Richard I. Duclos

The total syntheses of D-erythro-sphingosine, N-palmitoylsphingosine (ceramide), and glucosylceramide (cerebroside) via an azidosphingosine analog.

The total synthesis of D-erythro-sphingosine (9) was performed by a chirospecific method starting from D-galactose via an azidosphingosine intermediate to give highly homogeneous (>99.9% C18:1) sphingosine base (9) which contained no observable olefin isomerization by product and was demonstrated to be optically pure by a novel method utilizing Moshers acid. Ceramide (10) was prepared from this sphingosine (9) with highly homogeneous (99.8% C16:0) palmitic acid by two methods. The cerebroside glucosylceramide (23) was the next sphingolipid in this series to be synthesized in a highly homogeneous form. These three sphingolipids are currently being used for biophysical studies of the structures of their hydrated bio-molecular assemblies.

Integrity of 111In-radiolabeled superparamagnetic iron oxide nanoparticles in the mouse☆

INTRODUCTION Iron-oxide nanoparticles can act as contrast agents in magnetic resonance imaging (MRI), while radiolabeling the same platform with nuclear medicine isotopes allows imaging with positron emission tomography (PET) or single-photon emission computed tomography (SPECT), modalities that offer better quantification. For successful translation of these multifunctional imaging platforms to clinical use, it is imperative to evaluate the degree to which the association between radioactive label and iron oxide core remains intact in vivo. METHODS We prepared iron oxide nanoparticles stabilized by oleic acid and phospholipids which were further radiolabeled with (59)Fe, (14)C-oleic acid, and (111)In. RESULTS Mouse biodistributions showed (111)In preferentially localized in reticuloendothelial organs, liver, spleen and bone. However, there were greater levels of (59)Fe than (111)In in liver and spleen, but lower levels of (14)C. CONCLUSIONS While there is some degree of dissociation between the (111)In labeled component of the nanoparticle and the iron oxide core, there is extensive dissociation of the oleic acid component.

Bornyl- and Isobornyl-Δ8-tetrahydrocannabinols: A Novel Class of Cannabinergic Ligands

Structure-activity relationship studies of classical cannabinoid analogues have established that the C3 aliphatic side chain plays a pivotal role in determining cannabinergic potency. In earlier work, we provided evidence for the presence of subsites within the CB1 and CB2 cannabinoid receptor binding domains that can accommodate bulky conformationally defined substituents at the C3 alkyl side chain pharmacophore of classical cannabinoids. We have now extended this work with the synthesis of a series of Delta (8)-THC analogues in which bornyl substituents are introduced at the C3 position. Our results indicate that, for optimal interactions with both CB1 and CB2 receptors, the bornyl substituents need to be within close proximity of the tricyclic core of Delta (8)-THC and that the conformational space occupied by the C3 substituents influences CB1/CB2 receptor subtype selectivity.

The Cannabinergic System as a Target for Anti-inflammatory Therapies

Habitual cannabis use has been shown to affect the human immune system, and recent advances in endocannabinoid research provide a basis for understanding these immunomodulatory effects. Cell-based experiments or in vivo animal testing suggest that regulation of the endocannabinoid circuitry can impact almost every major function associated with the immune system. These studies were assisted by the development of numerous novel molecules that exert their biological effects through the endocannabinoid system. Several of these compounds were tested for their effects on immune function, and the results suggest therapeutic opportunities for a variety of inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, allergic asthma, and autoimmune diabetes through modulation of the endocannabinoid system.

Assay and inhibition of diacylglycerol lipase activity.

A series of N-formyl-α-amino acid esters of β-lactone derivatives structurally related to tetrahydrolipstatin (THL) and O-3841 were synthesized that inhibit human and murine diacylglycerol lipase (DAGL) activities. New ether lipid reporter compounds were developed for an in vitro assay to efficiently screen inhibitors of 1,2-diacyl-sn-glycerol hydrolysis and related lipase activities using fluorescence resonance energy transfer (FRET). A standardized thin layer chromatography (TLC) radioassay of diacylglycerol lipase activity utilizing the labeled endogenous substrate [1″-(14)C]1-stearoyl-2-arachidonoyl-sn-glycerol with phosphorimaging detection was used to quantify inhibition by following formation of the initial product [1″-(14)C]2-arachidonoylglycerol and further hydrolysis under the assay conditions to [1-(14)C]arachidonic acid.

Synthesis and biological properties of the fluorescent ether lipid precursor 1-O-[9′-(1″-pyrenyl)]nonyl-sn-glycerol

The synthesis of an ω-pyrene-labeled 1-O-alkyl-sn-glycerol was performed using a chirospecific method starting from R-(−)-2,3-O-isopropylidene-sn-glycerol. The product, 1-O-[9′-(1″-pyrenyl)]nonyl-sn-glycerol (pAG), is a fluorescent ether lipid that has a pyrene moiety covalently attached at the alkyl chain terminus. pAG was taken into CHO-K1 cells and a plasmalogen-deficient variant of CHO-K1, NRel-4. This variant is defective in dihydroxyacetonephosphate acyltransferase, which catalyzes the first step in plasmenylethanolamine (PlsEtn) biosynthesis. pAG was incorporated primarily into ethanolamine and choline phospholipids as well as a neutral lipid fraction tentatively identified as alkyldiacylglycerol. NRel-4 accumulated more fluorescence in the phospholipid fraction than CHO-K1, specifically in the ethanolamine phospholipids. Analysis of the fluorescent lipids showed that 93% of the pAG was incorporated into glycerolipids with the ether bond intact. Although the addition of 20 μM 1-O-hexadecyl-sn-glycerol to the medium fully restored PlsEtn biosynthesis in NRel-4 cells, pAG only partially restored PlsEtn synthesis. Incubation of cells with pAG followed by irradiation with long-wavelength (>300 nm) ultraviolet light resulted in cytotoxicity. NRel-4 cells displayed an increased sensitivity to this treatment compared with CHO-K1 cells. This photodynamic cytotoxicity approach could be used to select for mutants that are defective in downstream steps in ether lipid biosynthesis.

Unusual Hydration Properties of C16:0 Sulfatide Bilayer Membranes

After deacylation of bovine brain sulfatide under mild alkaline conditions and reacylation using palmitoyl chloride (, Chem. Phys. Lipids. 34:41-53), the anionic glycosphingolipid N-palmitoyl galactosulfatide (C16:0-GalSulf) has been synthesized. By differential scanning calorimetry (DSC), anhydrous C16:0-GalSulf exhibits an endothermic transition, T(M) = 93 degrees C (DeltaH = 5. 5 kcal/mol C16:0-GalSulf) on heating. With increasing hydration (50 mM sodium phosphate buffer, pH 7.0; 50 mM NaCl), T(M) decreases, reaching a limiting value of 49 degrees C (DeltaH = 8.2 kcal/mol C16:0-GalSulf) at 20 wt% buffer. X-ray diffraction data have been recorded over the hydration range 0-62% at temperatures below (20 degrees C) and above (60 degrees C) T(M). At 20 degrees C, sharp wide-angle reflections at approximately 1/4.4 A(-1), approximately 1/4.1 A(-1), and approximately 1/3.8 A(-1) indicate the presence of an ordered-chain gel phase, whereas at 60 degrees C a broad reflection at 1/4.5 A(-1) characteristic of a melted-chain phase is observed. Lamellar diffraction patterns consistent with the presence of bilayer phases are observed at both temperatures. At 60 degrees C, in the liquid-crystalline L(alpha) phase, the bilayer periodicity increases with hydration, in both water and 100 mM Na(+) buffer. Interestingly, in the gel phase at 20 degrees C, the bilayer periodicity (d = 64 A) is insensitive to hydration (over the range 30-60 wt%) with either water or buffer. The continuous swelling behavior exhibited by the L(alpha) bilayer phase of C16:0-GalSulf is typical of lipids bearing a net negative charge and confirms that the presence of 100 mM Na(+) is insufficient to shield the charge contributed by the sulfate group. In contrast, the lack of continuous swelling behavior of the bilayer gel phase of C16:0-GalSulf is unusual and resembles that of Na(+) soaps. Thus, presumably, alterations in the surface charge characteristics of the C16:0-GalSulf bilayer occur on hydrocarbon chain melting and lead to major changes in lipid hydration.

Synthesis and preliminary evaluation of N-(16-18F-fluorohexadecanoyl)ethanolamine (18F-FHEA) as a PET probe of N-acylethanolamine metabolism in mouse brain.

N-Acylethanolamines are lipid signaling molecules found throughout the plant and animal kingdoms. The best-known mammalian compound of this class is anandamide, N-arachidonoylethanolamine, one of the endogenous ligands of cannabinoid CB1 and CB2 receptors. Signaling by N-acylethanolamines is terminated by release of the ethanolamine moiety by hydrolyzing enzymes such as fatty acid amide hydrolase (FAAH) and N-acylethanolamine-hydrolyzing amidase (NAAA). Herein, we report the design and synthesis of N-(16-(18)F-fluorohexadecanoyl)ethanolamine ((18)F-FHEA) as a positron emission tomography (PET) probe for imaging the activity of N-acylethanolamine hydrolyzing enzymes in the brain. Following intravenous administration of (18)F-FHEA in Swiss Webster mice, (18)F-FHEA was extracted from blood by the brain and underwent hydrolysis at the amide bond and incorporation of the resultant (18)F-fluorofatty acid into complex lipid pools. Pretreatment of mice with the FAAH inhibitor URB-597 (1 mg/kg IP) resulted in significantly slower (18)F-FHEA incorporation into lipid pools, but overall (18)F concentrations in brain regions were not altered. Likewise, pretreatment with a NAAA inhibitor, (S)-N-(2-oxo-3-oxytanyl)biphenyl-4-carboxamide (30 mg/kg IV), did not significantly affect the uptake of (18)F-FHEA in the brain. Although evidence was found that (18)F-FHEA behaves as a substrate of FAAH in the brain, the lack of sensitivity of brain uptake kinetics to FAAH inhibition discourages its use as a metabolically trapped PET probe of N-acylethanolamine hydrolyzing enzyme activity.

Capturing Unknown Substrates via in Situ Formation of Tightly Bound Bisubstrate Adducts: S-Adenosyl-vinthionine as a Functional Probe for AdoMet-Dependent Methyltransferases

Identifying an enzymes substrates is essential to understand its function, yet it remains challenging. A fundamental impediment is the transient interactions between an enzyme and its substrates. In contrast, tight binding is often observed for multisubstrate-adduct inhibitors due to synergistic interactions. Extending this venerable concept to enzyme-catalyzed in situ adduct formation, unknown substrates were affinity-captured by an S-adenosyl-methionine (AdoMet, SAM)-dependent methyltransferase (MTase). Specifically, the electrophilic methyl sulfonium (alkyl donor) in AdoMet is replaced with a vinyl sulfonium (Michael acceptor) in S-adenosyl-vinthionine (AdoVin). Via an addition reaction, AdoVin and the nucleophilic substrate form a covalent bisubstrate-adduct tightly complexed with thiopurine MTase (2.1.1.67). As such, an unknown substrate was readily identified from crude cell lysates. Moreover, this approach is applicable to other systems, even if the enzyme is unknown.

The total synthesis of 2-O-arachidonoyl-1-O-stearoyl-sn-glycero-3-phosphocholine-1,3,1'-13C3 and -2,1'-13C2 by a novel chemoenzymatic method

2-O-Arachidonoyl-1-O-stearoyl-sn-glycero-3-phosphocholine was synthesized with carbon-13 enrichment of the three glycerol carbons and the carbonyl of the stearoyl group. Phospholipase A(2) was utilized to give optically pure lyso-PC, and only 3% acyl migration occurred during reacylation with arachidonic acid anhydride. This phospholipid is an important biosynthetic precursor of arachidonic acid metabolites as well as the endocannabinoid 2-arachidonoylglycerol (2-AG), and is now available for NMR studies.