis, not susceptible to cleavage by phos-
pholipases A1, A2, and D. The resistance of DEPN-8
to chemical degradation by phospholipases has been
directly demonstrated in previous studies (Lin et al.,
1997; Wang et al., 2003). Results here documented that
synthetic surfactants containing SO2-lipid or DEPN-
8 + 0.75% SP-B + 0.75% SP-C were not impaired in
surface activity by PLA2 (Fig. 6), and that sulfur-
containing phosphonolipids were chemically resistant
to degradation by this enzyme (Table 5). In contrast, the
active bovine lung surfactant extract CLSE was signif-
icantly impaired in surface tension lowering ability by
PLA2 (Fig. 6).
The most widely used current exogenous sur-
factants for treating RDS in premature infants are
animal-derived. Laboratory research and clinical trials
have documented that exogenous surfactant drugs
derived from animals (Infasurf (CLSE), Survanta,
Curosurf) have greater biophysical and physiological
activity than first-generation synthetic surfactants like
Exosurf and ALEC (Cummings et al., 1992; Egan et al.,
1983; Hall et al., 1992b; Hudak et al., 1996, 1997;
Mizuno et al., 1995; Notter, 2000; Seeger et al., 1993;
Vermont–Oxford Neonatal Network, 1996). However,
synthetic lung surfactants have significant potential
advantages over animal-derived surfactants as pharma-
ceutical products. Synthetic surfactants manufactured
in vitro have greater compositional reproducibility,
and also can incorporate novel molecular compo-
nents and properties such as structural resistance to
phospholipases as emphasized here. The composi-
tional complexity and batch-to-batch variability of
animal surfactants increases the scope and expense
of required quality control during manufacture. At
least in principle, synthetic drugs become increasingly
cost-effective over time once initial development
costs are recovered. Synthetic surfactants are also free
from concerns about prion-caused diseases like bovine
spongioform encephalitis that potentially affect animal
lung supplies. In addition to synthetic lipid components
like DEPN-8 or SO2-lipid as studied here, synthetic
lung surfactants can also in principle be formulated to
contain human-sequence peptide components, making
them less subject to cultural and religious considera-
tions compared to bovine or porcine preparations.
In summary, this paper has examined the sur-
face activity of several new synthetic phosphonolipids
including two C16:0 disaturated sulfur-containing
compounds (SO2-lipid and S-lipid) and a C16:1 unsat-
urated diether analog (UnDEPN-8). Also studied were
DEPN-8, a highly active disaturated ether-linked phos-
phonolipid synthesized in our prior work, as well as
the major lung surfactant glycerophospholipid DPPC
and the clinically relevant bovine surfactant extract
CLSE. Phosphonolipids were studied for adsorption,
film behavior, and overall dynamic surface activity
alone and in combination with purified SP-B and/or
SP-C from bovine lung surfactant. Synthetic lung sur-
factants containing DEPN-8 or SO2-lipid + 1.5% SP-
B or 0.75% SP-B + 0.75% SP-C had surface activity
approaching or equaling that of CLSE, with maxi-
mal activity found when both apoproteins were present
together. Mixtures of DEPN-8 or SO2-lipid + 0.75%
SP-B + 0.75% SP-C maintained high surface activity
when incubated with PLA2, while CLSE was inhibited
inactivitybythislyticenzyme. Sulfur-containingphos-
phonolipids (SO2-lipid and S-lipid) were also directly
shown to be chemically resistant to PLA2, as demon-
strated previously for DEPN-8.
Acknowledgements
The authors gratefully acknowledge the financial
support of the National Institutes of Health through
grant HL-56176 (RHN, BAH, ZW, AS) plus Pul-
monary Training Grant HL-66988 (YC) at the Univer-
sity of Rochester.

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Y. Chang et al. / Chemistry and Physics of Lipids 137 (2005) 77–93
Appendix A. Details on lipid starting materials
for preparing S-lipid, SO2-lipid, and UnDEPN-8
56–58 ◦C (literature 60.5–61.5 ◦C, Morris-Natschke et
al., 1986).
Detailed reaction intermediates and methods used
in the synthesis and purification of DEPN-8 have
been published previously by Notter, Turcotte and co-
workers (e.g., Turcotte et al., 1991; Wang et al., 2003).
In addition, some synthesis details for S-lipid and SO2-
lipid have also recently been reported (Chang et al.,
2004). A summary of the starting lipid compounds
used for preparing S-lipid, SO2-lipid, and UnDEPN-
8 is given below.
A.3. Synthesis of 1-S-hexadecyl-2-O-hexadecyl-3-
O-trityl-rac-thioglycerol
DMSO (20 ml) and powered KOH (2.62 g,
46.7 mmol) were mixed and stirred for 10 min,
followed by addition of 1-S-hexadecyl-3-O-
trityl-rac-thioglycerol (6.72 g, 11.7 mmol) and
1-bromohexadecane (7.13 g, 23.4 mmol) and stirring
for 8 h at 55 ◦C. Water (40 ml) was added, and the
mixture was then extracted with EtOAc (3 × 50 ml).
The combined organic layers were washed with
water, dried over MgSO4, filtered, evaporated and
purified by column chromatography (Al2O3, hexane)
to provide 1-S-hexadecyl-2-O-hexadecyl-3-O-trityl-
rac-thioglycerol, 94% yield; mp: 34–35 ◦C. 1H NMR
(CDCl3, 400 MHz): 7.20–7.45 (m, 15H), 3.46–3.54
(m, 3H), 3.21 (dd, J = 1.6, 4.8 Hz, 2H), 2.69 (ABX,
J = 5.2, 6.4, 13.6 Hz, 2H), 2.48 (t, J = 7.4 Hz, 2H),
1.49–1.58 (m, 4H), 1.26–1.32 (m, 52H), 0.88 (t,
J = 6.6 Hz, 6H); 13C NMR (CDCl3, 100 MHz): 144.1,
128.7, 127.7, 126.9, 86.6, 79.3, 70.7, 64.8, 34.2, 33.1,
31.9, 30.1, 29.8, 29.7, 29.6, 29.4, 29.3, 28.9, 26.2,
22.7, 14.1; IR (CH2Cl2): 3059, 3033, 2924, 1449,
1264 cm−1. Analysis, calculated for C54H86O2S: C,
81.10; H, 10.76; found: C, 81.28; H, 10.62.
A.1. Synthesis of 1-S-hexadecyl-rac-thioglycerol
1-Thioglycerol (11.59 g, 105 mmol) and 1-
bromohexadecane (30.5 g, 100 mmol) were dissolved
in 95% EtOH (180 ml). Ethanolic KOH solution
(1 M, 105 ml) was added dropwise over a period of
60 min with stirring under N2. The reaction mixture
was then stirred overnight (18 h), diluted with 1 l
of water, and cooled. The white crystalline sulfide
was collected, washed with cooled water (10 ◦C) and
recrystallized from methanol to obtain 1-S-hexadecyl-
rac-thioglycerol, 95% yield; mp: 65–66 ◦C (literature
69–70 ◦C, Morris-Natschke et al., 1986).
A.2. Synthesis of 1-S-hexadecyl-3-O-
trityl-rac-thioglycerol
A.4. Synthesis of 1-S-hexadecyl-2-O-
hexadecyl-rac-thioglycerol
1-S-hexadecyl-rac-thioglycerol(15.0 g,45.1 mmol),
triphenylmethyl chloride (15.1 g, 54.1 mmol) and
Et3N (5.47 g, 54.1 mmol) were dissolved in dry THF
(150 ml) and CH3CN (48 ml) at room temperature
and the mixture was refluxed for 8 h and cooled. The
resulting salt was filtered and washed with EtOAc
(100 ml). The combined organic phases were washed
with water (2 × 40 ml), 1% HCl (2 × 40 ml), saturated
NaHCO3 (100 ml), water and brine. After drying
over MgSO4, the mixture was filtered, evaporated,
and purified by column chromatography using 15:1
EtOAc:hexane as the elution solvent to give 1-S-
hexadecyl-3-O-trityl-rac-thioglycerol, 93% yield, mp:
1-S-hexadecyl-2-O-hexadecyl-3-O-trityl-rac-
thioglycerol (7.70 g, 9.63 mmol) and p-TsOH
(414 mg, 2.4 mmol) were dissolved in 95% methanol
(140 ml) and refluxed for 14 h. After cooling, the mix-
ture was extracted with 10:1 hexane:EtOAc, washed
with water, dried over MgSO4, filtered, evaporated,
and purified by flash chromatography (silica gel,
15:1 hexane:EtOAc) to provide known (Nali et al.,
1986) 1-S-hexadecyl-2-O-hexadecyl-rac-thioglycerol
in 85% yield, mp: 35–36 ◦C.

Y. Chang et al. / Chemistry and Physics of Lipids 137 (2005) 77–93
91
A.5. Synthesis of 1-SO2-hexadecyl-2-O-
hexadecyl-rac-sulfonylglycerol
extracted with 10:1 hexane:EtOAc, washed with water,
dried over MgSO4, filtered, evaporated, purified by
flash chromatography (silica gel, eluent 15:1 hexane:
EtOAc) to give 1-O-hexadecyl-2-O-Z-hexadec-9-enyl-
rac-glycerol in 85% yield, mp: 35–36 ◦C. H NMR
1
(CDCl3, 400 MHz): 5.29 (m, 2H), 3.66 (m, 1H), 3.55
(m, 2H), 3.47 (m, 4H), 3.39 (dt, J = 6.8 and 0.9 Hz,
2H), 2.34 (d, J = 5.8 Hz, 1H), 1.97 (m, 4H), 1.51 (m,
4H), 1.25 (m, 44H), 0.84 (m, 6H); 13C NMR (CDCl3,
100 MHz): 129.85, 127.74, 78.28, 71.77, 70.82, 70.33,
62.96, 31.89, 31.75, 30.03, 29.69, 29.66, 29.58, 29.46,
29.43, 29.41, 29.21, 28.94, 27.17, 27.14, 26.05, 22.64,
22.61, 14.0; IR (neat, cm−1): 3449, 3059, 3004, 2926,
2854, 1465, 1117; EIMS, m/z (%): 538 (M+, 15),
317 (92), 260 (53), 222 (78), 183 (100), 105 (64),
83 (82); HRMS, m/z: calculated for C35H70O3 [M]+:
538.5328; Found: 538.5338.
Synthesis procedures for final phosphonolipids
used 700–1100 mg of doubly functionalized glycerols
and generally followed previously published methods
(Chang et al., 2004; Wang et al., 2003) with the excep-
tion that crystallization was from 2:1 CHCl3/acetone
after flash chromatography. Final phosphonolipid com-
pounds were as follows.
1-SO2-hexadecyl-2-O-hexadecyl-rac-
sulfonylglycerol was prepared as described in
Chang et al. (2004).
A.6. 1-O-hexadecyl-2-O-Z-hexadec-9-enyl-3-O-
trityl-rac-glycerol
1-O-hexadecyl-3-O-trityl-rac-glycerol
(Stewart
and Kates, 1989) (810 mg, 1.45 mmol) and powdered
KOH (337 mg, 6 mmol) were dissolved in dry DMSO
(6 ml) at room temperature. C16H31OMs (500 mg,
1.54 mmol) in DMSO (3 ml) was added, and the
reaction mixture stirred at room temperature overnight
followed by heating to 60 ◦C for 2.5 h. After cooling
to room temperature, 5% NH4Cl (aqueous, 10 ml)
was added and the mixture was extracted with EtOAc,
washed with brine, dried over MgSO4 and purified by
flash chromatography (silica gel, 1:30 EtOAc:hexane)
to provide 1-O-hexadecyl-2-O-Z-hexadec-9-enyl-3-
O-trityl-rac-glycerol in 95% yield. 1H NMR (CDCl3,
400 MHz): 7.45–7.40 (m, 5H), 7.29–7.17 (m, 10H),
5.38–5.31 (m, 2H), 3.52 (m, 4H), 3.39 (t, J = 6.8 Hz,
2H), 3.17 (m, 2H), 2.01 (m, 4H), 1.61–1.49 (m, 4H),
1.26 (m, 45H), 0.88 (t, J = 6.8 Hz, 6H); 13C NMR
(CDCl3, 100 MHz): 144.1, 129.8, 129.0, 128.8, 128.7,
128.6, 127.7, 127.6, 126.8, 126.7, 86.5, 78.3, 71.6,
71.1, 70.6, 63.6, 31.9, 31.7, 30.1, 29.8, 29.7, 29.5,
29.4, 29.3, 28.9, 27.2, 26.1, 22.7, 22.6, 14.1; IR (neat,
cm−1): 3086, 3059, 3032, 3003, 2925, 2853.
S-lipid [( )-trimethyl(3-phosphonopropyl)ammo-
nium,
mono(2-hexadecyloxy-3-hexadecylsulfanyl-
propyl)ester]: 48% yield, mp: 203–204 ◦C (decompo-
sition). Spectral data have been reported previously
(Chang et al., 2004).
SO2-lipid [( )-trimethyl(3-phosphonopropyl)am-
monium, mono(2-hexadecyloxy-3-hexadecylsulfonyl-
propyl)ester]: 59% yield, mp: 234–235 ◦C (decompo-
sition). Spectral data have been reported previously
(Chang et al., 2004).
A.7. 1-O-hexadecyl-2-O-Z-hexadec-9-
enyl-rac-glycerol
UnDEPN-8 [( )-trimethyl(3-phosphonopropyl)
ammonium, mono(2-hexadec-9-enyloxy-3-hexadecyl-
oxypropyl)ester]: 51% yield as a waxy material.
1-O-hexadecyl-2-O-Z-hexadec-9-enyl-3-O-trityl-
rac-glycerol (3.97 g, 5.08 mmol) and p-TsOH (218 mg,
1.27 mmol) were dissolved in 95% methanol (140 ml)
and refluxed for 15 h. After cooling, the mixture was

92
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5.25 (m, 2H), 3.80 (m, 2H), 3.62–3.48 (m, 4H), 3.38
(m, 5H), 3.06 (s, 9H), 1.92 (m, 6H), 1.48 (m, 6H),
1.18 (m, 44H), 0.79 (t, J = 6.8 Hz, 6H); 13C NMR
(CDCl3:CD3OD = 1:1, 100 MHz): 129.2, 129.1, 77.6
(d, J = 6.8 Hz), 71.0, 70.0 (d, J = 10.7 Hz), 66.3 (d,
J = 12.3 Hz), 62.7, 52.1, 31.1, 31.3, 29.4, 29.1, 29.0,
28.9, 28.8, 28.7, 28.6, 28.3, 26.5, 25.5, 25.4, 21.9, 16.9,
13.1; 31P NMR (CDCl3:CD3OD = 1:1, 162 MHz): δ,
23.2; IR (CH2Cl2): 3051, 2926, 2854, 1467, 1264,
1052, 741 cm−1; LRMS, TOF MS CI (+ve ion), m/z
(%): 703 ((M + 1)+, 24), 397 (35), 381 (100); HRMS:
calculated for C41H85NO5P ([M + H]+): 702.6152;
found: 702.6165.
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