Synthesis of macrocyclic diacyl/dialkyl glycerols containing disulfide tether and studies of their effects upon incorporation in DPPC membranes. Implications in the design of phospholipase A2 modulators

Article abstract of DOI:10.1021/jo980866b

A general method for the preparation of novel disulfide-tethered macrocyclic diacylglycerols (DAGs) has been described. Overall synthesis involved stepwise protection, acylation, and deprotection to yield the bis(ω-bromoacyl) glycerols. In the crucial macrocyclization step, a unique reagent, benzyltriethylammonium tetrathiomolybdate (BTAT), has been used to convert individual bis(ω-bromoacyl) glycerols to their respective macrocyclic disulfides. DAG 6, which had ether linkages between hydrocarbon chains and the glycerol backbone, was also synthesized from an appropriate precursor using a similar protocol. One of the DAGs (DAG 5) had a carbon- carbon tether instead of a disulfide one and was synthesized using modified Glaser coupling. Preparation of α-disulfidetethered DAG (DAG 4) required an alternative method, as treatment of the bisbromo precursor with BTAT gave a mixture of several compounds from which separation of the target molecule was cumbersome. To avoid this problem, the bisbromide was converted to its corresponding dithiocyanate, which on further treatment with BTAT yielded the desired DAG (DAG 4) in good yield. Upon treatment with the reducing agent dithiothreitol (DTT), the DAGs that contain a disulfide tether could be quantitatively converted to their 'open-chain' thiol analogues. These macrocyclic DAGs and their reduced 'open-chain' analogues have been incorporated in DPPC vesicles to study their effect on model membranes. Upon incorporation of DAG 1 in DPPC vesicles, formation of new isotropic phases was observed by ³¹P NMR. These isotropic phases disappeared completely on opening the macrocyclic ring by a reducing agent. The thermotropic properties of DPPC bilayers having DAGs (1-6) incorporated at various concentrations were studied by differential scanning calorimetry. Incorporation of DAGs in general reduced the cooperativity unit (CU) of the vesicles. Similar experiments with reduced 'open-chain' DAGs incorporated in a DPPC bilayer indicated a recovery of CU with respect to their macrocyclic 'disulfide' counterparts. The effect of inclusion of these DAGs on the activity of phospholipase A₂ (PLA₂) was studied in vitro. Incorporation of DAG 1 in DPPC membranes potentiated both bee venom and cobra venom PLA₂ activities.

Full text of DOI:10.1021/jo980866b

9232
J . Org. Chem. 1998, 63, 9232-9242
Syn th esis of Ma cr ocyclic Dia cyl/Dia lk yl Glycer ols Con ta in in g
Disu lfid e Teth er a n d Stu d ies of Th eir Effects u p on In cor p or a tion
in DP P C Mem br a n es. Im p lica tion s in th e Design of P h osp h olip a se
A2 Mod u la tor s
Santanu Bhattacharya,*,† Sangita Ghosh,†,‡ and Kalpathy R. K. Easwaran‡
Department of Organic Chemistry and Molecular Biophysics Unit, Indian Institute of Science,
Bangalore 560 012, India
Received May 7, 1998
A general method for the preparation of novel disulfide-tethered macrocyclic diacylglycerols (DAGs)
has been described. Overall synthesis involved stepwise protection, acylation, and deprotection to
yield the bis(ω-bromoacyl) glycerols. In the crucial macrocyclization step, a unique reagent,
benzyltriethylammonium tetrathiomolybdate (BTAT), has been used to convert individual bis(ω-
bromoacyl) glycerols to their respective macrocyclic disulfides. DAG 6, which had ether linkages
between hydrocarbon chains and the glycerol backbone, was also synthesized from an appropriate
precursor using a similar protocol. One of the DAGs (DAG 5) had a carbon-carbon tether instead
of a disulfide one and was synthesized using modified Glaser coupling. Preparation of R-disulfide-
tethered DAG (DAG 4) required an alternative method, as treatment of the bisbromo precursor
with BTAT gave a mixture of several compounds from which separation of the target molecule was
cumbersome. To avoid this problem, the bisbromide was converted to its corresponding dithiocy-
anate, which on further treatment with BTAT yielded the desired DAG (DAG 4) in good yield.
Upon treatment with the reducing agent dithiothreitol (DTT), the DAGs that contain a disulfide
tether could be quantitatively converted to their “open-chain” thiol analogues. These macrocyclic
DAGs and their reduced “open-chain” analogues have been incorporated in DPPC vesicles to study
their effect on model membranes. Upon incorporation of DAG 1 in DPPC vesicles, formation of
new isotropic phases was observed by 31P NMR. These isotropic phases disappeared completely
on opening the macrocyclic ring by a reducing agent. The thermotropic properties of DPPC bilayers
having DAGs (1-6) incorporated at various concentrations were studied by differential scanning
calorimetry. Incorporation of DAGs in general reduced the cooperativity unit (CU) of the vesicles.
Similar experiments with reduced “open-chain” DAGs incorporated in a DPPC bilayer indicated a
recovery of CU with respect to their macrocyclic “disulfide” counterparts. The effect of inclusion of
these DAGs on the activity of phospholipase A2 (PLA2) was studied in vitro. Incorporation of DAG
1 in DPPC membranes potentiated both bee venom and cobra venom PLA2 activities.
In tr od u ction
These changes within the membrane are thought to
create “defects” within the bilayer,5 which in turn make
the substrate (phospholipids) more accessible to the
membrane-bound enzymes.6 Such processes may there-
fore be relevant to the understanding of the pathways
through which the DAGs exert their biological activities.
Previous studies have shown that selected structural
modifications in DAGs lead to pronounced changes in
their membrane associated regulatory enzymic properties
in vitro.6d All naturally occurring DAGs containing two
saturated acyl chains are known to perturb the mem-
brane at the lipid-water interface, while their unsatur-
Diacylglycerols (DAGs) are the precursors of phospho-
lipids and have been shown to play an important role in
controlling the activity of membrane-bound enzymes such
as protein kinase C (PKC) and phospholipase A2 (PLA2).1
Certain DAGs have the ability to modify the organiza-
tional properties of the membrane, such as inducing
bilayer to hexagonal or other nonbilayer phase formation2
or membrane fusion3 affecting membrane curvature.4
* To whom correspondence should be addressed. Also at the Chemi-
cal Biology Unit, J awaharlal Nehru Centre for Advanced Scientific
Research, Bangalore 560 012, India. E-mail: sb@orgchem.iisc.ernet.in.
Fax: +91-80-3341683, +91-80-3443529 (direct). Tel: +91-80-3092664.
† Department of Organic Chemistry, Indian Institute of Science.
‡ Molecular Biophysics Unit, Indian Institute of Science.
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10.1021/jo980866b CCC: $15.00 © 1998 American Chemical Society
Published on Web 11/14/1998

Synthesis of Macrocyclic Diacyl/Dialkyl Glycerols
J . Org. Chem., Vol. 63, No. 25, 1998 9233
Ch a r t 1
glycerols. In the present study, we present the full
details of the syntheses of altogether six macrocyclic
DAGs, including the ones described in earlier com-
munication. Newly synthesized DAGs include one with
a short chain (C6) macrocyclic disulfide unit. Another one
contains an ether type of hydrocarbon chain-glyceryl
backbone connector (dialkylglycerol), and the third con-
tains a carbon-carbon tethering at the chain termini
instead of a disulfide connection. Upon completion of the
synthesis and adequate characterization of the newly
described DAG analogues, we included a few of them in
DPPC membranes and determined their abilities to
modulate the activities of cobra venom and bee venom
PLA2 enzymes. We also describe herein the results of
incorporation of a few of these macrocyclic DAGs into
DPPC membranes as revealed from the examination of
calorimetric and 31P spectroscopic studies of various
DAG-DPPC mixed coaggregates.
Resu lts a n d Discu ssion
Ba ck gr ou n d . The DAGs (1-6) possess unique struc-
tural features as the acyl or alkyl chains are connected
to form a macrocyclic ring and could therefore be con-
sidered as putative precursors of lipids of archaebacterial
origin. The role of macrocyclic rings in archaebacterial
lipids in providing thermal stability to the membranes
has not been established yet but are expected to provide
a very different lipid packing and overall membrane
property, which might have bearing in membrane stabil-
ity and permeability and in the activities of various
membrane-bound enzymes.
Very few reports of macrocyclic models of archaebac-
terial lipids are known9 because any macrocyclization
reaction is inevitably accompanied by unwanted inter-
molecular reactions thereby giving polymer products.
Menger et al. synthesized9d a number of macrocyclic
phospholipids in which the two acyl chains are joined at
the end by a carbon-carbon single bond and showed that
such restrictions in chain mobility result in the formation
of thermally stable membranes. To understand the effect
of restriction of chain mobility on overall membrane
structure and its correlation to enzyme activity, we chose
a disulfide connector between the acyl chains of the
diacylglycerols in the present study.
To make sure that the effect of incorporation of these
DAGs (if any) is due to the imposed restriction in chain
mobility and not due to the presence of bulkier sulfur
atoms, which might lead to size incompatibility and
therefore packing differences when incorporated within
the membrane bilayers, we also synthesized a control
macrocyclic DAG 5 where instead of a disulfide link-
age a carbon-carbon tether is introduced by following
modified Glaser coupling conditions developed by Menger
et al.9d
ated counterparts such as dioleoylglycerol (DOG) perturb
the membrane only deep within the bilayer. Zidovetzki
et al.6d have also shown that activation of PLA2 due to
DAG incorporation in model membranes depends on
subtle perturbations at different depths of the membrane
bilayer. Hence it would be desirable to develop systems
in which the hydrocarbon part is modified to cause
perturbation at a specific position within the bilayer.
With such systems, one can look for the effect of such a
perturbation to find out if any correlation could be made
between PLA2 activation and the exact location within
the membrane at which perturbation is required for an
enhanced activity of the enzyme.
Keeping these facts in mind, we synthesized macrocy-
clic 1,2-DAG analogues (1-6) in which the acyl/alkyl
chains are connected at different positions to restrict the
extents of chain motions within the membrane (Chart
1). Because of the use of a disulfide connection, these
macrocycles can be conveniently opened up from disulfide
to the corresponding DAG-containing bisthiols via an in
situ reductive step.7 This also enables us to compare the
effect of macrocyclization with respect to that for its
analogue in which chain motions are not restricted.
In a preliminary communication,8 we reported the first
synthesis of three disulfide-tethered macrocyclic diacyl-
Syn th esis. When we initiated this study, a survey of
the available methods for the synthesis of lipids revealed
that there were limitations inherent in macrocyclization
(8) Ghosh, S.; Easwaran, K. R. K.; Bhattacharya, S. Tetrahedron
Lett. 1996, 37, 5769-5772.
(9) (a) Eguchi, T.; Arakawa, K.; Terachi, T.; Kakinuma, K. J . Org.
Chem. 1997, 62, 1924-1933. (b) Eguchi, T.; Kano, H.; Kakinuma, K.
Chem. Commun. 1996, 365. (c) Herbert, N.; Beck, A.; Lennox, R. B.;
J ust, G. J . Org. Chem. 1992, 57, 1777. (d) Menger, F. M.; Chen, X. Y.;
Brocchini, S.; Hopkins, H. P.; Hamilton, D. J . Am. Chem. Soc. 1993,
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114, 9227. (f) Kim, J .-M.; Thompson, D. H. Langmuir 1992, 8, 637-
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Chem. Soc. 1985, 107, 42-47.

9234 J . Org. Chem., Vol. 63, No. 25, 1998
Bhattacharya et al.
Ta ble 1. Su m m a r y of Sa lien t F ea tu r es of
Dia cylglycer ols Syn th esized a n d Isola ted Yield s of th e
F in a l Ma cr ocycliza tion Step
dithiocyanate in quantitative yields by stirring 11d with
excess NH4SCN in acetone at ambient temperature. Bis-
(2-thiocyanatopalmitoyl)glycerol was subsequently con-
verted to the corresponding DAG R,R-disulfide by treating
the bisthiocyanate with 2.2 equiv of the same reagent
(BTAT), which in this case acts via reductive dimerization
rather than by simple sulfur transfer.12
DAGa
macrocycle ring size depthb (Å) time (h) % yield
1
2
3
4
5
6
38
30
18
10
26
30
20.5
14.7
7.0
2.8
12.7
16.5
3
5
4
2
55
62
65
70
75
45
DAG 5, which consists of a macrocycle with an all-
carbon backbone, was synthesized as shown in Scheme
2. Briefly, benzylglycerol was obtained from isopropyli-
dene glycerol. This was then acylated with the corre-
sponding R,ω-alkynoic acid to yield bis(10,10′-undecyn-
oyl)benzylglycerol. This was then subjected to Glaser
coupling conditions (CuCl, TMEDA, xylene, O2) to obtain
bis(10,11-docosandiyne-1,22-dioyl) benzylglycerol 16 in
∼75% yield. This was then hydrogenated to remove the
benzyl group to afford DAG 5 in excellent yield. The
other DAG 6 that has been reported here has a different
structural feature. This contains an ether type hydro-
carbon-glycerol backbone linkage instead of a diester
connection, and the two chains in this molecule are
tethered via a disulfide bridge at the chain termini. The
macrocyclization step was similar to that adopted in
Scheme 1 for other disulfide formation. However, syn-
thesis of the precursor bis(ω-bromoalkyl)glycerol required
etherification as shown in Scheme 2.
Red u ction of th e Disu lfid es. Since our idea was to
see the effect of restriction of chain mobility within the
membrane bilayers of DPPC, it became imperative for
us to convert the disulfide tether to the corresponding
free chain DAG (containing thiols) by opening the mac-
rocycle and look for any change in the properties at the
membrane level. This would involve first the reduction
of the intramolecular disulfide bridge of the diacylglyc-
erols and then its incorporation in DPPC membranes.
Hence, we decided to perform prior reduction of the
macrocyclic disulfides in solution and characterize the
resulting product to ensure a quantitative extent of
disulfide to thiol conversion. First, reduction of DAG 3
(0.6 mmol) was attempted in ethanol upon incubation
6
a
b
For actual structure, see Chart 1. Depth indicates the length
at which the tether is likely to be present within the membrane.
procedures.10 Therefore, development of new procedures
for giant ring closures was necessary as a prerequisite
to the studies described herein.
Thus, the crucial step was the intramolecular disulfide
formation to give the macrocyclic product. The major
problem in this regard is the competing intermolecular
disulfide formation resulting in polymeric products.
Mindful of this limitation in widely adopted procedures
for disulfide formation, we chose to employ a unique
reagent, benzyl triethylammonium tetrathiomolybdate
(BTAT),11 in the key macrocyclization step. This reagent
acts as a sulfur transfer agent and, due to its templating
mechanism, unambiguously gives the desired intramo-
lecular disulfide-containing DAG in good yields in a single
step (Table 1). Although a number of procedures for
preparing simple saturated 1,2-DAGs are known, the
approach we adopted is direct, and the protection and
deprotection steps are less cumbersome. At the same
time, this affords good yields of the intermediates before
the last macrocyclization step is reached.
Multistep syntheses of various disulfide-tethered di-
acylglycerols (Scheme 1) are detailed in the Experimental
Section. Following this protocol, the bis(ω-bromoacyl)
glycerols that were candidates for macrocyclization were
obtained in good yield in only three steps starting from
the commercially available starting material 1,2-isopro-
pylideneglycerol. Once these bis(ω-bromoacyl)glycerols
were synthesized, treatment with 2 equiv of the reagent
benzyl triethylammonium tetrathiomolybdate (BTAT)
gave us the desired intramolecular disulfide as the only
isolable product. The reaction mixture in the sulfur-
transfer step was first subjected to a workup. Upon
workup, the material on a silica gel coated TLC plate
showed a major (>90%) spot at Rf ≈ 0.5 in addition to a
minor spot at the base in hexane/EtOAc (4:1). By column
chromatography, the material corresponding to the Rf ≈
0.5 was isolated. Subsequent spectral and analytical
characterization established the identity of this material
as the intramolecular disulfide. The material corre-
sponding to the minor spot at the base of the TLC plate
originated from the formation of inorganic tetrathiomo-
lybdate related material.
with dithiothreitol (DTT) (13.2 mmol) for 30 min.
A
qualitative inspection of thin layer chromatographic
plates (silica gel) eluted in hexane/EtOAc (4:1) indicated
the presence of components lying at Rf ≈ 0.2, with
complete disappearance of spot at Rf ≈ 0.5, which corre-
sponds to unreduced disulfide DAG. Their 1H NMR
spectra also confirmed the conversion of disulfide to thiol
(Figure 1D). Thus the -CH2-CH2-S-S- peak which
1
appears in the H NMR spectrum at 2.7 ppm as a triplet
in the disulfide DAG changes to a multiplet at 2.55 ppm
due to formation of -CH2-CH2-SH upon treatment with
the reducing agent. To further confirm and quantify the
extent of reduction of the disulfide DAG to the corre-
sponding dithiol analogue, Ellman’s reaction13 was also
performed on the product isolated after DTT treatment.
The disulfide DAG at a concentration of 0.07 mM showed
a residual absorbance of 0.043 with no maximum at 412
nm, whereas the corresponding reduced product showed
an absorbance of ∼0.6 with the maximum at 412 nm
(Figure 1B). All other disulfide-containing macrocyclic
DAGs could be reduced to their open chain thiol-contain-
ing analogues by the above procedure.
It is noteworthy that the MEM deprotection was also
equally successful on the macrocyclic disulfide-containing
MEM-protected DAG. For DAG 4, the corresponding
bromide being at the R carbon of the carbonyl group gave,
however, an inseparable mixture of mono- and disulfides
upon treatment with BTAT. This problem was overcome
by converting the dibromo precursor to the corresponding
(10) Breitenback, J .; Boosfeld, J .; Vo¨gtle, F. In Comprehensive
Supramolecular Chemistry; Vo¨gtle, F., Eds.; Elsevier Science Ltd:
Oxford, U.K. 1996, Vol. 2, pp 29-67.
(11) Ramesha, A. R.; Chandrasekaran, S. J . Org. Chem. 1994, 59,
1354-1357.
(12) Prabhu, K. R.; Ramesha, A. R.; Chandrasekaran, S. J . Org.
Chem. 1995, 60, 7142-7143.
(13) Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70.

Synthesis of Macrocyclic Diacyl/Dialkyl Glycerols
J . Org. Chem., Vol. 63, No. 25, 1998 9235
Sch em e 1a
a
Reagents and conditions: (a) NaH, THF, MEMCl, 0 °C, N2, 1.5 h, 80%; (b) p-TsOH, MeOH-H2O, RT, 2 h, 91%; (c) RCO2H, DCC,
DMAP, CCl4, 4 h, [R ) R1, 65%; R ) R2, 87%; R ) R3, 60%; R ) R4, 93%]; (d) TiCl4, CH2Cl2, N2, 0 °C, 1 h, 80%; (e) NH4SCN, acetone, RT,
6h, 75%; (f) (BnNEt3)2MoS4, DMF, RT, [R ) R1, 3 h, 55%; R ) R2, 3 h, 62%; R ) R3, 5 h, 62%; R ) R4, 2 h, 69%].
Ca lcu la tion of En er gy-Min im ized Con for m a tion s.
Molecular mechanics calculations using the INSIGHT II
2.3.5 package (DISCOVER, Biosym. Technologies) show
energy-minimized, preferred conformations of the mac-
rocyclic DAGs and also give a reasonable idea about the
depth at which the tether is positioned within the
membrane (Table 1).
For the purpose of calculation of energy-minimized
conformations of the DAGs, the basic glyceryl backbone-
hydrocarbon skeleton of each of these DAGs has been
taken from the known crystal structure of phospholip-
ids.14 This structure was then modified by inserting a
disulfide bond at an appropriate position on the hydro-
carbon chains along with removal of the phosphatidyl-
choline portion of the phospholipid. Then each of these
structure was subjected to the minimization as described
above. After several iterations, the energy of the con-
formation did not vary, and we considered this minimized
structure for measuring the depth of the tether even if
this might not be the exact global minima for the DAG.
It is gratifying to mention here that the energy-min-
imized structure for the corresponding dilauryl glycerol
obtained by the above-described procedure is in close
agreement with that of the reported crystal structure14b
of the same compound. Since the rationale behind this
calculation was to have an idea about the comparative
positions of the tether, we feel that this calculation serves
our purpose.
Figure 2 shows energy-minimized structure of DAGs
(1-6) along with the structures of naturally occurring
dipalmitoylglycerol (DPG). In the case of the open chain
DPG, the two acyl (palmitoyl) chains are seen to move
away from each other. J oining the two chains at the end
makes the molecule adopt a rodlike structure. At the
same time, tethering introduces a “bulge” at the level of
the disulfide linkage at a depth of ca. 20.5 Å. A similar
rodlike appearance is also observed for the -CH2-CH2-
end-linked structure in DAG 5, although in this molecule
there is no significant distortion at the tethering sites.
However, this DAG being the one with shorter acyl
chains, the tethering is at a depth of ∼12.7 Å, which when
incorporated in DPPC membranes should be around the
middle of the hydrocarbon part of the bilayer leaflets. In
the case of DAG 2, where chain tethering is around the
middle (∼14.7 Å) of the chain (C-12), a rodlike shape is
formed up to the level where the macrocyclic ring is
joined. The portions of the two acyl chains that are not
tethered remain free and move away from each other.
For DAG 4, in which the disulfide is located at the R
position with respect to the ester carbonyl group, the
(14) (a) Pearson, R. H.; Pascher, I. Nature 1979, 281, 499. (b)
Pascher, I.; Sundell, S.; Hauser, H. J . Mol. Biol. 1981, 153, 791.

9236 J . Org. Chem., Vol. 63, No. 25, 1998
Bhattacharya et al.
Sch em e 2a
anisotropic line shape characteristic of phospholipids in
a typical bilayer arrangement. As the temperature was
increased beyond Tm to ∼50 °C, the asymmetric line
shape narrowed indicating that a gel to liquid-crystalline
phase transition had taken place.16 A noteworthy feature
of this spectrum is the appearance of an additional
isotropic peak even with a very small concentration of
DAG (∼1 mol %), which is also relatively small in terms
of total integrated area. The presence of this isotropic
component is indicative of a situation where, due to the
presence of DAGs, some of the DPPC molecules undergo
rapid motion thereby completely averaging the chemical
shift anisotropy (csa). This peak corresponds to either
formation of regions of higher curvature or nonlamellar
phases within the bilayer surface.17 This means that the
incorporation of macrocyclic DAG having tethering at the
end of the hydrocarbon chain within DPPC bilayers
produces local disturbances in packing, which appear as
different entities only on melting of the main bilayer. As
mentioned earlier, it might be these regions of nonlamel-
lar phases that play a role in increasing the susceptibility
of the bilayer to PLA2 attack. However, on incorporation
of a high concentration of DAG (>20 mol %), the line
shape narrows showing reduced anisotropy with a de-
crease in the ∆σcsa to 16 ppm from that of 30 ppm in case
of parent DPPC vesicle (not shown). Interestingly, on
incorporation of the corresponding reduced DAG at the
same concentration (1-3 mol %) in the DPPC bilayer,
no such isotropic phases were observed even beyond the
phase transition temperature. Reduction of the disulfide
opens up the macrocycle and thereby relieves the mol-
ecules of the gauche conformations, which are created
as a consequence of chain tethering in macrocycles. This
renders the reduced DAGs more “acceptable” to the
parent DPPC bilayer, indicating that a packing distur-
bance in the hydrocarbon part of the bilayer determines
the formation of nonamellar phases. The results of
incorporation of most of the other disulfide DAGs in
DPPC membranes in their disulfide were similar. Thus,
upon reduction to their open chain nonmacrocyclic forms,
no isotropic phases could be seen. All the other macro-
cylic disulfide-tethered DAGs showed isotropic phases
above the phase transition temperature. Such isotropic
phases disappeared when similar experiments were done
with their reduced analogues. It is tempting to speculate
that these macrocyclic DAGs might be potential activa-
tors of phospholipase A2. It was, however, intriguing to
find that DAG 5, which had an all-carbon macrocycle,
did not show an isotropic peak at 50 °C. Since the length
of the hydrocarbon chain is smaller in this DAG 5, it may
be possible that the packing arrangement of this DAG
within the DPPC membrane is totally different from that
of DAG 1, which is unable to induce isotropic phase.15
a
Reagents and conditions: (a) (i) PhCH2Cl, KOH, toluene,
reflux, 16 h, 56%; (ii) p-TsOH, MeOH-H2O, RT, 2 h, 95%; (b)
t-(CH2)8CO2H, DCC, DMAP, CCl4, RT, 4 h, 80%; (c) CuCl,
TMEDA, xylene, 140 °C, O2, 6 h, 75%; (d) Pd/C, THF-EtOH, H2,
40 psi, 3 h, 60%; (e) NaH, THF, R/Br, reflux, 24 h, 45%; (f) TiCl4,
CH2Cl2, N2, 0 °C, 58%; (g) (BnNEt3)2MoS4, DMF, RT, 7 h, 45%.
energy-minimized structure is quite different from those
of the other DAGs. Here, the disulfide linkage, in order
to accommodate itself, adopts a totally different confor-
mation around the glycerol backbone region.
31P NMR Sp ectr oscop y. Naturally occurring DAGs
when incorporated within bilayer membranes produce
nonlamellar phases, which appear as isotropic signals in
31P NMR.6c,15,16 It is these nonlamellar phases that are
believed to expose the substrates (phospholipids) to the
membrane-bound enzyme PLA2. To ascertain the effect
of the macrocyclic DAGs on the phase polymorphism of
dipalmitoylphosphocholine (DPPC) membranes, we in-
vestigated specific DAG/DPPC mixed aggregates by 31P
NMR spectroscopy. The 31P NMR spectra were recorded
both above (50 °C) and below (25 °C) the phase transition
temperatures for aqueous dispersions of DPPC codis-
persed with different concentrations of DAG 1 (Figure
3). These studies show that in the gel phase, i.e., below
the phase transition temperature of DPPC membranes,
the spectra are similar for concentrations ranging from
1 to 30 mol % of DAG 1 and consist of broad, axially
Th er m otr op ic P h a se Beh a vior . The temperature
at which a phospholipid bilayer is half converted from a
solid gel phase into a fluid phase is the gel to liquid
crystalline phase transition temperature (Tm). Such
phase transitions are in general a highly cooperative
event. The cooperativity unit (CU)18 gives an idea about
the number of lipid molecules that undergo melting
(15) (a) Ortiz, A.; Aranda, F. J .; Villa´lain, J .; San Martin, C.; Micol,
V.; Go´mez-Fernandez, J . C. Chem. Phys. Lipids 1992, 62, 215-224.
(b) Basa´n˜ez, G.; Nieva, J . L.; Rivas, E.; Alonso, A.; Gon˜i, F. M. Biophys.
J . 1996, 70, 2299-2306.
(16) Ghosh, S.; Easwaran, K. R. K.; Bhattacharya, S. Unpublished
results.
(17) (a) Heimburg, T.; Weurz, U.; Marsh, D. Biophys. J . 1992, 63,
1369-1378. (b) Lopez-Garcia, F.; Villa´lain, J .; Gomez-Fernandez, J .
C.; Quinn, P. J . Biophys. J . 1994, 66, 1991-2004.
(18) The cooperative unit is defined as follows: cooperative unit )
(∆Hv/∆Hc), where ∆Hv is the van’t Hoff enthalpy and ∆Hc is the
calorimetric enthalpy.

Synthesis of Macrocyclic Diacyl/Dialkyl Glycerols
J . Org. Chem., Vol. 63, No. 25, 1998 9237
F igu r e 1. Comparison of the UV-vis and 1H-NMR (300 MHz) spectra of DAG 3 and its reduced analog. Panel A shows the
UV-vis spectrum of DAG 3 in ethanol upon treatment with Ellman’s reagent. Panel B shows the UV-vis spectrum of DAG 3 in
ethanol after treatment with DTT followed by addition of Ellman’s reagent. Panel C shows the 1H-NMR spectrum of DAG 3.
Panel D shows the 1H-NMR spectrum of DAG 3 upon DTT treatment followed by purification of the product.
together. Hence, when the DAGs are more homoge-
neously distributed, the CU of transition should be high.
A summary of the calorimetric data that characterize
these phase transitions for various DPPC/DAG mixed
aggregates is presented in Table 2. It is clear that the
solid to fluid bilayer melting temperatures of DPPC in
different coaggregates are little affected upon incorpora-
tion of various DAGs, even up to ∼10 mol %. Comparison
of the enthalpies of transition as well as the cooperativity
unit of a DAG 1/DPPC bilayer with its reduced DAG/
DPPC bilayer clearly indicates a recovery of these
parameters on reduction of the macrocyclic DAG to its
corresponding free chain thiol analogue. The same is the
case for other disulfide DAGs and their reduced ana-
logues. This observation again substantiates our obser-
vation in 31P NMR spectroscopy that opening of the
macrocylic DAG by reduction makes it more “adaptable”
to the DPPC bilayer thereby making a more homoge-
neous bilayer and hence showing the higher cooperativity
value. The observation of disappearance of the isotropic
peak at 50 °C on adding higher concentrations of DAG
is also substantiated by the increased cooperativity value
at higher DAG concentrations.
initial rates of reaction obtained in the absence or in the
presence of DAGs. In Figure 4, a comparison of the
relative activities of two different PLA2s in DAG 1 doped
DPPC membranes is shown. As is seen from the figure,
increasing concentrations of DAG 1 within DPPC mem-
branes steadily enhances the activity of the two venom
PLA2s. Incorporation of 30 mol % DAG 1 enhanced the
activity of bee venom ∼6-fold and the activity of cobra
venom ∼4.5-fold with respect to that of the parent DPPC
membrane. These results are similar to those for other
macrocylic DAGs.
Many investigators propose that certain activators
perturb membrane structure such that phospholipid
substrates become more “exposed” to the membrane-
bound PLA2 enzymes. Most activators of PLA2 that have
been already reported in the literature act not by activat-
ing the membrane bound PLA2 but by allowing more of
the enzyme in the water layer to bind to the membrane
interface. Generally, most of the PLA2s, including the
ones examined in the present study, bind not so strongly
to the vesicles of phosphatidylcholine. Thus, most of the
PLA2 remains in the water layer, and the reaction
velocity is slow. Under such conditions, many compounds
that can partition into the vesicle interface can lead to
more enzyme-membrane binding, and such agents will
function as putative activators of PLA2. This type of
activation may not be specific activation in the usual
sense of the activator binding directly to the enzyme and
P h osp h olip a se A2 Assa y. We also present herein the
results on PLA2 activity due to incorporation of one
representative tethered DAG 1 into DPPC bilayer mem-
brane. This result has been shown in terms of relative
activities of the PLA2s, which is actually a ratio of the

9238 J . Org. Chem., Vol. 63, No. 25, 1998
Bhattacharya et al.
F igu r e 2. Ball and stick model representations of energy-
minimized (INSIGHT) structures of the macrocyclic DAGs (1,2
and 4-6). Also shown is the structure of naturally occurring
DAG, dipalmitoylglycerol (DPG), for comparison.
altering its catalytic properties. It is likely that the DAG
analogues prepared in the present study cause activation
of PLA2 indirectly by making the phosphatidylcholine
substrates more available to the PLA2. Hence, it is
evident that macrocyclization in the DAGs does perturb
the DPPC membrane bilayer in a way such that increas-
ing amounts of the PLA2 enzyme can now be at the lipid-
water interface, thereby showing enhanced activity.
F igu r e 3. 31P-NMR spectra of DPPC bilayer membranes
incorporated with (a) DAG 1 and (b) DAG 1 that has been
reduced by prior DTT treatment.
Con clu sion s
In summary, we have synthesized a series of macro-
cyclic diacylglycerols (1-6) where a disulfide linkage has
been used to “stitch” the two hydrocarbon chains. Our
synthetic strategy is general and involves convenient
protection and deprotection reactions as well as mild
reaction conditions. We have been able to join the two
hydrocarbon chains by disulfide linkage at different
depths thereby providing systems with different extents
of restriction in chain mobility. Interchain linking
through a disulfide tether also provided the extra ad-
vantage of converting these macrocycles into the corre-
sponding open chain thiol analogues via a simple dithio-
threitol-mediated reduction step. The access to the open
chain analogue allows further investigation to systemati-
cally compare the effects of macrocyclization. The syn-
thesis outlined herein convincingly establish the utility
of this intramolecular disulfide coupling protocol for the
efficient synthesis of this important class of molecules.
Though the overall yield for each of these macrocyclic
DAGs are moderate to good, we feel that this method
would find application in the synthesis of related mac-
rocyclic compounds. One notable strength of the syn-

Synthesis of Macrocyclic Diacyl/Dialkyl Glycerols
J . Org. Chem., Vol. 63, No. 25, 1998 9239
Ta ble 2. Th er m otr op ic Tr a n sition Da ta on th e
DAG-Dop ed DP P C Coa ggr ega tesa
PLA2s examined herein. However, their effect on PLA2
activation might be phenomenological. It is likely that
the DAG 1 analogues function to activate PLA2 by
causing an increasing fraction of enzyme to be at the
membrane surface.
The fact that until now only a few “structure-activity”
comparisons between appropriately designed synthetic
DAGs with differing extents of hydrocarbon modifications
have been carried out reflects the inaccessibility of the
required hydrocarbon-modified DAGs. In the present
work, we demonstrate how effective design and synthesis
of large macrocyclic diacylglycerols influences phase
transition and other properties of the DPPC membranes.
We are currently examining further details of the effects
of incorporation of these diacylglycerol derivatives in
other membranes.
conc
C. U.
DAG
(mol %) Tm (°C)b ∆Hc × E3c ∆Hv × E3c (∆Hv/∆Hc)
1
3
10
3
10
10
10
5
10
5
10
5
40.9
40.9
41.3
41.2
39.8
39.3
39.04
38.9
40.1
39.6
39.5
39.5
41.6
44.6
5.08
4.57
3.6
421
282
330
257
213
262
617
423
809
546
254
170
359
230
82.9
61.7
91.7
67.8
40.04
42.6
90.07
64.1
114.6
78.7
33.7
23.7
48.2
27.8
reduced
3.8
2
5.32
6.15
6.9
6.6
7.06
6.9
7.5
7.2
7.5
8.3
reduced
3
reduced
4
15
5
15
reduced
a
See text for preparation of individual coaggregates. b Tm refers
to the main transition temperature. c ∆Hc and ∆Hv values are
Exp er im en ta l Section
expressed as kcal mol-1
.
Gen er a l. Unless noted otherwise, all starting materials
were obtained from the best known commercial suppliers and
were used without further purification. THF was distilled
from sodium benzophenone under N2, and CH2Cl2 was distilled
from P2O5 under N2 immediately prior to use. DMF was
distilled under reduced pressure over P2O5 and stored over
molecular sieves (4 Å). Reactions involving air- and/or moisture-
sensitive reagents were executed under an inert atmosphere
of dry N2. Column chromatography was performed using silica
gel (Merck, 60-120 mesh). Benzyltriethylammonium tetra-
thiomolybdate was prepared as reported earlier.19 N-(7-
Nitrobenz-2-oxa-1,3-diazol-4-yl)aminohexanoic acid (NBD-ami-
nohexanoic acid), dipalmitoylphosphocholine (DPPC), 1-hexa-
decanoyl-2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminohexanoyl]-
sn-glycero-3-phosphocholine (NBD-PC), phospholipases A2
(cobra venom Naja mocambique and bee venom), and TRIZMA
base were obtained from Sigma, and 5,5′-dithiobis(2-nitroben-
zoic acid) (Ellman’s reagent) was purchased from Aldrich and
1
used as received. Descriptions of analytical instruments, H
NMR, IR, fluoroscence, mass and UV-vis spectrometers have
been previously published.20 1H and 13C NMR spectra were
obtained at the indicated field for each compound as solutions
in CDCl3. Chemical shifts for 1H and 13C NMR spectra are
reported in parts per million (ppm δ) downfield relative to the
internal standard tetramethylsilane (TMS); for the 13C spectra
TMS was referenced to the central line of the CDCl3 triplet (δ
77.0). Low-voltage ionization (30 eV) was used to record the
mass spectra.
Gen er a l P r oced u r e for P r ep a r in g 1,2-O-Dia cyl-3-O-(2-
m eth oxyeth oxym eth yl)glycer ol.21 The diol, 9 (0.263 g, 1.49
mmol), was esterified with the requisite fatty acid by adding
a solution of diol in dry CCl4 (0.1 g/mL) to a solution of 2.2
mol equiv of the acid in dry CCl4 (0.1 g/mL) and a catalytic
amount of DMAP at 0 °C. To the resulting mixture was added
N,N-dicyclohexylcarbodiimide (0.71 g, 2.99 mmol), and the
mixture was stirred for 4 h. DCU formed during the reaction
was filtered off, and the precipitate was washed with 2 mL of
CCl4. The filtrate and washings were taken together, and the
solvent was evaporated to give a crude product, which was
purified by column chromatography eluting with the solvent
indicated for each specific compound.
F igu r e 4. Histogram showing the activity of PLA2s on DAG
1 incorporated DPPC vesicles. Hollow bar corresponds to bee
venom PLA2, while shaded bar corresponds to cobra venom
PLA2.
thetic strategy adopted herein is that this unambiguously
gives the intramolecular macrocyclic disulfide as the only
isolable product, thus making the separation and the
purification a much easier task.
The macrocyclic DAGs (1-6) and their reduced open
chain analogues have been used to investigate the effect
of imposing chain restriction within the membrane
bilayer. Preliminary results of these investigation have
been reported here. 31P NMR of DAG 1 incorporated
DPPC vesicles indicates formation of isotropic phases.
These phases disappear upon the opening of the macro-
cycle by the reduction of the disulfide tether. Moreover,
thermotropic properties have been studied for DPPC
membranes containing various amounts of individual
restricted DAGs (1-6) as well as their nonmacrocyclic
analogues. These studies indicate that the cooperativity
of the vesicular system is enhanced upon relaxation of
the macrocyclic DAG to the open chain version. Prelimi-
nary studies establish that macrocyclization of DAG 1
results in an enhancement of the activity of both types of
1,2-B is (16-b r o m o h e x a d e c a n o y l)-3-O -(2-m e t h o x y -
eth oxym eth yl)glycer ol (10a ). 10a was obtained as a color-
less, viscous oil in 65% yield from 9 by column chromatography
upon elution with hexane/EtOAc (12:1): IR (neat) ν 1740 cm-1
;
(19) Ramesha, A. R.; Chandrasekaran, S. Synth. Commun. 1992,
22, 3277.
(20) (a) Bhattacharya, S.; Snehalatha, K.; George, S. K. J . Org.
Chem. 1998, 63, 27. (b) Bhattacharya, S.; Snehalatha, K. J . Org. Chem.
1997, 62, 2198. (c) Bhattacharya, S.; Haldar, S. Biochim. Biophys. Acta
1996, 1283, 21-30.
(21) Yamauchi, K.; Hihara, M.; Kinoshita, M. Bull. Chem. Soc. J pn.
1987, 60, 2169-2172.

9240 J . Org. Chem., Vol. 63, No. 25, 1998
Bhattacharya et al.
1H NMR (200 MHz) δ 1.25 (s, 52H), 2.32 (t, 4H), 3.4-3.8
(complex m, 13H), 4.11-4.3 (m, 2H), 4.7 (s, 2H), 5.23 (m, 1H);
13C NMR (22.5 MHz ) δ 24.87, 28.12, 28.66, 29.53, 32.78,33.87,
58.89, 62.47, 65.94, 66.91, 69.94, 71.57, 95.5, 172.88, 173.2.
1,2-Bis(6-br om oh exa n oyl)-3-O-(2-m eth oxyeth oxym eth -
yl)glycer ol (10c). 10c was obtained as a colorless, viscous
oil in 92.4% yield from 9 by column chromatography eluting
1,2-Bis(2-br om oh exa d eca n oyl)glycer ol (11d ). 11d was
obtained as a gum in 57% yield by column chromatography
eluting with hexane/EtOAc (8:1): IR (neat) ν 3600-3300, 1780
cm-1; 1H NMR (80 MHz) δ 0.88 (t, 6H), 1.25 (s, 52H), 3.75 (d,
2H), 4.0-4.4 (m, 4H), 5.0 (m, 1H).
1,2-Bis(12-br om od od ecyl)glycer ol (18). 18 was obtained
as a viscous oil in 58% yield by column chromatography eluting
with hexane/EtOAc (12:1): IR (neat) ν 1740 cm-1 1H NMR
;
with hexane/EtOAc (8:1): IR (neat) ν 3600-3400 cm-1 1H
;
(80 MHz) δ 1.25 (m,12H), 2.25 (t, 4H), 3.25 (t, 4H), 3.5-3.75
(m, 9H), 4.0-4.3 (m, 2H), 4.65 (s, 2H), 5.0-5.2 (m, 1H); 13C
NMR (22.5 MHz ) δ 23.9, 27.47, 32.24, 33.32, 33.76, 58.89,
62.58, 65.83, 70.05, 71.57, 95.51, 172.55, 172.88.
NMR (80 MHz) δ 1.25 (s, 40H), 3.25-3.6 (complex m, 13H).
1,2-Bis(2-th iocyan atoh exadecan oyl)glycer ol (12). Com-
pound 11d (0.592 g, 0.868 mmol) was dissolved in 5 mL of
acetone and to it was added a ∼20-fold excess of NH4SCN
(1.345 g, 17.6 mmol), and the mixture was stirred for 6 h. The
acetone was evaporated, and the residue after aqueous workup
gave a light-yellow oil in 75% yield via column chromatography
using hexane/EtOAc (9:1): IR (neat) ν 3600-3300, 2140, 1760
1,2-B i s (12-b r o m o o c t a d e c a n o y l)-3-O -(2-m e t h o x y -
eth oxym eth yl)glycer ol (10b). 10b was obtained as a vis-
cous oil in 86.5% yield from 9 by column chromatography
1
eluting with hexane/EtOAc (10:1): IR (neat) ν 1730 cm-1; H
NMR (200 MHz) δ 0.88 (t, 6H),1.25 (s, 56H), 2.30 (t, 4H), 3.39
(s, 3H), 3.1-4.31 (complex m, 10H), 4.71 (s, 2H), 5.22 (m, 1H);
13C NMR (22.5 MHz ) δ 13.82, 22.38, 24.65, 26.17, 27.36, 28.88,
29.42, 31.05, 31.70, 33.76, 38.96, 41.02, 58.24, 58.68, 62.25,
65.72, 66.69, 69.73, 71.46, 95.30, 172.447, 172.77.
cm-1 1H NMR (80 MHz) δ 0.88 (t, 6H), 1.25 (s, 48H), 3.6-
;
3.75 (m, 4H), 4.25 (m, 6H), 5.1 (m, 1H); LRMS, EI, m/z (%)
682 (M+)(2).
Gen er a l P r oced u r e for P r ep a r in g Ma cr ocyclic Disu l-
fid e-Con ta in in g 1,2-Dia cylglycer ol. To a solution of the
appropriate bis(ω-bromo) or bis(R-thiocyanato)acylglycerol
(0.277 mmol) in DMF (20 mL) was added benzyltriethylam-
monium tetrathiomolybdate (0.4 g, 0.657 mmol), and the
mixture was stirred for a requisite time (Table 1). The
resulting mixture was concentrated, poured into water, and
extracted with CHCl3. The organic layer was washed with
water, and the solvent was evaporated.
1,2-Bis(2-b r om oh e xa d e ca n oyl)-3-O-(2-m e t h oxye t h -
oxym eth yl)glycer ol (10d ). 10d was obtained as a viscous
oil in 60% yield from 9 by column chromatography eluting with
hexane/EtOAc (20:1): IR (neat) ν 1740 cm-1 1H NMR (200
;
MHz) δ 0.88 (t, 6H), 1.25 (s, 52H), 3.36 (s, 3H), 3.3-3.7 (m,
6H), 4.0-4.35 (m, 4H), 4.6 (s, 2H), 5.2 (m, 1H).
1,2-Bis(12-br om od od ecyl)-3-O-(2-m eth oxyeth oxym eth -
yl)glycer ol (17). NaH (0.34 g, 14.04 mmol) was added to a
stirred dry THF solution (40 mL) of diol 9 (0.4 g, 2.27 mmol),
and the solution was stirred at room temperature for 30 min.
To it 1,12-dibromododecane (4.1 g, 12.5 mmol) was added, and
the mixture was refluxed for 24 h. It was cooled and
concentrated, and the residue was extracted with CHCl3. The
extract was washed with water, dried over anhyd Na2SO4, and
concentrated. A colorless oil in 45% yield was obtained by
column chromatography upon elution initially with hexane
until excess dibromide was separated and then with hexane/
EtOAc (6:1): 1H NMR (90 MHz) δ 1.25 (s, 40H), 3.2-3.7
(complex m, 17H), 3.3 (s, 3H), 4.7 (s, 2H); 13C NMR (22.5 MHz)
δ 25.98, 28.06, 28.72, 29.5, 30.02, 32.75, 33.79, 70.46, 71.63,
75.66, 95.69, 114.04.
1,2-Bis(16-th iotetr a tr ia con ta -1,34-d ioyl)glycer ol (1). 1
was obtained as a white semisolid in 55% yield by column
chromatography eluting with hexane/EtOAc (6:1): IR (neat)
ν 3500-3100, 1720 cm-1; 1H NMR (200 MHz) δ 1.25 (s, 52H),
2.23 (t, 4H), 2.69 (t, 4H), 3.72 (d, 2H), 4.1-4.37 (m, 2H), 5.11-
(m, 1H);13C NMR (22.5 MHz) δ 24.4, 24.8, 27.4, 28.4, 28.8,
33.14, 33.89, 39.55, 61.44, 67.7, 73.11, 173.45, 173.7; LRMS,
EI, m/z (%) 630 (M+) (85); HRMS, EI, calcd for C35H66O5S2
630.4351, found 630.435
1,2-Bis(6-th iotetr a d eca -1,14-d ioyl)glycer ol (3). 3 was
obtained as a colorless oil in 65% yield by column chromatog-
raphy eluting with hexane/EtOAc (6:1): IR (neat) ν 3500-
3100, 1720 cm-1 1H NMR (80 MHz) δ 1.25-1.75 (m, 12H),
;
Gen er a l P r oced u r e for Dep r otection of a Meth oxy-
eth oxym eth yl (MEM) Gr ou p fr om 1,2-O-Dia cyl/d ia lk yl-
3-O-(2-m eth oxyeth oxym eth yl)glycer ol. The MEM pro-
tecting group could be conveniently removed under aprotic
conditions by stirring (0.713 mmol) of the appropriate 1,2-O-
diacyl/dialkyl-3-O-(2-methoxyethoxymethyl)glycerol in dry
CH2Cl2 with 2.1 mol equiv of TiCl4 (1.4 mmol) under an N2
blanket at 0 °C for 1 h. To it 1 M NaHCO3 was added, and
the resulting mixture was stirred vigorously and finally
extracted with CHCl3. The organic layer was washed with
water, dried over anhyd Na2SO4, and concentrated.
2.25 (t, 4H), 2.65 (t, 4H), 3.6 (d, 2H), 4.0-4.3 (m, 2H), 5.1 (m,
1H); 13C NMR (22.5 MHz,) δ 24.44, 24. 76, 25.41, 27.47, 28.45,
28.66, 33.11, 33.87, 39.28, 39.50, 61.39, 62.79, 64.53, 67.67,
72.22, 173.53; LRMS, EI, m/z (%) 350 (M+) (85); HRMS, EI,
calcd for C15H26O5S2 350.1215, found 350.1208.
1,2-Bis(12-th ioocta d eca n oyl)glycer ol (2). 2 was ob-
tained as a colorless oil in 62% yield by column chromatogra-
phy eluting with hexane/EtOAc (6:1): IR (neat) ν 3500-3100
1
cm-1, 1740; H NMR (200 MHz) δ 0.88 (t, 6H), 1.25(s, 56H),
2.26 (t, 4H), 2.6 (m, 1H), 2.8 (m, 1H), 3.72 (d, 2H), 4.1-4.4
(complex m, 2H), 5.06 (m, 1H); 13C NMR (22.5 MHz) δ 14.15,
22.7, 24.98, 26.82, 27.15, 29.53, 31.92, 34.19, 39.072, 41.23,
65.07, 173.42, 173.85; LRMS, EI, m/z (%) 686 (M+) (20);
HRMS, EI, calcd for C39H74O5S2 686.4995, found 686.4978.
1,2-Bis(16-br om oh exa d eca n oyl)glycer ol (11a ). 11a was
obtained as a white semisolid in 40% yield by column chro-
matography eluting with hexane/EtOAc (9:1): IR (neat) ν
3600-3300, 1740 cm-1; 1H NMR (80 MHz) δ 1.25 (s, 52H), 2.25
(t, 4H), 3.32 (t, 4H), 3.62 (d, 2H), 4.12(m, 2H), 5.0(m, 1H); 13
C
1,2-Bis(12-th ioh exa cosyl)glycer ol (6). 6 was obtained as
a white semisolid in 45% yield by column chromatography
NMR (22.5 MHz) δ 24.98, 28.23, 28.88, 29.64, 32.89, 34.08,
65.07, 68.43, 173.96.
eluting with hexane/EtOAc (6:1): IR (neat) ν 3600-3240 cm-1
;
1H NMR (200 MHz) δ 1.21 (s, 20H), 2.63 (t, 2H, CH2-S-S-,
J ) 7.47 Hz), 3.25-3.6 (complex m, 8H); 13C NMR (22.5 MHz)
δ 14.15, 22.7, 27.26, 28.89, 29.43, 29.75, 32.03, 34.74, 45.57,
65.94, 169.52, 169.85; LRMS, EI, m/z (%) 490 (M+) (10);
HRMS, EI, calcd for C27H54O3S2 requires 490.4111, found
490.3516.
1,2-Bis(6-br om oh exa n oyl)glycer ol (11c). 11c was ob-
tained as an oil in 50% yield by column chromatography
eluting with hexane/EtOAc (9:1): IR (neat) ν 3600-3200, 1740
cm-1; 1H NMR (80 MHz) δ 1.25-1.5 (m, 12H), 2.3 (t, 4H), 3.25
(t, 4H), 3.65 (d, 2H), 4.0-4.25 (m, 2H), 5.0(m, 1H); 13C NMR
(22.5 MHz) δ 23.79, 25.31, 27.36, 32.13, 33.32, 33.65, 60.95,
62.25, 64.85, 67.67, 72.00, 173.31, 176.88.
1,2-Bis(12-br om oocta d eca n oyl)glycer ol (11b). 11b was
obtained as an oil in 72% yield by column chromatography
eluting with hexane/EtOAc (9:1): IR (neat) ν 3400-3160, 1760
cm-1; 1H NMR (200 MHz) δ 0.88 (t, 6H), 1.25 (s, 52H), 3.65 (d,
2H), 3.8-4.2 (m, 2H), 5.0 (m, 1H); 13C NMR (22.5 MHz) δ
14.04, 22.6, 24.76, 27.15, 27.47, 28.99, 29.42, 31.16, 31.81,
33.11, 33.97, 39.07, 61.28, 62.14, 64.96, 68.10, 72.00, 173.31,
173.74.
1,2-Bis(2-th ioh exa d eca n oyl)glycer ol (4). 4 was obtained
as a colorless oil in 70% yield by column chromatography
eluting with hexane/EtOAc (8:1): IR (neat) ν 3600-3300, 1740
1
cm-1; H NMR (400 MHz) δ 0.81 (t, 6H), 1.18 (m, 52H), 3.2-
3.4 (complex m, 2H), 3.7-4.6 (complex m, 4H), 5.25 (m, 1H);
13C NMR (22.5 MHz) δ 14.04, 22.7, 27.25, 28.77, 29.31, 29.64,
31.92, 34.62, 45.57, 60.95, 63.23, 65.83, 67.67, 73.3, 169.73;
LRMS, EI, m/z (%) 630 (M+) (5); HRMS, EI, calcd for
C
35H66O5S2 requires 630.4384, found 630.4352.

Synthesis of Macrocyclic Diacyl/Dialkyl Glycerols
J . Org. Chem., Vol. 63, No. 25, 1998 9241
1,2-O-Dia cyl-3-O-ben zylglycer ol. The diol22 (0.263 g, 1.49
mmol) was condensed with the requisite acid by adding a
solution of diol in dry CCl4 (0.1 g/mL) to 2.2 mol equiv of the
acid in dry CCl4 (0.1 g/mL) and a catalytic amount of DMAP
at 0 °C. To the resulting mixture was added N,N-dicyclohex-
ylcarbodiimide (0.71 g, 2.99 mmol), and the mixture was
stirred for 4 h. DCU formed during the reaction was filtered
off, and the precipitate was washed with 2 mL of CCl4. The
solvent was evaporated from the filtrate and washings to give
a crude product, which was purified by column chromatogra-
to prepare a stock reagent solution. An amount of 0.04 mg of
the DTT-induced reaction product was taken in 3 mL of the
same buffer solution, and 0.1 mL of the DTNB reagent solution
was added and kept for 30 min at room temperature with
stirring. The absorbance of the resulting mixture was then
measured at 412 nm. However, when the parent disulfide-
containing DAG was treated with Ellman reagent without
prior treatment with DTT, no absorption maximum (λmax) at
412 nm was observed. This is consistent with the conclusion
that more than a 2-fold molar excess of DTT ensures quantita-
tive reduction of disulfide.
Vesicle P r ep a r a tion . In a typical protocol used in this
study, several mixtures of DPPC and an appropriate amount
of DAG were mixed in a final volume of 0.3 mL of chloroform
in a small glass tube. In these mixtures, the concentrations
of DAG varied from 1 to 30 mol %. These solutions were
evaporated to dryness under a stream of oxygen-free dry N2.
For the preparation of vesicles containing reduced DAGs, 2.2
equiv of DTT was also added as an ethanolic solution along
with the DAG and DPPC. The remaining traces of the solvent
were removed by keeping the samples under high vacuum for
3-4 h. The lipid films were then hydrated with water (Milli-
Q) and were subjected to three freeze-thaw cycles. The
resulting suspension was used for DSC experiments.
31P NMR Sp ectr oscop y. D2O (0.5 mL) was added to the
dry lipid film as described in the preceding section, and the
samples were heated at 70 °C for 30 min to ensure optimal
hydration of the samples. The samples were then vortexed
and put through three freeze-thaw cycles. The samples were
then transferred to 5 mm NMR tubes. The final concentration
of the phospholipid was 80 mM. Proton dipolar decoupled
242.8 MHz 31P NMR spectra were recorded in Fourier trans-
form mode using a Varian Unity plus 600 MHz spectrometer
interfaced with a SPARC LX station computer. Temperature
was controlled using a temperature control unit. All chemical
shift values are quoted in parts per million (ppm), positive
values referring to low-field shifts. Accumulated free induction
decays were obtained from 5000 transients. A spectral width
of 100 kHz, a memory of 8000 data points, a 1 s interpulse
time, and a 90° radio frequency pulse were the parameters
used to record the spectra. Before Fourier transformation, an
exponential multiplication was applied resulting in a 100 Hz
line broadening.
Differ en tia l Sca n n in g Ca lor im etr y (DSC). Multilamel-
lar vesicles were prepared by dispersing a thin lipid film of
DPPC (1.47 mg) containing different amounts of DAGs in 2
mL of water (Milli-Q). DSC measurements were carried out
using a Microcal MC-2 model heat conduction differential
scanning microcalorimeter that consisted of a reference cell
and a sample cell. The melting behavior was measured using
the same water as a reference. Heating scans were recorded
between 30 and 60 °C at a scan rate of 1.5 deg/min. The
calorimetric data were analyzed using Origin data analysis
software for DSC supplied by the manufacturer. Tm is the
temperature of the half of the transition peak area. The ratio
of the van’t Hoff enthalpy (∆Hv) to the calorimetric enthalpy
(∆Hc), i.e, ∆Hv/ ∆Hc provides the cooperativity (CU) of the
transition. Analysis of DPPC using this protocol gave ther-
modynamic data, which were in agreement with those that
were previously reported.23
phy eluting with
following.
a solvent mixture as indicated in the
1,2-Bis(10-u n d ecyn oyl)-3-O-ben zylglycer ol (15). 15 was
obtained as a colorless oil in 80% yield by column chromatog-
raphy eluting with hexane/EtOAc (8:1): IR (neat) ν 3300, 2120,
1760 cm-1 1H NMR (80 MHz) δ 1.25 (s, 24H), 1.93 (t, 2H),
;
2.15-2.3 (complex m, 8H), 3.5 (d, 2H), 4.15-4.25 (m, 2H), 4.5
(s, 2H), 5.2 (q like, 1H), 7.3 (s, 5H); 13C NMR (22.5 MHz) δ
18.06, 24.58, 27.56, 28.34, 28.67, 32.65, 33.76, 33.98, 62.39,
68.03, 69.80, 73.01, 84.28, 103.85, 116.01, 127.29, 128.06,
134.59, 137.46, 137.79, 172.62, 172.84; LRMS, EI, m/z (%) 510
(M+) (5).
1,2-(10,11-Docosa n d iyn e-1,22-d ioyl)-3-O-ben zylglycer -
ol (16). To a three-necked 500 mL round-bottom flask fitted
with a condenser and a rubber stopper with a glass tube
reaching the bottom of the flask were added CuCl (1.0 g, 10
mmol), N,N,N′,N′-tetramethyl ethylenediamine (20 mmol), and
300 mL of xylene, and the mixture was stirred. This was then
heated gradually to 140 °C with the passage of bubbling O2
through the reaction mixture. When the reaction mixture
reached 140 °C, 3 mL of a solution of compound 15 (1.4 g, 2.75
mmol) in 50 mL of xylene was added to the reaction mixture
slowly. The solution finally became dark brown in color. After
this, the heating and O2 bubbling were stopped, xylene was
filtered off, the reaction mixture was acidified with concen-
trated HCl and extracted with CHCl3 solvent was evaporated.
The pure product from this material was isolated as a
semisolid in 75% yield by column chromatography upon eluting
with hexane/EtOAc (16:1): IR (neat) ν 1720 cm-1; 1H NMR (80
MHz) δ 1.25 (s, 24H), 1.6 (m, 4H), 2.25 (t, 4H), 3.5 (d, 2H),
4.0-4.25 (m, 3H), 4.5 (s, 2H), 7.3 (s, 5H); 13C NMR (22.5 MHz)
δ 18.82, 25.67, 28.49, 28.92, 29.36, 34.78, 34.99, 63.71, 66.74,
68.91, 70.75, 74.00, 128.39, 129.15, 130.45, 138.47, 173.79,
174.11; LRMS, EI, m/z (%) 508 (M+) (27).
1,2-(Docosa n e-1,22-d ioyl)glycer ol (5). A solution of com-
pound 24 (0.5 g, 0.98 mmol) in THF-ethanol (20 mL, 4:1 v/v)
was hydrogenated in the presence of 10% Pd/C (0.25 g, 50%
w/w) at 40 psi for 3 h. The catalyst was filtered off, the solvent
was evaporated, and the pure product was obtained as a solid
in 60% yield by column chromatography eluting with hexane/
1
EtOAc (5:1): IR (neat) ν 3600-3200, 1760 cm-1; H NMR (80
MHz) δ 1.25 (s, 36H), 2.25 (t, 4H), 3.6 (d, 2H), 4.1-4.25 (m,
2H), 5.0-5.1 (m, 1H); 13C NMR (22.5 MHz) δ 24.87, 27.69,
28.12, 28.88, 34.08, 34.30, 61.17, 62.47, 64.85, 67.89, 72.12,-
173.42, 173.64; LRMS, EI, m/z (%) 426 (M+) (40); HRMS, EI,
calcd for C25H46O5S2 426.3341, found 426.3337.
Th iol An a lysis. To a solution of a given disulfide contain-
ing DAG (1 equiv) in ethanol was added 2.2 equiv of dithiotrei-
tol (DTT), and the mixture was stirred for 30 min at ambient
temperature. Ethanol was evaporated, and the product was
extracted with CHCl3, washed with water, and dried with
anhydrous Na2SO4. TLC indicated complete removal of DTT
from the reaction mixture after four extractions. The resulting
solution was concentrated upon evaporation of CHCl3, and the
residue was examined spectroscopically to ensure the quan-
P h osp h olip a se A2 Assa y. Multilamellar vesicles consist-
ing of substrates DPPC and NBD-PC and doped with different
concentrations of DAG were prepared in the same manner as
decribed earlier. The concentration of each substrate in the
vesicular suspension was 5 mM and 5 µM, respectively.
A
1
specific DAG derivative was incorporated in the MLVs at
concentrations ranging from 5 to 30 mol %. In the case of
phospholipase A2 assay, however, a buffer was used to make
the vesicular dispersions instead of water, which contained
50 mM Tris-HCl, 10 mM Ca2+, and 1 mM NaN3 at pH 7.4.
Phospholipase A2 activity was measured according to the
method of Stubbs et al.24
titative conversion of disulfide to thiols. A representative H
NMR spectra for the product obtained upon treatment of DAG
3 with DTT is shown in Figure 1. A 0.5 M Tris buffer (pH
8.3) was prepared and diluted with an equal volume of ethanol
containing 1% EDTA. To 20 mL of this solution was added
40 mg (0.1 mmol) of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)
(22) Kates, M.; Chan, T. H.; Stanacev, N. Z. Biochemistry 1963, 2,
394-397.
(23) Blume, A. Biochemistry 1983, 22, 5436.

9242 J . Org. Chem., Vol. 63, No. 25, 1998
Bhattacharya et al.
Typically, to a 0.4 mL vesicular solution was added 100 µL
of phospholipase A2 (0.5 µg of either cobra venom or bee venom)
to start the reaction. The reaction mixtures were kept for 5
min with respective venom PLA2s at 45 °C, the temperature
at which host lipid matrix, DPPC, remains in its fluid state
(>Tm of DPPC). After the reaction mixture was incubated for
the requisite time, the reaction was stopped by the addition
of 1.88 mL of a mixture of CHCl3/MeOH (1:2 v/v). The re-
sulting suspension was separated into discrete aqueous and
organic phases by further addition of 625 µL each of CHCl3
and water. An aliquot of the aqueous phase was then removed,
and its fluorescence intensity at 530 nm with excitation at 470
nm was measured. A standard curve was obtained using
known quantities of NBD-hexanoic acid. Background fluo-
rescence was determined from the identical measurements in
the absence of PLA2.
Ack n ow led gm en t. This work was supported by the
Department of Science and Technology Grant DST/INT/
M-6/97. S.G. thanks CSIR for a fellowship.
Su p p or tin g In for m a tion Ava ila ble: Copies of 1H, 13C
NMR, and mass spectra for compounds 1-6 (23 pages). This
material is contained in libraries on microfiche, immediately
follows this article in the microfilm version of the journal, and
can be ordered from the ACS; see any current masthead page
for ordering information.
(24) Stubbs, C. D.; Wesley Williams, B.; Pryor, C. L.; Rubin, E. Arch.
Biochem. Biophys. 1988, 262, 560-573.
J O980866B