Formal Synthesis of (+)-Laurencin by Gold(I)-Catalyzed Intramolecular Dehydrative Alkoxylation.

8-Membered cyclic ethers are found in a wide range of natural products; however, they are challenging synthetic targets due to enthalpic and entropic barriers. The gold(I)-catalyzed intramolecular dehydrative alkoxylation of ω-hydroxy allylic alcohols was explored to stereoselectively construct α,α'-cis-oxocenes and further applied in a formal synthesis of (+)-laurencin. The gold(I)-catalyzed intramolecular dehydrative alkoxylation may constitute an alternative method for the synthesis of molecular building blocks and natural products that contain highly functionalized 8-membered cyclic ethers.

Identification of a cyclic nucleotide as a cryptic intermediate in molybdenum cofactor biosynthesis.

The molybdenum cofactor (Moco) is a redox cofactor found in all kingdoms of life, and its biosynthesis is essential for survival of many organisms, including humans. The first step of Moco biosynthesis is a unique transformation of guanosine 5'-triphosphate (GTP) into cyclic pyranopterin monophosphate (cPMP). In bacteria, MoaA and MoaC catalyze this transformation, although the specific functions of these enzymes were not fully understood. Here, we report the first isolation and structural characterization of a product of MoaA. This molecule was isolated under anaerobic conditions from a solution of MoaA incubated with GTP, S-adenosyl-L-methionine, and sodium dithionite in the absence of MoaC. Structural characterization by chemical derivatization, MS, and NMR spectroscopy suggested the structure of this molecule to be (8S)-3',8-cyclo-7,8-dihydroguanosine 5'-triphosphate (3',8-cH2GTP). The isolated 3',8-cH2GTP was converted to cPMP by MoaC or its human homologue, MOCS1B, with high specificities (Km < 0.060 µM and 0.79 ± 0.24 µM for MoaC and MOCS1B, respectively), suggesting the physiological relevance of 3',8-cH2GTP. These observations, in combination with some mechanistic studies of MoaA, unambiguously demonstrate that MoaA catalyzes a unique radical C-C bond formation reaction and that, in contrast to previous proposals, MoaC plays a major role in the complex rearrangement to generate the pyranopterin ring.

A phosphoethanolamine-modified glycosyl diradylglycerol in the polar lipids of Clostridium tetani.

The polar lipids of the anaerobic bacterium Clostridium tetani, the causative agent of tetanus, have been examined by two-dimensional thin layer chromatography, ESI mass spectrometry, and NMR spectroscopy. Plasmalogen and di- and tetra-acylated species of phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, and N-acetylglucosaminyl diradylglycerol were the major lipids present in most strains examined except for strain ATCC 10779, the parent of strain E88, the first C. tetani strain to have its genome sequenced. This strain contained the same di- and tetra-acylated species but did not contain plasmalogens. All strains contained a novel derivative of N-acetylglucosaminyl diradylglycerol in which a phosphoethanolamine unit is attached to the 6'-position of the sugar, as judged by selective 31P-decoupled, 1H-detected NMR difference spectroscopy. The N-acetylglucosamine (GlcNAc) residue is presumably linked to the 3-positon of the diradylglycerol moiety, and it has the beta-anomeric configuration. Very little plasmalogen component was detected by mass spectrometry in the precursors phosphatidic acid and phosphatidylserine, consistent with the idea that plasmalogens are formed from diacylated phospholipids at a late stage of phospholipid assembly in anaerobic clostridia.

Identification of undecaprenyl phosphate-beta-D-galactosamine in Francisella novicida and its function in lipid A modification.

Francisella tularensis is a highly infectious pathogen that causes tularemia. Francisella lipid A contains an unusual galactosamine (GalN) unit, attached to its 1-phosphate moiety. Two genes, flmF2 and flmK, are required for the addition of GalN to Francisella lipid A, but the relevant enzymes and the GalN donor substrate have not been characterized. We now report the purification and identification of a novel minor lipid from Francisella novicida that functions as the GalN donor. On the basis of electrospray ionization mass spectrometry (ESI/MS) and NMR spectroscopy, we propose that this compound is undecaprenyl phosphate-beta-d-GalN. Approximately 0.5 mg of pure lipid was obtained from 10 g of F. novicida by chloroform/methanol extraction, followed by DEAE-cellulose chromatography, mild alkaline hydrolysis, and thin-layer chromatography. ESI/MS in the negative mode revealed a molecular ion [M - H](-) at m/z 1006.699, consistent with undecaprenyl phosphate-GalN. (31)P NMR spectroscopy showed a single phosphorus atom in the phosphodiester linkage. Selective inverse decoupling difference spectroscopy demonstrated that the undecaprenyl phosphate group is attached to the anomeric carbon of the sugar. (1)H NMR studies showed the presence of a polyisoprene chain and a sugar consistent with a beta-d-GalN unit. Heteronuclear multiple-quantum coherence (HMQC) analysis confirmed that nitrogen is attached to C-2 of the sugar. Purified undecaprenyl phosphate-beta-d-GalN supports the in vitro modification of lipid IV(A) by membranes of Escherichia coli cells expressing FlmK, an orthologue of E. coli ArnT, the enzyme that transfers 4-amino-4-deoxy-l-arabinose to lipid A in polymyxin-resistant strains. The discovery of undecaprenyl phosphate-beta-d-GalN suggests Francisella modifies lipid A with GalN on the periplasmic surface of the inner membrane.

Palladium-Catalyzed Synthesis of α-Trifluoromethyl Benzylic Amines via Fluoroarylation of gem-Difluoro-2-azadienes Enabled by Phosphine-Catalyzed Formation of an Azaallyl-Silver Intermediate.

We report the synthesis of α-trifluoromethyl benzylic amines through the vicinal fluoroarylation of gem-difluoro-2-azadienes. Our studies indicate that XPhos plays an important role as a phase transfer catalyst that promotes the addition of AgF to the difluoroazadiene, generating an α-trifluoromethyl azaallyl-silver intermediate that we have characterized by NMR spectroscopy. This intermediate likely transmetallates to Pd, coupling several aryl iodides to deliver products in up to 90% yield. Modification of the azadiene's activating group facilitates challenging cross-couplings.

Isolation and characterization of karlotoxin 1, a new amphipathic toxin from Karlodinium veneficum.

The karlotoxins (KmTxs) are a family of compounds produced by the dinoflagellate Karlodinium veneficum that cause membrane permeabilization. The structure of KmTx 1, determined using extensive 2D NMR spectroscopy, is very similar to the amphidinols and related compounds, though KmTx 1 features unique structural modifications of the conserved core region. The structure of KmTx 1 differs from that reported for KmTx 2, the only other reported karlotoxin to date, in lacking chlorination at its terminal alkene and possessing a hydrophobic arm that is two carbons longer.

Biochemical analysis of inositol phosphate kinases.

Lipid-derived inositol phosphates (IPs) are a complex group of second messengers generated by the sequential phosphorylation of inositol 1,4,5-trisphosphate (IP(3)). Synthetic pathways leading from IP(3) to the formation of inositol tetrakisphosphate IP(4), inositol pentakisphosphate IP(5), inositol hexakisphosphate IP(6), and inositol pyrophosphates PP-IPs have been elucidated in eukaryotes from yeast to human. Studies have attributed a variety of cellular functions to IPs, highlighting the importance of understanding how the pathways for their synthesis are regulated. This chapter summarizes experimental techniques for the biochemical characterization of the key inositol phosphate kinases IPKs necessary for producing the diverse array of IP species.

Application of a Radical Methodology toward the Synthesis of d,l-5alpha-Pregnanes and Related Steroids: A Stereoselective Radical Cascade Approach.

A stereoselective radical cascade cyclization to 5alpha-pregnanes is presented. Oxidative free radical cyclization of an appropriately substituted chloro cyano ester polyene was used to introduce the all trans stereochemistry in the steroid nucleus. The cyano group was utilized to introduce a C-8beta angular hydrogen, while the chloro ester moiety served as an entry to the geminal hydrogens at C-4.

Structure and Conformations of Monoacyl and Diacyl 1,2,4-Triazolidine-3,5-diones.

A series of monoacyl and diacyl 1,2,4-triazolidine-3,5-diones substituted at position 4 with either a phenyl or a tert-butyl group was prepared. Both acetyl and benzoyl groups were utilized as the acyl substituents. The diacylated compounds containing one or two acetyl groups were somewhat unstable to moisture. The acylated compounds were studied by (1)H, (13)C and (15)N NMR spectroscopy and X-ray crystallography to determine if they were acylated on nitrogen or ring carbonyl oxygen. The results indicated that the acylations occurred on nitrogen. The NMR spectra and molecular modeling computations were used to assign conformations to several of the diacylated compounds.

Total Synthesis of d,l-Isospongiadiol: An Intramolecular Radical Cascade Approach to Furanoditerpenes.

A stereoselective oxidative free-radical cyclization of beta-keto ester polyenes 7 and 19 has been accomplished as a one-step entry to the tricarbocyclic synthons 8and 21 which contain five and six stereogenic centers, respectively. These key synthons possessing an axial carboethoxy group at C-4 were ultimately converted to the spongian skeleton (8--> 14 and 21 --> 25 -->14). The synthesis of d,l-isospongiadiol (3) from the common intermediate 14 was realized after introduction of the 2alpha-hydroxy group in the spongian A-ring via epoxidation of silyl enol ether 28 and subsequent desilylation.

Structural analysis and detection of biological inositol pyrophosphates reveal that the family of VIP/diphosphoinositol pentakisphosphate kinases are 1/3-kinases.

We have characterized the positional specificity of the mammalian and yeast VIP/diphosphoinositol pentakisphosphate kinase (PPIP5K) family of inositol phosphate kinases. We deployed a microscale metal dye detection protocol coupled to a high performance liquid chromatography system that was calibrated with synthetic and biologically synthesized standards of inositol pyrophosphates. In addition, we have directly analyzed the structures of biological inositol pyrophosphates using two-dimensional 1H-1H and 1H-31P nuclear magnetic resonance spectroscopy. Using these tools, we have determined that the mammalian and yeast VIP/PPIP5K family phosphorylates the 1/3-position of the inositol ring in vitro and in vivo. For example, the VIP/PPIP5K enzymes convert inositol hexakisphosphate to 1/3-diphosphoinositol pentakisphosphate. The latter compound has not previously been identified in any organism. We have also unequivocally determined that 1/3,5-(PP)2-IP4 is the isomeric structure of the bis-diphosphoinositol tetrakisphosphate that is synthesized by yeasts and mammals, through a collaboration between the inositol hexakisphosphate kinase and VIP/PPIP5K enzymes. These data uncover phylogenetic variability within the crown taxa in the structures of inositol pyrophosphates. For example, in the Dictyostelids, the major bis-diphosphoinositol tetrakisphosphate is 5,6-(PP)2-IP4 ( Laussmann, T., Eujen, R., Weisshuhn, C. M., Thiel, U., Falck, J. R., and Vogel, G. (1996) Biochem. J. 315, 715-725 ). Our study brings us closer to the goal of understanding the structure/function relationships that control specificity in the synthesis and biological actions of inositol pyrophosphates.

Assessment of citrate concentrations in citrus fruit-based juices and beverages: implications for management of hypocitraturic nephrolithiasis.

BACKGROUND AND PURPOSE: Dietary intake of citrate in the form of citrus juices (eg, lemonade, orange juice) will enhance urinary citrate excretion, a valuable benefit for patients with hypocitraturic calcium oxalate nephrolithiasis. While information on citrate concentrations in select citrus juices is available, data on citrate concentrations of commercially available beverages (juice and otherwise) are limited. Using nuclear magnetic resonance spectroscopy (1H NMR), we report citrate concentrations of several beverages to help guide dietary recommendations aimed at increasing urinary citrate excretion and correcting hypocitraturia. METHODS: Citrate concentrations of a squeezed lemon, several fruit juices, and common beverages were measured using 1H NMR. Spectra for each sample were obtained in duplicate; citrate peak was identified, measured, and quantified and compared with the citrate concentration in the juice of 1 medium lemon. RESULTS: Quantitative analysis revealed the highest concentration of citrate was in grapefruit juice (64.7 mmol/L), followed in decreasing concentrations by lemon juice (47.66 mmol/L), orange juice (47.36 mmol/L), pineapple juice (41.57 mmol/L), reconstituted lemonade (38.65 mmol/L), lemonade flavored Crystal Light (38.39 mmol/L), ready to consume not from concentrate lemonade (38.24 mmol/L), cranberry juice (19.87 mmol/L), lemon-flavored Gatorade (19.82 mmol/L), homemade lemonade (17.42 mmol/L), Mountain Dew (8.84 mmol/L), and Diet 7Up (7.98 mmol/L), respectively. CONCLUSIONS: According to 1H NMR, all of the tested "natural" citrus juices have high concentrations of citrate (38.3-67.4 mmol/L), with grapefruit juice having the highest concentration of the beverages chosen. Lemonade flavored Crystal Light had the highest concentration of citrate in the nonjuice category of tested beverages. In patients with mild to moderate hypocitraturia, dietary supplementation with citrus-based juices may be an effective alternative to medical management while not requiring large serving sizes. Further prospective studies are warranted to evaluate the clinical significance of these findings.

Attenuated virulence of a Francisella mutant lacking the lipid A 4'-phosphatase.

Francisella tularensis causes tularemia, a highly contagious disease of animals and humans, but the virulence features of F. tularensis are poorly defined. F. tularensis and the related mouse pathogen Francisella novicida synthesize unusual lipid A molecules lacking the 4'-monophosphate group typically found in the lipid A of Gram-negative bacteria. LpxF, a selective phosphatase located on the periplasmic surface of the inner membrane, removes the 4'-phosphate moiety in the late stages of F. novicida lipid A assembly. To evaluate the relevance of the 4'-phosphatase to pathogenesis, we constructed a deletion mutant of lpxF and compared its virulence with wild-type F. novicida. Intradermal injection of 10(6) wild-type but not 10(8) mutant F. novicida cells is lethal to mice. The rapid clearance of the lpxF mutant is associated with a stronger local cytokine response and a greater influx of neutrophils compared with wild-type. The F. novicida mutant was highly susceptible to the cationic antimicrobial peptide polymyxin. LpxF therefore represents a kind of virulence factor that confers a distinct lipid A phenotype, preventing Francisella from activating the host innate immune response and preventing the bactericidal actions of cationic peptides. Francisella lpxF mutants may be useful for immunization against tularemia.

A conserved family of enzymes that phosphorylate inositol hexakisphosphate.

Inositol pyrophosphates are a diverse group of high-energy signaling molecules whose cellular roles remain an active area of study. We report a previously uncharacterized class of inositol pyrophosphate synthase and find it is identical to yeast Vip1 and Asp1 proteins, regulators of actin-related protein-2/3 (ARP 2/3) complexes. Vip1 and Asp1 acted as enzymes that encode inositol hexakisphosphate (IP6) and inositol heptakisphosphate (IP7) kinase activities. Alterations in kinase activity led to defects in cell growth, morphology, and interactions with ARP complex members. The functionality of Asp1 and Vip1 may provide cells with increased signaling capacity through metabolism of IP6.

Structure and biosynthesis of free lipid A molecules that replace lipopolysaccharide in Francisella tularensis subsp. novicida.

Francisella tularensis subsp. novicida U112 phospholipids, extracted without hydrolysis, consist mainly of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, and two lipid A species, designated A1 and A2. These lipid A species, present in a ratio of 7:1, comprise 15% of the total phospholipids, as judged by 32Pi labeling. Although lipopolysaccharide is detectable in F. tularensis subsp. novicida U112, less than 5% of the total lipid A is covalently linked to it. A1 and A2 were analyzed by electrospray ionization and matrix-assisted laser desorption ionization mass spectrometry, gas chromatography/mass spectrometry, and NMR spectroscopy. Both compounds are disaccharides of glucosamine, acylated with primary 3-hydroxystearoyl chains at positions 2, 3, and 2' and a secondary palmitoyl residue at position 2'. Minor isobaric species and some lipid A molecules containing a 3-hydroxypalmitoyl chain in place of 3-hydroxystearate are also present. The 4'- and 3'-positions of A1 and A2 are not derivatized, and 3-deoxy-d-manno-octulosonic acid (Kdo) is not detectable. The 1-phosphate groups of both A1 and A2 are modified with an alpha-linked galactosamine residue, as shown by NMR spectroscopy and gas chromatography/mass spectrometry. An alpha-linked glucose moiety is attached to the 6'-position of A2. The lipid A released by mild acid hydrolysis of F. tularensis subsp. novicida lipopolysaccharide consists solely of component A1. F. tularensis subsp. novicida mutants lacking the arnT gene do not contain a galactosamine residue on their lipid A. Formation of free lipid A in F. tularensis subsp. novicida might be initiated by an unusual Kdo hydrolase present in the membranes of this organism.

An outer membrane enzyme encoded by Salmonella typhimurium lpxR that removes the 3'-acyloxyacyl moiety of lipid A.

The Salmonella and related bacteria modify the structure of the lipid A portion of their lipopolysaccharide in response to environmental stimuli. Some lipid A modifications are required for virulence and resistance to cationic antimicrobial peptides. We now demonstrate that membranes of Salmonella typhimurium contain a novel hydrolase that removes the 3'-acyloxyacyl residue of lipid A in the presence of 5 mM Ca2+. We have identified the gene encoding the S. typhimurium lipid A 3'-O-deacylase, designated lpxR, by screening an ordered S. typhimurium genomic DNA library, harbored in Escherichia coli K-12, for expression of Ca2+-dependent 3'-O-deacylase activity in membranes. LpxR is synthesized with an N-terminal type I signal peptide and is localized to the outer membrane. Mass spectrometry was used to confirm the position of lipid A deacylation in vitro and the release of the intact 3'-acyloxyacyl group. Heterologous expression of lpxR in the E. coli K-12 W3110, which lacks lpxR, resulted in production of significant amounts of 3'-O-deacylated lipid A in growing cultures. Orthologues of LpxR are present in the genomes of E. coli O157:H7, Yersinia enterocolitica, Helicobacter pylori, and Vibrio cholerae. The function of LpxR is unknown, but it could play a role in pathogenesis because it might modulate the cytokine response of an infected animal.

Kdo2-Lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4.

The LIPID MAPS Consortium (www.lipidmaps.org) is developing comprehensive procedures for identifying all lipids of the macrophage, following activation by endotoxin. The goal is to quantify temporal and spatial changes in lipids that occur with cellular metabolism and to develop bioinformatic approaches that establish dynamic lipid networks. To achieve these aims, an endotoxin of the highest possible analytical specification is crucial. We now report a large-scale preparation of 3-deoxy-D-manno-octulosonic acid (Kdo)(2)-Lipid A, a nearly homogeneous Re lipopolysaccharide (LPS) sub-structure with endotoxin activity equal to LPS. Kdo(2)-Lipid A was extracted from 2 kg cell paste of a heptose-deficient Escherichia coli mutant. It was purified by chromatography on silica, DEAE-cellulose, and C18 reverse-phase resin. Structure and purity were evaluated by electrospray ionization/mass spectrometry, liquid chromatography/mass spectrometry and (1)H-NMR. Its bioactivity was compared with LPS in RAW 264.7 cells and bone marrow macrophages from wild-type and toll-like receptor 4 (TLR-4)-deficient mice. Cytokine and eicosanoid production, in conjunction with gene expression profiling, were employed as readouts. Kdo(2)-Lipid A is comparable to LPS by these criteria. Its activity is reduced by >10(3) in cells from TLR-4-deficient mice. The purity of Kdo(2)-Lipid A should facilitate structural analysis of complexes with receptors like TLR-4/MD2.

A formyltransferase required for polymyxin resistance in Escherichia coli and the modification of lipid A with 4-Amino-4-deoxy-L-arabinose. Identification and function oF UDP-4-deoxy-4-formamido-L-arabinose.

Modification of the phosphate groups of lipid A with 4-amino-4-deoxy-L-arabinose (L-Ara4N) is required for resistance to polymyxin and cationic antimicrobial peptides in Escherichia coli and Salmonella typhimurium. We previously demonstrated that the enzyme ArnA catalyzes the NAD+-dependent oxidative decarboxylation of UDP-glucuronic acid to yield the UDP-4''-ketopentose, uridine 5'-diphospho-beta-(L-threo-pentapyranosyl-4''-ulose), which is converted by ArnB to UDP-beta-(L-Ara4N). E. coli ArnA is a bi-functional enzyme with a molecular mass of approximately 74 kDa. The oxidative decarboxylation of UDP-glucuronic acid is catalyzed by the 345-residue C-terminal domain of ArnA. The latter shows sequence similarity to enzymes that oxidize the C-4'' position of sugar nucleotides, like UDP-galactose epimerase, dTDP-glucose-4,6-dehydratase, and UDP-xylose synthase. We now show that the 304-residue N-terminal domain catalyzes the N-10-formyltetrahydrofolate-dependent formylation of the 4''-amine of UDP-L-Ara4N, generating the novel sugar nucleotide, uridine 5'-diphospho-beta-(4-deoxy-4-formamido-L-arabinose). The N-terminal domain is highly homologous to methionyl-tRNA(f)Met formyltransferase. The structure of the formylated sugar nucleotide generated in vitro by ArnA was validated by 1H and 13C NMR spectroscopy. The two domains of ArnA were expressed independently as active proteins in E. coli. Both were required for maintenance of polymyxin resistance and L-Ara4N modification of lipid A. We conclude that N-formylation of UDP-L-Ara4N is an obligatory step in the biosynthesis of L-Ara4N-modified lipid A in polymyxin-resistant mutants. We further demonstrate that only the formylated sugar nucleotide is converted in vitro to an undecaprenyl phosphate-linked form by the enzyme ArnC. Because the L-Ara4N unit attached to lipid A is not derivatized with a formyl group, we postulate the existence of a deformylase, acting later in the pathway.

A Leptospira interrogans enzyme with similarity to yeast Ste14p that methylates the 1-phosphate group of lipid A.

Distinct from other spirochetes, cells of Leptospira interrogans contain orthologues of all the Escherichia coli lpx genes required for lipid A biosynthesis, but they synthesize a modified form of lipopolysaccharide that supposedly activates toll-like receptor 2 (TLR2) instead of TLR4. The recent determination of the L. interrogans lipid A structure revealed an unprecedented O-methylation of its 1-phosphate group (Que-Gewirth, N. L. S., Ribeiro, A. A., Kalb, S. R., Cotter, R. J., Bulach, D. M., Adler, B., Saint Girons, I., Werts, C., and Raetz, C. R. H. (2004) J. Biol. Chem. 279, 25420-25429). The enzymatic activity responsible for selective 1-phosphate methylation has not been previously explored. A membrane enzyme that catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to the 1-phosphate moiety of E. coli Kdo2-[4'-(32)P]lipid A has now been discovered. The gene encoding this enzyme was identified based on the hypothesis that methylation of a phosphate group is chemically analogous to methylation of a carboxylate moiety at a membrane-water interface. Database searching revealed a candidate gene (renamed lmtA) in L. interrogans showing distant homology to the yeast isoprenylcysteine carboxyl methyltransferase, encoded by sterile-14, which methylates the a-type mating factor. Orthologues of lmtA were not present in E. coli, the lipid A of which normally lacks the 1-phosphomethyl group, or in other spirochetes, which do not synthesize lipid A. Expression of the lmtA gene behind the lac promoter on a low copy plasmid resulted in the appearance of SAM-dependent methyltransferase activity in E. coli inner membranes and methylation of about 30% of the endogenous E. coli lipid A. Inactivation of the ABC transporter MsbA did not inhibit methylation of newly synthesized lipid A. Methylated E. coli lipid A was analyzed by mass spectrometry and NMR spectroscopy to confirm the location of the phosphomethyl group at the 1-position. In human cells, engineered to express the individual TLR subtypes, 1-phosphomethyl-lipid A purified from lmtA-expressing E. coli potently activated TLR4 but not TLR2.

Synthesis and characterization of carbohydrate-based phospholipids.

Novel carbohydrate-based phospholipids containing two saturated C(12) (dilauroyl ribo-phosphocholine) (DLRPC), C(14) (dimyristoyl ribo-phosphocholine) (DMRPC), and C(20) (diarachadonyl ribo-phosphocholine) (DARPC) carboxylic acid chains were synthesized. The physical properties of the supramolecular structures formed by these compounds were compared to those formed by their direct glycerol analogues dilauroyl phosphocholine (DLPC), dimyristoyl phosphocholine (DMPC), and diarachadonyl phosphocholine (DAPC). Modulated differential scanning calorimetry (MDSC) and X-ray diffraction data indicated that with chain lengths < or =14 carbons, the carbohydrate backbone increased the thermal stability of the bilayer below the phase-transition temperature (T(m)) as compared to the glycerol-based lipids. With longer chains (C(20)), the bilayer structure was destabilized as compared to glycerol-based lipids. NMR studies of a DMRPC vesicle dispersion reveal split choline headgroup signals and distinct magnetization transfer effects arising from the "inner" and "outer" surfaces of the bilayer vesicle. Modulated differential scanning calorimetry also demonstrated that glycerol- and carbohydrate-based lipids mix, as evidenced by a single intermediate T(m). In addition, carbohydrate-based lipid/cholesterol mixtures exhibited a decrease in enthalpy with an increase in cholesterol concentration. Unlike glycerol phospholipids, carbohydrate lipids were resistant to enzymatic degradation by phospholipase A(2) (PLA(2)).