Synthesis and surface activity of diether-linked phosphoglycerols: Potential applications for exogenous lung surfactants

Article abstract of DOI:10.1016/j.bmcl.2006.09.083

The synthesis of three phosphoglycerols is described, one of which contains the previously unknown phosphonoglycerol headgroup. The surface tension-lowering capabilities of synthetic lung surfactant mixtures containing the PG analogs were measured on the

Full text of DOI:10.1016/j.bmcl.2006.09.083

Bioorganic & Medicinal Chemistry Letters 17 (2007) 113–117
Synthesis and surface activity of diether-linked phosphoglycerols:
Potential applications for exogenous lung surfactants
Robert H. Notter,a Zhongyi Wang,b Zhengdong Wang,a
Jason A. Davyb and Adrian L. Schwanb,*
aDepartment of Pediatrics, University of Rochester, Rochester, NY 14642, USA
bDepartment of Chemistry, University of Guelph, Guelph, Ont., Canada N1G 2W1
Received 11 August 2006; revised 24 September 2006; accepted 27 September 2006
Available online 30 September 2006
Abstract—The synthesis of three phosphoglycerols is described, one of which contains the previously unknown phosphonoglycerol
headgroup. The surface tension-lowering capabilities of synthetic lung surfactant mixtures containing the PG analogs were mea-
sured on the pulsating bubble surfactometer and compared to known controls. The PG-containing mixtures exhibited superior sur-
face tension-lowering properties indicating the significant potential of these analogs as components in synthetic exogenous lung
surfactants.
ꢀ 2006 Elsevier Ltd. All rights reserved.
A life-threatening deficiency of pulmonary surfactant is
the cause of the neonatal respiratory distress syndrome
(RDS), a common disease among preterm infants less
than 32 weeks gestation. In addition, dysfunction (inac-
tivation) of pulmonary surfactant is a major contributor
to the pathophysiology of clinical acute lung injury
(ALI) and the acute respiratory distress syndrome
(ARDS), which affect patients of all ages and have sub-
stantial mortality rates of 30–50 percent despite sophis-
ticated medical intensive care.1,2 Exogenous surfactant
replacement therapy is currently being used with tre-
mendous success to improve survival and minimize mor-
bidity in premature infants with RDS, and there is great
interest in extending this therapy to patients suffering
from ALI/ARDS.1–3
released into the pulmonary interstitium during inflam-
matory injury in vivo.4–7 Phospholipase A2 (PLA2) is
also present in meconium,8 a fetal product that can be
aspirated to cause lethal lung injury in newborns. Sever-
al studies have shown that phospholipases are directly
inhibitory to lung surfactant activity.5,8–11 These en-
zymes not only degrade glycerophospholipids, but also
produce reaction products such as free fatty acids and
lysophosphatidylcholines that biophysically inhibit lung
surfactant function12–14 and damage the integrity of the
alveolocapillary membrane.15,16 The current paper deals
with novel ether-linked phosphatidylglycerol (PG)-relat-
ed phospholipids or phosphonolipids that are designed
to be structurally resistant to degradation by phospho-
lipases A1, A2 (and also phospholipase D in the case
of PG phosphonolipids).
Surfactant therapy for ALI/ARDS is complicated by the
presence of inhibitory substances in injured inflamed
lungs, and requires that exogenous surfactants having
maximal surface activity and resistance to inactivation
be delivered effectively to the alveoli. In addition to bio-
physical inhibitors such as plasma proteins, lytic en-
zymes including phospholipases in injured lung tissue
can also detrimentally affect endogenous and exogenous
pulmonary surfactants. Phospholipases are known to be
Despite the vulnerability of glycerophospholipids to
phospholipase activity, almost all current mainstream
exogenous surfactants depend on 1,2-dipalmitoyl-sn-3-
phosphatidylcholine (DPPC) as a predominant surface
tension-lowering lipid component.1,17 A promising alter-
native to DPPC is its diether phosphonolipid analog 1,
designated DEPN-8, whose synthesis and high surface
activity have been reported previously.11,18–24 DEPN-8
reaches minimum surface tensions of <1 mN/m in
dynamically compressed surface films, and has superior
adsorption and respreading capabilities compared to
DPPC.20,21,23,24 When combined with purified lung sur-
factant proteins (SP)-B/C, DEPN-8 forms exogenous
Keywords: Lung surfactant therapy; Phosphoglycerols; Organic syn-
thesis; Surface tension.
*
Corresponding author. E-mail: schwan@uoguelph.ca
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.083

114
R. H. Notter et al. / Bioorg. Med. Chem. Lett. 17 (2007) 113–117
surfactants of very high activity that are resistant to deg-
radation or inhibition if exposed to PLA2.11,19 The pres-
ent study reports new PG-related analogs that further
increase the activity of synthetic lung surfactants con-
taining DEPN-8 (Fig. 1).
O
P
i
ii
C16H33
O
OH
Cl
Cl
C16H33
O
O
RO
RO
O OH
P
iii
2,3
C16H33
O
O
O
O
Although zwitterionic phospholipids like DPPC are key
tensoactive components in endogenous lung surfactant,
anionic lipids including PGs also play important roles in
surface-active behavior. In particular, PG compounds
have been shown to selectively interact with SP-B, the
most biophysically active apoprotein in endogenous sur-
factant.1,25–30 Accordingly, we have targeted surface-ac-
tive phospholipase-resistant PG analogs to combine
with DEPN-8 in novel lung surfactants. In this communi-
cation, we report the synthesis of three diether PG targets
and provide preliminary results indicating the high sur-
face activity of mixtures containing these compounds.
O
RO
Figure 2. Reagents and conditions for synthesis of PG analogs 2 and 3.
(i) POCl3, Et3N, THF; (ii) 1—rac-solketal, Et3N, THF, 2—1 M
Na2CO3 (aq); (iii) 70% HOAc in H2O, 48% over three steps for 2; 43%
for 3.
O
P
EtO
O
EtO
O
5
Based on available 1,2-disubstituted glycerols,11,19 the
laboratory synthesis of lipids 2 and 3 proceeded smooth-
ly using well-established protocols.31–33 Phosphorylation
with POCl3 was followed by treatment with solketal to
complete the installation of the PG headgroup. Deace-
talization using 70% HOAc in water and then flash chro-
matography offered lipids 2 and 3 in 48% and 43%
yields, respectively (see footnote to data and experi-
ment).34 The double bond of 3 was not perturbed during
its synthesis, as indicated by 1H and 13C NMR spectros-
copy. Unsaturated lipid 3 is a new entity, and although
lipid 2 has been prepared in the past,35–38 the chemistry
herein represents the first fully chemical synthesis of the
acid form of 2. Prior syntheses of 2 have largely been
based on the work of Comfurius,39 who demonstrated
the introduction of the glycerol unit by the enzymatic
action of phospholipase D (Fig. 2).
Recognizing that dealkylation of the phosphonate must
not perturb the protecting group of the 3,4-diol, we
turned to benzoate groups for the latter. To this end,
lithium dimethyl methanephosphonate was reacted with
glycidyl benzoate (6) in the presence of BF3ÆOEt2, using
a protocol already described for glycidyl ethers.43 The
free hydroxyl group of 7 was readily converted to the
second benzoate, giving 8. The phosphonate was then
converted to the bis(trimethylsilyl) ester (9) using
TMSBr for immediate conversion to 10.44 Dichloride
10 was then captured with 1,2-bis(hexadecyl)glycerol
affording protected lipid 11. Phosphonoglycerol 4 was
obtained through a methanolysis reaction and was iso-
lated in 65% yield after chromatography and recrystalli-
zation. Lipid
4
represents the first known
phosphonoglycerol bearing the 3,4-dihydroxybutyl
hydrogen phosphonate headgroup, regardless of the
functional groups linking the fatty alkyl chains to the
glycerol backbone (Fig. 3).45
The preparation of phosphonoglycerol 4 proved more
challenging than for compounds 2 or 3. It was initially
believed that phosphonate 5, prepared by the Michae-
lis–Arbuzov reaction of triethyl phosphite with 4-(2-iod-
oethyl)-2,2-dimethyl-1,3-dioxolane,40 could be used as a
source of the phosphonoglycerol headgroup, and that
eventual 1,2-disubstituted glycerol attachment would
form the basis of the preparation. However, in accord
with literature precedent,41,42 phosphonate 5 could not
be selectively dealkylated to the phosphonic acid with
TMSBr or related reagents without competitive
deacetalization.
The overall dynamic surface tension-lowering ability of
synthetic lung surfactants containing 9:1 (molar ratio)
DEPN-8/PG-2, DEPN-8/PG-3, or DEPN-8/PG-4 with
and without added 1.5% (by wt.) of bovine SP-B/C
were measured with a pulsating bubble surfactometer
(Table 1).46 Assessments of overall surface activity on
O
P
MeO
O
P
Y
O
P
MeO
i
ii
MeO
Y
MeO
OBz
OBz
7
OH
OBz
8 (Y = OMe)
9 (Y = OTMS)
10 (Y = Cl)
iii
iv
O O-
N+
O OH/O-
P
P
C16H33
O
O
O OH
P
C16H33
O
1, DEPN-8
C16H33
O
O
X
OH
v
vi
RO
OH
C16H33
O
O
OBz
4
O O-
P
C16H33
O
OBz
2, R = hexadecyl, X = O
3, R = hexadec-9-enyl, X = O
4, R = hexadecyl, X = CH2
N+
11
C15H31CO2
O
O
Figure 3. Reagents and conditions for synthesis of PG phosphonolipid
4. (i) 1—nBuLi, THF, then BF3ÆOEt2 and 6, À78 ꢁC to rt, 75%; (ii)
BzCl, Et3N, DMAP, CH2Cl2, 92%; (iii) 2.2 equiv TMSBr, CH2Cl2; (iv)
3 equiv (COCl)2, cat DMF, CH2Cl2; (v) 1—rac-1,2-bis(hexadecyl)glyc-
erol, Et3N, CHCl3, 2—Amberliteꢂ resin, 65% from 7; (vi) 1—MeOH/
CH2Cl2 (1:2), K2CO3 (anhyd), 2—H3O+, 65%.
C15H31CO2
DPPC
Figure 1. Schematic diagram of the previously synthesized diether
phosphonolipid compound DEPN-8 (compound 1) and its relation to
DPPC and to the phosphoglycerol analogs (compounds 2–4) of the
current paper.

R. H. Notter et al. / Bioorg. Med. Chem. Lett. 17 (2007) 113–117
115
Table 1. Dynamic surface activities of synthetic lung surfactants containing DEPN-8 and a PG analog (PG-2, -3 or -4) with and without 1.5% bovine
SP-B/C
Surfactant mixture
Surface tension (mN/m) at minimum bubble radius at time (min)
0.25
0.5
1
2
5
10
15
20
DEPN-8
32
28
25
29
2
1
1
1
27
26
24
28
2
1
1
1
21
19
17
19
2
1
2
1
14
12
10
11
2
2
2
1
6
5
2
4
1
2
1
1
2
1
<1
9:1 DEPN-8/PG-2
9:1 DEPN-8/PG-3
9:1 DEPN-8/PG-4
<1
<1
<1
DEPN-8 + 1.5% SP
13
10
8
1
1
0
1
7
3
3
4
1
1
0
1
4
2
2
2
1
1
0
1
3
0
<1
9:1 DEPN-8/PG-2 + 1.5% SP
9:1 DEPN-8/PG-3 + 1.5% SP
9:1 DEPN-8/PG-4 + 1.5% SP
<1
<1
<1
11
DPPC
9:1 DPPC/POPG
9:1 DPPC/POPG + 1.5% SP
66
45
17
1
2
2
64
36
15
1
2
2
60
28
11
1
2
2
41
25
5
1
2
1
29
22
2
1
1
1
25
21
<1
1
1
23
19
0
1
21
18
0
1
Data are means SEM for n = 3–5. All surfactants were studied at a uniform total lipid concentration of 2.5 mg/ml on a pulsating bubble
surfactometer (General Transco, Largo, FL) at 37 ꢁC, 20 cycles/min, and 50% area compression. Surfactants were dispersed in 0.15 M NaCl + 2 mM
CaCl2, and a tiny air bubble was formed and pulsated between maximum and minimum radii of 0.55 and 0.4 mm while the pressure drop across the
air–water interface of the bubble was measured with a precision transducer. Surface tension at minimum bubble radius (minimum surface tension)
was calculated from the measured pressure drop using the Laplace equation.46 Abbreviations: DPPC, dipalmitoyl phosphatidylcholine; POPG,
palmitoyl-oleoyl phosphatidylglycerol; SP, mixture of surfactant proteins B/C isolated chromatographically from lavaged calf lung surfactant.11
this instrument reflect the combined effects of adsorp-
tion and dynamic film compression at a cycling rate
(20 cycles/min), temperature (37 ꢁC), and area compres-
sion (50% from maximum to minimum area) relevant
for the mammalian lungs in vivo.1,46,47 The mixed bo-
vine SP-B/C used in activity studies was isolated on an
LH-20 column from extracted calf lung surfactant, as
described previously by Wang et al.11 The presence of
a PG analog (PG-2, -3, or -4) was beneficial for overall
surface tension lowering in synthetic lung surfactants
with DEPN-8. The overall surface activities of 9:1 mix-
tures of DEPN-8 with PG-2, -3, or -4 were increased
slightly but consistently compared to DEPN-8 alone
(Table 1). Similarly, the overall surface activities of syn-
thetic lung surfactants containing 9:1 DEPN-8/PG-2, -3,
or -4 + 1.5% bovine SP-B/C all showed a small but con-
sistent increase relative to DEPN-8 + 1.5% bovine SP-B/
C. The high surface activities of mixtures of 9:1 DEPN-
8/PG-2, -3, or -4 with and without 1.5% bovine SP-B/C
also greatly exceeded the activity of corresponding con-
trol mixtures containing the ester-linked glycerophos-
pholipids DPPC and 1-palmitoyl-2-oleoyl-PG (POPG).
In particular, the surface activities of mixtures of 9:1
DEPN-8/PG-2, -3, or -4 on the pulsating bubble were
all much greater than the activity of 9:1 DPPC/POPG,
and the surface activities of 9:1 DEPN-8/PG-2, -3, or -
4 + 1.5% bovine SP-B/C were also all substantially
greater than the activity of 9:1 DPPC/POPG + 1.5% bo-
vine SP-B/C (Table 1).
potential to interact with amphipathic lung surfactant
proteins or related synthetic peptides in exogenous sur-
factants. Anionic PG glycerophospholipids in native
lung surfactant have previously been reported to have
specific interactions with surfactant apoproteins or syn-
thetic peptides.1,25–30 Preliminary biophysical assess-
ments of model lung surfactants containing 9:1
DEPN-8/PG-2, 9:1 DEPN-8/PG-3, or 9:1 DEPN-8/
PG-4 with or without added bovine SP-B/C indicated
that the presence of the PG component enhanced the
already high surface activity of corresponding surfac-
tant mixtures containing DEPN-8. Moreover, the sur-
face activities of mixtures containing 9:1 DEPN-8/PG-
2, -3, or -4 were greater than corresponding mixtures
containing the ester-linked glycerophospholipids 9:1
DPPC/POPG. These results indicate that mixtures of
ether-linked PG analogs combined with DEPN-8 or
related zwitterionic phosphonolipids plus surfactant
proteins/peptides have significant potential utility as
synthetic exogenous lung surfactants. Further research
detailing the interfacial biophysics and physiological
activity of synthetic lung surfactants of this kind is
clearly warranted. Because of the high surface activity
and molecular structural phospholipase-resistance of
phosphonolipids, synthetic surfactants containing these
compounds may be particularly applicable for use in
clinical syndromes like ALI/ARDS where inflammatory
pulmonary injury is present.
In terms of significance relating to organic chemistry,
the preparation of the diether PG compounds reported
in this paper includes novel contributions to phospho-
lipid synthesis. This is particularly true for the synthesis
of the phosphonoglycerol analog 4, which is noteworthy
in its potential to incorporate chirality in both the 3-car-
bon glycerol-based fragment and the 4-carbon butanol-
based fragment. The preparation of additional phospho-
noglycerol compounds for use in synthetic lung surfac-
tants is currently underway.
To summarize, this paper has reported the synthesis of
three ether-linked PG lipids designed to have structural
resistance to cleavage by phospholipase A1 and A2
(PG-2, -3, and -4) as well as to phospholipase D (PG-
4). The anionic nature of these compounds at neutral
pH affords the potential for enhanced intermolecular
interactions with zwitterionic phosphonolipids and
phospholipids in synthetic lung surfactants. These new
phospholipase-resistant PG compounds also have the

116
R. H. Notter et al. / Bioorg. Med. Chem. Lett. 17 (2007) 113–117
24. Skita, V.; Chester, D. W.; Oliver, C. J.; Turcotte, J. G.;
Acknowledgments
Notter, R. H. J. Lipid Res. 1995, 36, 1116.
25. Biswas, N.; Shanmukh, S.; Waring, A. J.; Walther, F.;
Wang, Z.; Chang, Y.; Notter, R. H.; Dluhy, R. A.
Biophys. Chem. 2005, 113, 223.
26. Shanmukh, S.; Biswas, N.; Waring, A. J.; Walther, F.;
Wang, Z.; Chang, Y.; Notter, R. H.; Dluhy, R. A.
Biophys. Chem. 2005, 113, 233.
27. Baatz, J. E.; Elledge, B.; Whitsett, J. A. Biochemistry 1990,
29, 6714.
28. Yu, S.-H.; Possmayer, F. Biochim. Biophys. Acta 1992,
1126, 26.
The authors gratefully acknowledge the financial sup-
port of the National Institutes of Health through Grant
HL-56170.
Supplementary data
1
Supplementary data is available showing copies of H
and 13C NMR spectra of lipids 2, 3, 4 (6 pages). Supple-
mentary data associated with this article can be found,
in the online version, at doi:10.1016/j.bmcl.2006.09.083.
29. King, R. J.; MacBeth, M. C. Biochim. Biophys. Acta 1981,
647, 159.
30. Perez-Gil, J.; Casals, C.; Marsh, D. Biochemistry 1995, 34,
3964.
31. Harlos, K.; Eibl, H. Biochemistry 1980, 19, 895.
32. Ishihara, M.; Sano, A. Chem. Pharm. Bull. 1996, 44,
1096.
33. Stepanov, A. E.; Shvets, V. I. Chem. Phys. Lipids 1986, 41,
1.
34. Preparative procedures and characterization data for PG’s 2
and 3. rac-1,2-Dihexadecyl-3 phosphoglycerol (2). Freshly
distilled POCl3 (410 mg, 2.64 mmol) was cooled in an ice-
References and notes
1. Notter, R. H. Lung Surfactants: Basic Science and Clinical
Applications; Marcel Dekker,: New York, 2000.
2. Notter, R. H.; Finkelstein, J. N.; Holm, B. A. Lung Injury:
Mechanisms, Pathophysiology and Therapy; Taylor Fran-
cis Group, Inc.: Boca Raton, 2005.
3. Lewis, J. F.; Brackenbury, A. Crit. Care Med. 2003,
31(suppl), S324.
bath.
A solution of 1,2-dihexadecylglycerol (1.19 g,
2.20 mmol) in 15 mL THF and Et3N (334 mg, 3.3 mmol)
was added dropwise to the phosphorus oxychloride while
stirring continuously. Stirring was continued at rt for 2 h.
After recooling to 0 ꢁC, a solution of solketol (349 mg,
2.64 mmol) and Et3N (668 mg, 6.6 mmol) in 10 mL THF
was added dropwise. After 2 h at rt, the reaction mixture
was filtered by suction and 1 M Na2CO3 (aq) was added to
the filtrate and stirring continued for 15 h at rt. Acetone
(30 mL) was added to the upper THF phase and the
precipitate was removed by filtration. The filtrate was
evaporated to dryness. The residue was dissolved in a
solution of HOAc/H2O (70:30, v/v) and was stirred for 2 h.
The lipid was extracted with CHCl3 and purified by flash
chromatography (eluent CHCl3:MeOH = 10:1) to yield
730 mg of 2, 48% yield. Characterization data for rac-1,2-
dihexadecyl-3 phosphoglycerol (2): mp 157–159 ꢁC; IR
(neat, mmax): 3396, 2917, 2850, 1467, 1384, 1226, 1116, and
4. Kim, D. K.; Fukuda, T.; Thompson, B. T.; Cockrill, B.;
Hales, C.; Bonventre, J. V. Am. J. Physiol. 1995, 269,
L109.
5. Holm, B. A.; Kelcher, L.; Liu, M.; Sokolowski, J.;
Enhorning, G. J. Appl. Physiol. 1991, 71, 317.
6. Touqui, L.; Arbibe, L. Mol. Med. Today 1999, 5, 244.
7. Vadas, P.; Pruzanski, W. Lab. Invest. 1986, 55, 391.
8. Schrama, A. J. J.; de Beufort, A. J.; Sukul, Y. R. M.;
Jansen, S. M.; Poorthuis, B. J. H. M.; Berger, H. M. Acta
Paediatr. 2001, 90, 412.
9. Duncan, J. E.; Hatch, G. M.; Belik, J. Can. J. Physiol.
Pharmacol. 1996, 74, 957.
10. Enhorning, G.; Shumel, B.; Keicher, L.; Sokolowski, J.;
Holm, B. A. J. Appl. Physiol. 1992, 73, 941.
11. Wang, Z.; Schwan, A. L.; Lairson, L. L.; O’Donnell, J. S.;
Byrne, G. F.; Foye, A.; Holm, B. A.; Notter, R. H. Am. J.
Physiol. 2003, 285, L550.
12. Hall, S. B.; Lu, Z. R.; Venkitaraman, A. R.; Hyde, R. W.;
Notter, R. H. J. Appl. Physiol. 1992, 72, 1708.
13. Holm, B. A.; Wang, Z.; Notter, R. H. Pediatr. Res. 1999,
46, 85.
14. Wang, Z.; Notter, R. H. Am. J. Respir. Crit. Care Med.
1998, 158, 28.
15. Hall, S. B.; Notter, R. H.; Smith, R. J.; Hyde, R. W. J.
Appl. Physiol. 1990, 69, 1143.
16. Niewoehner, D.; Rice, K.; Sinha, A.; Wangensteen, D. J.
Appl. Physiol. 1987, 63, 1979.
17. Chess, P.; Finkelstein, J. N.; Holm, B. A.; Notter, R. H. In
Ref. 2; 17 pp 617.
18. Chang, Y.; Wang, Z.; Notter, R. H.; Wang, Z.; Long, Q.;
Schwan, A. L. Bioorg. Med. Chem. Lett. 2004, 14, 5983.
19. Chang, Y.; Wang, Z.; Schwan, A. L.; Wang, Z.; Holm, B.
A.; Baatz, J. E.; Notter, R. H. Chem. Phys. Lipids 2005,
137, 77.
20. Liu, H.; Lu, R. Z.; Turcotte, J. G.; Notter, R. H. J. Colloid
Interface Sci. 1994, 167, 378.
21. Liu, H.; Turcotte, J. G.; Notter, R. H. J. Colloid Interface
Sci. 1994, 167, 391.
22. Liu, H.; Turcotte, J. G.; Notter, R. H. Langmuir 1995, 11,
101.
23. Turcotte, J. G.; Lin, W. H.; Pivarnik, P. E.; Sacco, A. M.;
Bermel, M. S.; Lu, Z.; Notter, R. H. Biochim. Biophys.
Acta 1991, 1084, 1.
1060 cmÀ1 1H NMR (CDCl3/CD3OD = 4:1, 400 MHz):
;
3.92 (m, 4H), 3.80 (m, 1H), 3.54–3.62 (m, 6H), 3.35–3.50
(m, 3H), 1.56 (m, 4H), 1.26–1.36 (m, 52H), 0.88 (t,
J = 6.8 Hz, 6H) ppm; 13C NMR (CDCl3/CD3OD = 4:1,
100 MHz): 77.4, 71.6, 70.7, 70.4, 70.2, 66.3, 64.7, 62.2,
31.7, 29.7, 29.5, 29.3, 29.1, 25.8 (d, J = 6.1 Hz), 22.4,
13.8 ppm; 31P NMR (CDCl3/CD3OD = 4:1, 162 MHz):
5.8 ppm; HRMS, ESI TOF (Àve), m/z: calcd for
C38H78O8P [MÀH]À: 693.5434; found: 693.5395.Charac-
terization data for rac-1-hexadecyl-2-hexadec-9-encyl-3-
phosphoglycerol (3): IR (neat, mmax): cmÀ1: 3420, 3195,
3010, 2921, 2851, 1467, 1256, 1240, 1132, 1101, 1058 cmÀ1
;
1H NMR (CDCl3/CD3OD = 4:1, 400 MHz): 5.30 (m, 2H),
3.90–3.84 (m, 4H), 3.74 (pent, J = 4.9 Hz, 1H), 3.63–3.49
(m, 6H), 3.45–3.38 (m, 3H), 1.98 (m, 4H), 1.51 (m, 4H),
1.23 (s, 44H), 0.84 (m, 6H), ppm; 13C NMR (CDCl3/
CD3OD = 4:1, 100 MHz): 129.6, 129.4, 77.6 (d,
J = 8.2 Hz), 71.4, 70.9 (d, J = 5.1 Hz), 70.3, 70.2, 66.1 (d,
J = 5.7 Hz), 64.7, 62.2, 57.2, 31.6, 31.4, 29.7, 29.4, 29.3,
29.2, 29.0, 28.9, 28.6, 26.8, 25.7 (d,J = 3.2 Hz), 22.3 (d,
J = 3.1 Hz), 13.2 ppm; 31P NMR (CDCl3/CD3OD = 4:1,
162 MHz): 5.3 ppm; HRMS, ESI TOF (+ve), m/z: calcd
for C38H78O8P [M+H]+: 693.5434; found: 693.5430.
35. Bittova, L.; Sumandea, M.; Cho, W. J. Biol. Chem. 1999,
274, 9665.
36. Cevc, G. Biochemistry 1987, 26, 6305.
37. Das, S.; Cho, W. J. Biol. Chem. 2002, 277, 23838.

R. H. Notter et al. / Bioorg. Med. Chem. Lett. 17 (2007) 113–117
117
38. Kertscher, H. P.; Gawrisch, K.; Brezesinski, G. Pharmazie
1984, 39, 150.
39. Comfurius, P.; Zwaal, R. F. A. Biochim. Biophys. Acta
1977, 488, 36.
40. Chen, J.; Prestwich, G. D. J. Org. Chem. 1998, 63, 430.
41. Arth, H.-L.; Sinerius, G.; Fessner, W.-D. Liebigs Ann.
1995, 2037.
42. Lalinde, N.; Tropp, B. E.; Engel, R. Tetrahedron 1983, 39,
2369.
43. Bittman, R.; Byun, H. S.; Mercier, B.; Salari, H. J. Med.
Chem. 1994, 37, 425.
44. Bhongle, N. N.; Notter, R. H.; Turcotte, J. G. Synth.
Commun. 1987, 17, 1071.
45. Key experimental procedures leading to PG 4. Preparation
of dibenzoyl protected lipid 11. Neat TMSBr (0.39 mL,
3.06 mmol) was added dropwise to a solution of com-
pound 8 (546 mg, 1.39 mmol) dissolved in CH2Cl2 (4 mL)
stirring at À30 ꢁC. After 40 min., the mixture was stirred
at rt for 3 h. Removal of the solvent under anhydrous
conditions, afforded a thick oil (9). To this oil at rt were
added CH2Cl2 (5 mL), DMF (two drops), and oxalyl
chloride (0.36 mL, 4.17 mmol). The solution was stirred at
rt for 30 min. during which time gas evolved. The solvent
was removed under anhydrous conditions and residue was
heated to 50 ꢁC under vacuum for 1 h. The resulting oil
(10) was diluted with CHCl3 (30 mL) and stirred at 0 ꢁC.
To this solution was added dropwise and slowly a solution
of Et3N (0.19 mL, 1.39 mmol) and 1,2-dihexadecylglycerol
(376 mg, 0.695 mmol) in CHCl3 (30 mL). The mixture was
allowed to warm to rt and was stirred for 48 h. After
addition of water (0.7 mL), the mixture was stirred for 1 h.
The majority of solvent removed under vacuum and to the
residue was added 15 mL of the CHCl3/MeOH/
H2O = 10:10:1 solvent system and 18 mL of Amberliteꢂ
ion exchange resin. The mixture was stirred for 1 h, was
filtered, and the resin washed with the same solvent
system. The combined solvents were removed under
vacuum. The resulting residue was dissolved in CH2Cl2,
washed with brine, and dried over MgSO4. After filtration
and concentration, the residue was triturated into hot
hexanes (3·). The hexanes were removed under vacuum
and crude compound 11 was purified by flash chromatog-
raphy (eluent with MeOH/CHCl3 = 1:20) to get 399 mg of
the protected phosphonolipid (11), 65% yield. Data for 11:
1H NMR (CDCl3, 400 MHz): 7.92 (m, 4H), 7.42 (m, 2H),
7.31 (m, 4H), 4.50–4.47 (m, 1H), 4.35 (m, 1H), 3.97–3.91
(m, 2H), 3.48-3.28 (m, 6H), 2.07 (m, 2H), 1.8 (m, 2H), 1.42
(m, 4H), 1.16 (m, 54H), 0.79 (t, J = 6.6 Hz, 6H) ppm; 13C
NMR (CDCl3, 100 MHz): 166.0, 165.9, 133.1, 132.9,
129.7, 129.6, 128.3, 77.5, 72.0, 71.7, 70.6, 70.1, 65.2, 64.2,
60.3, 31.9, 29.9, 29.7, 29.6, 29.5, 29.3, 26.0 (d, J = 4.3 Hz),
22.6, 14.1 ppm; 31P NMR (CDCl3, 162 MHz): 31.4 ppm;
IR (neat, mmax): 3300, 3063, 2917, 2850, 1723, 1265,
1071 cmÀ1.rac-2,3-Bis(hexadecyloxy)propyl hydrogen 3,4-
dihydroxybutylphosphonate (4). Phosphonate 11 (364 mg,
0.40 mmol) was dissolved in MeOH/CH2Cl2 (1:2, 20 mL).
K2CO3 (anhyd, 223 mg, 1.62 mmol) was added and the
mixture was stirred at rt for 18 h. Water (5 mL) was added
and the pH was adjusted to 2–3 with6 M HCl. The
mixture was extracted with CH2Cl2/MeOH = 2:1
(2 · 20 mL) and the organic extracts were washed with
brine, dried over MgSO4, and filtered through Celiteꢂ.
After removal of the solvent, the residue was purified by
flash chromatography (eluent from MeOH/CHCl3 = 1:10
to MeOH/CHCl3 = 1:4) to get 169 mg of phosphonolipid
4, 65% yield.Characterization data for 4: IR (neat, mmax):
3392, 2917, 2850, 1457, 1180, 1072 cmÀ1 1H NMR
;
(CDCl3, 400 MHz): 3.90 (m, 2H), 3.75 (m, 1H), 3.63–
3.56 (m, 5H), 3.51–3.46 (m, 4H), 1.75–1.69 (m, 4H), 1.57
(m, 4H), 1.29 (m, 52H), 0.89 (t, J = 6.6 Hz, 6H) ppm; 13C
NMR (CDCl3, 100 MHz): 77.7 (d, J = 6.0 Hz), 71.7 (d,
J = 9.1 Hz), 71.3, 70.2, 69.9, 65.1, 63.1, 31.4, 29.5, 29.2,
29.1, 29.0, 28.9, 25.6 (d, J = 4.4 Hz), 22.2, 13.4 ppm; 31P
NMR (CDCl3, 162 MHz): 29.6 ppm; HRMS, ESI TOF
(+ve), m/z: calcd for C39H82O7P [M+H]+: 693.5798; found:
693.5753.
46. Enhorning, G. J. Appl. Physiol. 1977, 43, 198.
47. Hall, S. B.; Bermel, M. S.; Ko, Y. T.; Palmer, H. J.;
Enhorning, G. A.; Notter, R. H. J. Appl. Physiol. 1993, 75,
468.