Synthesis of phospholipids with fatty acid chains containing aromatic units at various depths

A series of diacyl phosphatidylcholine lipid derivatives, which contain aromatic units at various depths of their fatty acid chains, have been synthesized. These lipids produced stable aqueous suspensions. Electron microscopy revealed the presence of vesicular aggregates in the suspensions of these newly synthesized lipids. These membranes were oxidatively stable and maintained fluid character at ambient temperature making them ideal candidates for membrane protein reconstitution studies.


Introduction
Phospholipids are important building blocks of the biological membranes that constitute the cellular bodies. 1 The lipophilic parts of such molecules are generally composed of fatty acid chains, which are often connected to the glycerol based backbones via ester or ether linkages. 2A number of phospholipid analogues have been synthesized incorporating modified lipophilic chains, due to their importance in the investigations of the structure and function of biological membranes.Examples include ones containing sulfur-substituted fatty acids, 3a vicinal-chain tethered phospholipids with large macrocyclic rings, 3b-d chains incorporating aromatic fluorenyl residues, 3e or chains with acetylenic residues, 3f or terminal acryloyl groups, 3g and phospholipids with methyl-substituted chains.3h-i Phospholipid analogues also exhibit useful pharmaceutical and medicinal activities.Towards this end, several phospholipids have been synthesized that exhibit significant antifungal, 4a antitumor, 4b antihypertensive, 4c and anti-inflammatory 4d properties.As a result there have been numerous attempts to find novel routes to achieve the synthesis of various phospholipid derivatives. 5

Results and Discussion
In the reconstitution of membrane bound proteins or enzymes in vitro, phospholipids bearing unsaturated hydrocarbon chains are particularly useful. 6However, in order to keep the protein in their functionally active, correctly folded state, it is essential to maintain the membrane in its disordered state with significant dynamic fluidity.Hydrocarbon chains with cis-olefin containing unsaturations allow preservation of the membrane in a fluidized state, which is necessary for optimal signal transduction activity by the membrane bound protein. 6Phospholipids containing naturally occurring unsaturated fatty acids bearing cis-double bonds such as oleic acid (cis-9octadecenoic acid), linoleic acid (cis,cis-9,12-octadecadienoic acid), or arachidonic acid (cis,cis,cis,cis-5,8,11,14-eicosatetraenoic acid) are generally therefore used for the studies of membrane protein reconstitution.However, the membranes prepared from such phospholipids, invariably suffer from facile lipid peroxidation 7 limiting their usefulness for such biochemical studies.In addition, these lipids are also photochemically sensitive, restricting the shelf life and utility of the resulting formulations even further.
It occurred to us that by designing phospholipids with chains bearing oxidatively stable unsaturation units one might provide a way to circumvent this problem.Unlike their olefinic counterparts, aromatic systems are known for their chemical stability.As a part of our ongoing chemical biology program 8 focused on optimizing lipid lead structures which offer suitable membranes for reconstitution of membrane proteins at ambient conditions, we embarked upon the syntheses of a series of phospholipids incorporating aromatic units in their hydrophobic fatty acid segments. 9In this paper we present in detail the synthesis of three such phospholipid analogues 1 -3, in which unsaturation in the form of aromatic units forms part of the acyl chains at specific locations along the chains.We also demonstrate that incorporation of such aromatic units into hydrocarbon chains leads to remarkable fluidization of the resulting membranes.

Synthesis
The synthesis of the individual phospholipids 13 started with the preparation of fatty acids containing aromatic units at specified locations of the hydrocarbon chain (Scheme 1).Friedel-Crafts acylation of benzene with caproyl chloride in the presence of anhydrous AlCl 3 , under reflux for ~10 h furnished n-pentyl phenyl ketone in ~58% yield.This molecule upon reduction under Huang-Minlon conditions 10 gave n-hexyl benzene, 6, which was isolated in ~60% yield as a colorless liquid, upon distillation under reduced pressure.The n-hexyl benzene was then acylated using methyl adipoyl chloride in the presence of anhydrous AlCl 3 in dry carbon disulfide under refluxing conditions to give 7 in ~58% isolated yield.The ketoester, 7 was finally converted to 6-(4-n-hexylphenyl)hexanoic acid, 8 in ~60% yield first upon reduction under refluxing conditions with NH 2 NH 2 /KOH in ethylene glycol followed by work up and acidification.
The key starting material for the synthesis of phospholipid, free sn-glycerophosphocholine was obtained 11 from natural lecithin.This was first extracted from egg yolk and then subjected to hydrolysis using 0.1M Bu 4 N + OH -in MeOH to afford free sn-glycerophosphocholine (GPC) after chromatographic purification.The resulting material was then converted to a complex with CdCl 2 (GPC.CdCl 2 ) to make it soluble in CHCl 3 . 9Finally the syntheses of the desired phospholipids from the newly synthesized fatty acids containing aromatic units were accomplished by full acylation of GPC.CdCl 2 with respective anhydride of the appropriate fatty acid in ethanol-free, freshly dry CHCl 3 in the presence of dicyclohexylcarbodiimide (DCC) and N,N-4-dimethylaminopyridine (DMAP).This is schematically presented in Scheme 2. All the new intermediates and final compounds were characterized by IR, NMR spectroscopy and elemental analysis.Spectral data for these compounds are presented in the Experimental Section.

Scheme 2
Brief bath sonication (10 min) above 50 o C, of the thin film prepared from each of the above phospholipid in aqueous HEPES (5 mM) buffer containing 1 mM EDTA at pH 7.4 afforded stable, translucent lipid suspensions.The existence of multi-walled vesicular organizations (MLVs) in such aggregates was evident from negative stain transmission electron microscopy studies (not shown) with each of these lipid samples. 8Differential scanning calorimetric studies of these aggregates from 5 to 90 o C did not show the presence of any peak due to main-chain melting transition suggesting that the membranes remained in their fluid state already at 5 o C. Inclusion of any of these lipids 13 into a sample containing dipalmitoyl phosphatidylcholine (DPPC) however, reduced the main chain melting transition of the DPPC by several degrees depending on the mol% of the synthetic phospholipids incorporated.Taken together it is evident that these newly synthesized phospholipids retain a fluid like melted state (liquid-crystalline like phase) at ambient temperatures in their membranes.These lipid suspensions remain unscathed in ambient conditions for several months demonstrating high chemical stability associated with such lipid aggregates.
In summary, a general and convenient method has been developed for the synthesis of phospholipid analogues bearing acyl chains with aromatic type unsaturations, which form the first examples of this class of compounds that are also oxidatively and photochemically stable.Such synthetic phospholipids are now being exploited for membrane protein reconstitution studies in our laboratory.

Experimental Section
General Procedures.Unless stated otherwise, all general chemicals were obtained from commercial grades of the highest purity available and used without further purification.Solvents were dried and distilled prior to use.Dicyclohexylcarbodiimide (DCC), N,N-4dimethylaminopyridine (DMAP), adipic acid, dodecanoic acid, hexanoic acid, were purchased from Aldrich.The sn-Glycero-3-phosphocholine-CdCl 2 complex was prepared from egg yolk by procedure as described. 11IR spectra were recorded using a Perkin-Elmer 781 IR spectrophotometer.NMR spectra were obtained on either a Bruker WH-400 (400 MHz) or Jeol-90Q (90 MHz) or Varian-T60 (60 MHz) spectrometers.The chemical shifts for NMR spectra are reported in parts per million (ppm) relative to SiMe 4 as internal standard and J values are reported in Hz.Column chromatography was performed using silica gel of 60-120 mesh procured from Merck.Elemental analyses were performed by using a Carlo Erba Elemental Analyzer Model 1106.
and to this added a solution of 6-(4-n-hexylphenyl) hexanoic 0.27 g, 1 mmol) and triethylamine (1 mL) dissolved in dry THF (12 mL) in a dropwise fashion using a pressure-equalizing dropping funnel and stirred overnight at room temperature.The solvent was removed under reduced pressure and the crude solid material obtained was extracted with solvent ether.Ether layer was washed with water and dried over anhydrous sodium sulphate.CH 2 -C 6 H 5 ), 7.19 (m, 10H, aromatic-H).Anal.Calcd.for C 36 H 54 O 3 : C, 80.85, H, 10.18.Found: C, 81.2, H, 10.12.