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 A(2) modulators

A general method for the preparation of novel disulfide-tethered macrocycli c diacylglycerols (DAGs) has been described. Overall synthesis involved ste pwise protection, acylation, and deprotection to yield the bis(omega-bromoa cyl) glycerols. In the crucial macrocyclization step, a unique reagent, ben zyltriethylammonium tetrathiomolybdate (BTAT), has been used to convert ind ividual bis(omega-bromoacyl) glycerols to their respective macrocyclic disu lfides. 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 ins tead of a disulfide one and was synthesized using modified Glaser coupling. Preparation of alpha-disulfide-tethered DAG (DAG 4) required an alternativ e method, as treatment of the bisbromo precursor with BTAT gave a mixture o f several compounds from which separation of the target molecule was cumber some. To avoid this problem, the bisbromide was converted to its correspond ing dithiocyanate, which on further treatment with BTAT yielded the desired DAG (DAG 4) in good yield. Upon treatment with the reducing agent dithioth reitol (DTT), the DAGs that contain a disulfide tether could be quantitativ ely converted to their "open-chain" thiol analogues. These macrocyclic DAGs and their reduced "open-chain" analogues have been incorporated in DPPC ve sicles to study their effect on model membranes. Upon incorporation of DAG 1 in DPPC vesicles, formation of new isotropic phases was observed by P-31 NMR, These isotropic phases disappeared completely on opening the macrocycl ic ring by a reducing agent. The thermotropic properties of DPPC bilayers h aving DAGs (1-6) incorporated at various concentrations were studied by dif ferential scanning calorimetry. Incorporation of DAGs in general reduced th e cooperativity unit (CU) of the vesicles. Similar experiments with reduced "open-chain" DAGs incorporated in a DPPC bilayer indicated a recovery of C U with respect to their macrocyclic "disulfide" counterparts. The effect of inclusion of these DAGs on the activity of phospholipase A(2) (PLA(2)) was studied in vitro. Incorporation of DAC 1 in DPPC membranes potentiated bot h bee venom and cobra venom PLA(2) activities.