Lamellar Gel (Lβ) Phases of Ternary Lipid Composition Containing Ceramide and Cholesterol

Lipid lateral segregation into specific domains in cellular membranes is associated with cell signaling and metabolic regulation. This phenomenon partially arises as a consequence of the very distinct bilayer-associated lipid physico-chemical properties that give rise to defined phase states at a given temperature. Until now lamellar gel (L_β) phases have been described in detail in single or two-lipid systems. Using x-ray scattering, differential scanning calorimetry, confocal fluorescence microscopy, and atomic force microscopy, we have characterized phases of ternary lipid compositions in the presence of saturated phospholipids, cholesterol, and palmitoyl ceramide mixtures. These phases stabilized by direct cholesterol-ceramide interaction can exist either with palmitoyl sphingomyelin or with dipalmitoyl phosphatidylcholine and present intermediate properties between raft-associated phospholipid-cholesterol liquid-ordered and phospholipid-ceramide L_β phases. The present data provide novel, to our knowledge, evidence of a chemically defined, multicomponent lipid system that could cooperate in building heterogeneous segregated platforms in cell membranes.     

Cholesterol Modulates the Interaction of β-Amyloid Peptide with Lipid Bilayers

The interaction of an amphiphilic, 40-amino acid β-amyloid (Aβ) peptide with liposomal membranes as a function of sterol mole fraction (X_sterol) was studied based on the fluorescence anisotropy of a site-specific membrane sterol probe, dehydroergosterol (DHE), and fluorescence resonance energy transfer (FRET) from the native Tyr-10 residue of Aβ to DHE. Without Aβ, peaks or kinks in the DHE anisotropy versus X_sterol plot were detected at X_sterol ≈ 0.25, 0.33, and 0.53. Monomeric Aβ preserved these peaks/kinks, but oligomeric Aβ suppressed them and created a new DHE anisotropy peak at X_sterol ≈ 0.38. The above critical X_sterol values coincide favorably with the superlattice compositions predicted by the cholesterol superlattice model, suggesting that membrane cholesterol tends to adopt a regular lateral arrangement, or domain formation, in the lipid bilayers. For FRET, a peak was also detected at X_sterol ≈ 0.38 for both monomeric and oligomeric Aβ, implying increased penetration of Aβ into the lipid bilayer at this sterol mole fraction. We conclude that the interaction of Aβ with membranes is affected by the lateral organization of cholesterol, and hypothesize that the formation of an oligomeric Aβ/cholesterol domain complex may be linked to the toxicity of Aβ in neuronal membranes.     

Hydrido-derivatives of [Ir4(CO)10(diphosphine)]: X-ray analyses of [Ir4H(CO)9(μ-Ph2PH(CH3)PPh2)] and [Ir4 H(CO)9(μ-Ph2P(CH2)nPPh2)] (n = 2, 3)

Some novel hydrido-anions of general formula [Ir₄H(CO)₉(μ-L-L)]⁻ (L-L = Ph₂PCH(CH₃)PPh₂, Ph₂P(CH₂)₂PPh₂, Ph₂P(CH₂)₃PPh₂ and Ph₂AsCH₂AsPh₂) have been obtained by the reaction of [Ir₄(CO)₁₀(μ-L-L)] with the base 1,8-diazabicyclo[5.4.0]undec-7-ene in wet dichloromethane. According to IR and ¹H, ³¹P and ¹³C NMR data at low temperature, these anionic derivatives display a single conformation in solution: three edge-bridging COs around the triangular basal face and both the hydride and the bidentate ligands located in axial positions relative to this face. The structures of four compounds were established by X-ray diffraction studies, which confirmed the configuration proposed on the basis of spectroscopic data.     

Synthesis, characterisation and structural studies of [Cu(dach)(μ-NCS)(NCS)]n and [Cu(dach)2(N3)]ClO4 (dach=1,4-diazacycloheptane)

A new singly bridging complex [Cu(dach)(μ-NCS)(NCS)]_n (dach=1,4-diazacycloheptane) has been synthesised and its crystal structure determined. There are many examples of double NCS bridged polymeric chains, but fewer singly bridged ones. IR, ESR and temperature variable magnetic studies are described but no magnetic interaction was found between the copper centres. [Cu(dach)₂(N₃)]ClO₄ has also been characterised by IR, ESR spectra and magnetic studies. The crystal structure determination shows that it is a penta-coordinated monomeric species with an axially coordinated azide linked to the perchlorate counterion by hydrogen bonding.     

Synthesis and characterisation of adducts of [Pt2(μ-S)2(PPh3)4] with organo-palladium and platinum-hydride substrates

The reactions of [Pt₂(μ-S)₂(PPh₃)₄] towards a range of palladium(II) complexes containing organometallic ligands (cyclopalladated N-donor ligands, η³-allyl, phenyl) have been explored, leading to the formation of a series of cationic, trinuclear sulfido-bridged aggregates containing {Pt₂PdS₂} cores. [Pt₂(μ-S)₂(PPh₃)₄] also reacts with the platinum(II) hydride complex trans-[PtHCl(PPh₃)₂] giving the adduct [Pt₂(μ-S)₂(PPh₃)₄PtH(PPh₃)]⁺. X-ray crystal structure determinations on the complexes [Pt₂(μ-S)₂(PPh₃)₄PdPh(PPh₃)]PF₆ and [Pt₂(μ-S)₂(PPh₃)₄PtH(PPh₃)]PF₆ are reported, and show the expected bis μ₃-sulfido aggregates with three square-planar metal centres.     

Thallium(III) complexes of the metalloligands [Pt2(μ-S)2(PPh3)4] and [Pt2(μ-Se)2(PPh3)4]

Reactions of [Pt₂(μ-S)₂(PPh₃)₄] with the diarylthallium(III) bromides Ar₂TlBr [Ar = Ph and p-ClC₆H₄] in methanol gave good yields of the thallium(III) adducts [Pt₂(μ-S)₂(PPh₃)₄TlAr₂]⁺, isolated as their salts. The corresponding selenide complex [Pt₂(μ-Se)₂(PPh₃)₄TlPh₂]BPh₄ was similarly synthesised from [Pt₂(μ-Se)₂(PPh₃)₄], Ph₂TlBr and NaBPh₄. The reaction of [Pt₂(μ-S)₂(PPh₃)₄] with PhTlBr₂ gave [Pt₂(μ-S)₂(PPh₃)₄TlBrPh]⁺, while reaction with TlBr₃ gave the dibromothallium(III) adduct [Pt₂(μ-S)₂(PPh₃)₄TlBr₂]⁺[TlBr₄]⁻. The latter complex is a rare example of a thallium(III) dihalide complex stabilised solely by sulfur donor ligands. X-ray crystal structure determinations on the complexes [Pt₂(μ-S)₂(PPh₃)₄TlPh₂]BPh₄, [Pt₂(μ-S)₂(PPh₃)₄TlBrPh]BPh₄ and [Pt₂(μ-S)₂(PPh₃)₄TlBr₂][TlBr₄] reveal a greater interaction between the thallium(III) centre and the two sulfide ligands on stepwise replacement of Ph by Br, as indicated by shorter Tl-S and Pt?Tl distances, and an increasing S-Tl-S bond angle. Investigations of the ESI MS fragmentation behaviour of the thallium(III) complexes are reported.     

Designed Fluorescent Probes Reveal Interactions between Amyloid-β(1-40) Peptides and GM1 Gangliosides in Micelles and Lipid Vesicles

A hallmark of the common Alzheimer's disease (AD) is the pathological conversion of its amphiphatic amyloid-β (Aβ) peptide into neurotoxic aggregates. In AD patients, these aggregates are often found to be tightly associated with neuronal G_M1 ganglioside lipids, suggesting an involvement of G_M1 not only in aggregate formation but also in neurotoxic events. Significant interactions were found between micelles made of newly synthesized fluorescent G_M1 gangliosides labeled in the polar headgroup or the hydrophobic chain and Aβ(1-40) peptide labeled with a BODIPY-FL-C1 fluorophore at positions 12 and 26, respectively. From an analysis of energy transfer between the different fluorescence labels and their location in the molecules, we were able to place the Aβ peptide inside G_M1 micelles, close to the hydrophobic-hydrophilic interface. Large unilamellar vesicles composed of a raftlike G_M1/bSM/cholesterol lipid composition doped with labeled G_M1 at various positions also interact with labeled Aβ peptide tagged to amino acids 2 or 26. A faster energy transfer was observed from the Aβ peptide to bilayers doped with 581/591-BODIPY-C₁₁-G_M1 in the nonpolar part of the lipid compared with 581/591-BODIPY-C₅-G_M1 residing in the polar headgroup. These data are compatible with a clustering process of G_M1 molecules, an effect that not only increases the Aβ peptide affinity, but also causes a pronounced Aβ peptide penetration deeper into the lipid membrane; all these factors are potentially involved in Aβ peptide aggregate formation due to an altered ganglioside metabolism found in AD patients.     

N-Heterocyclic carbene gold(I) and silver(I) adducts of [Pt2(μ-S)2(PPh3)4]

Reaction of the metalloligand [Pt₂(μ-S)₂(PPh₃)₄] with the N-heterocyclic carbene (NHC) complexes IPrAuCl, IMesAuCl and IMesAgCl in methanol gave the first examples of metal adducts of [Pt₂(μ-S)₂(PPh₃)₄] that contain NHC ligands, namely [Pt₂(μ-S)₂(PPh₃)₄AuL]⁺ (L = IPr, IMes) and [Pt₂(μ-S)₂(PPh₃)₄AgIMes]⁺. The complexes were isolated as hexafluorophosphate salts. Reaction of [Pt₂(μ-S)₂(PPh₃)₄] with excess IPrAuCl in refluxing methanol yielded only the mono-adduct, in contrast to the behaviour with the gold(I) phosphine complex Ph₃PAuCl, which undergoes double addition giving [Pt₂(μ-SAuPPh₃)₂(PPh₃)₄]²⁺. The X-ray structure of [Pt₂(μ-S)₂(PPh₃)₄AuIPr]PF₆ was determined and reveals that the ‘free’ sulfide is substantially sterically protected by the IPr ligand, accounting for the low reactivity towards addition of a second AgIPr⁺ moiety.     

Lipid and Peptide Dynamics in Membranes upon Insertion of n-alkyl-β-D-Glucopyranosides

The effect of nonionic detergents of the n-alkyl-β-D-glucopyranoside class on the ordering of lipid bilayers and the dynamics of membrane-embedded peptides were investigated with ²H- and ³¹P-NMR. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was selectively deuterated at methylene segments C-2, C-7, and C-16 of the two fatty acyl chains. Two trans-membrane helices, WALP-19 and glycophorin A_71-98, were synthesized with Ala-d₃ in the central region of the α-helix. n-Alkyl-β-D-glucopyranosides with alkyl chains with 6, 7, 8, and 10 carbon atoms were added at increasing concentrations to the lipid membrane. The bilayer structure is retained up to a detergent/lipid molar ratio of 1:1. The insertion of the detergents leads to a selective disordering of the lipids. The headgroup region remains largely unaffected; the fatty acyl chain segments parallel to the detergent alkyl chain are only modestly disordered (10-20%), whereas lipid segments beyond the methyl terminus of the detergent show a decrease of up to 50%. The change in the bilayer order profile corresponds to an increase in bilayer entropy. Insertion of detergents into the lipid bilayers is completely entropy-driven. The entropy change accompanying lipid disorder is equivalent in magnitude to the hydrophobic effect. Ala-d₃ deuterated WALP-19 and GlycA_71-97 were incorporated into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine at a peptide/lipid molar ratio of 1:100 and measured above the 1,2-dimyristoyl-sn-glycero-3-phosphocholine gel/liquid-crystal phase transition. Well-resolved ²H-NMR quadrupole splittings were observed for the two trans-membrane helices, revealing a rapid rotation of the CD₃ methyl rotor superimposed on an additional rotation of the whole peptide around the bilayer normal. The presence of detergent fluidizes the membrane and produces magnetic alignment of bilayer domains but does not produce essential changes in the peptide conformation or dynamics.     

Reaction of cyanamide and their derivatives with (η-C5H5)Mn(CO)3 and (η-C5H4CH3)Mn(CO)3

The reaction of cyanamide and its derivatives with the (η⁵-C₅H₅)Mn(CO)₂(THF) and (η⁵-C₅H₄CH₃)Mn(CO)₂(THF) complexes affords the cyanamide substituted complexes of types (η⁵-C₅H₅)Mn(CO)₂(NCN(R′)(R″)) (2a-d) and (η⁵-C₅H₄CH₃)Mn(CO)₂(NCN(R′)(R″)) (3a-e). All complexes were characterized by spectroscopy (¹H, ¹³C NMR, IR), elemental and mass spectroscopy analysis. Complex 2b (η⁵-C₅H₅)Mn(CO)₂(NCN(CH₃)₂) was additionally examined by single crystal X-ray structure determination.     

A one-dimensional copper (II) coordination polymer [Cu3(ampym)2(μ1,1-N3)4(μ1,3-N3)2(dmf)2]n (ampym = 2-aminopyrimidine) containing both end-on and end-to-end azido bridges

A new polynuclear copper (II) complex, derived from the azido-bridging ligand and 2-aminopyrimidine, has been synthesized and its 3-D structure has been determined by X-ray diffraction methods at two different temperatures. The compound crystallizes in the triclinic system space group, with the central copper atom lying on an inversion centre. The crystal structure is built up by trinuclear units (each of them contains two double end-on azido bridges) linked through two azide ions in an end-to-end (EE) fashion, to yield the polymer chain [Cu₃(ampym)₂(μ_1,1-N₃)₄(μ_1,3-N₃)₂(dmf)₂]_n. Magnetic susceptibility measurement shows a ferromagnetic interaction above 30 K, whereas a weak anti-ferromagnetic interaction prevails in the range of 30-2 K.     

Cholesterol Modulates the Dimer Interface of the β2-Adrenergic Receptor via Cholesterol Occupancy Sites

The β₂-adrenergic receptor is an important member of the G-protein-coupled receptor (GPCR) superfamily, whose stability and function are modulated by membrane cholesterol. The recent high-resolution crystal structure of the β₂-adrenergic receptor revealed the presence of possible cholesterol-binding sites in the receptor. However, the functional relevance of cholesterol binding to the receptor remains unexplored. We used MARTINI coarse-grained molecular-dynamics simulations to explore dimerization of the β₂-adrenergic receptor in lipid bilayers containing cholesterol. A novel (to our knowledge) aspect of our results is that receptor dimerization is modulated by membrane cholesterol. We show that cholesterol binds to transmembrane helix IV, and cholesterol occupancy at this site restricts its involvement at the dimer interface. With increasing cholesterol concentration, an increased presence of transmembrane helices I and II, but a reduced presence of transmembrane helix IV, is observed at the dimer interface. To our knowledge, this study is one of the first to explore the correlation between cholesterol occupancy and GPCR organization. Our results indicate that dimer plasticity is relevant not just as an organizational principle but also as a subtle regulatory principle for GPCR function. We believe these results constitute an important step toward designing better drugs for GPCR dimer targets.     

The complexes with end-to-end dicyanamide bridges: syntheses, characterization and crystal structures of [Cu(μ1,5-dca)2(phen)]n and [Cd(μ1,5-dca)2(py)2]n (phen=phenanthroline; py=pyridine; dca=dicyanamide, N(CN)2 )

Two new dicyanamide (dicyanamide=[N(CN)₂]⁻, dca) bridged complexes, [Cu(μ_1,5-dca)₂(phen)]_n (1) and [Cd(μ_1,5-dca)₂(py)₂]_n (2) have been synthesized and their structures have been determined by X-ray crystallography diffraction. Complex 1 crystallizes in the monoclinic space group P2(1)/c with a=10.1502(3), b=10.9815(4), c=14.5839(4) Å and Z=4. The adjacent copper atoms are connected by single end-to-end dca bridges to form a chain structure along the b axis. The chains are linked via Cu?N weak interactions to give rise to a 2D layer structure, which furthermore into a 3D structure by the π-π interaction between aromatic rings of adjacent layers. Complex 2 crystallizes in the monoclinic space group C2/m with a=6.6849(10), b=17.476(2), c=13.231(2) Å and Z=4. The cadmium(II) center is six-coordinated with a distorted octahedral geometry, bounded to four N atoms of four dca ligands and two N atoms of two-chelated py ligands. Neighbor Cd(II) atoms are linked by the double end-to-end dca bridges to generate a chain structure, which result in a 2D layer structure through the π-π interactions between the adjacent chains with distances 3.641 Å. EPR and magnetic results of 1 suggest that the complex exhibits a weak ferromagnetic interaction through CuNCNCNCu pathways.     

Sulfide-bridged aggregates from the metalloligand [Pt2(μ-S)2(PPh3)4] and β-diketonate complexes of cobalt(II) and zinc(II)

Reaction of [Pt₂(μ-S)₂(PPh₃)₄] with a range of zinc(II) and cobalt(II) complexes ML₂, where L is a β-diketonate ligand CH₃COCHCOCH₃, PhCOCHCOPh, CF₃COCHCOTh (Th = 2-thienyl)] permits the synthesis of adducts [Pt₂(μ-S)₂(PPh₃)₄M(diketonate)]⁺, isolated as their salts in moderate yields. The cobalt and zinc acetylacetonate complexes were characterised by single-crystal X-ray diffraction studies, which reveal isomorphous structures, with tetrahedral heterometal centres.     

Synthesis and characterization of triphenylphosphine stabilized silver α,β-unsaturated carboxylate: Crystal structure of [Ag(O2CCHC(CH3)2)(PPh3)2]

A series of triphenylphosphine coordinated silver α,β-unsaturated carboxylates of type [Ag(O₂CR)(PPh₃)_n: n = 1, R = CH₃CHCH (2a), (CH₃)₂CCH (2b), CH₃CH₂CHCH (2c), CH₃CH₂CH₂CHCH (2d), PhCHCH (2e), CH₂CH (2f); n = 2, CH₃CHCH (3a), (CH₃)₂CCH (3b), CH₃CH₂CHCH (3c), CH₃CH₂CH₂CHCH (3d)] were prepared by reaction of relative silver carboxylates (1a-1f) with triphenylphosphine in chloroform. These complexes were obtained in high yields and characterized by elemental analysis, ¹H NMR, ¹³C NMR, ³¹P NMR and IR spectroscopy. Thermal stability of the complexes has been determined by TG analysis. The molecular structure of [Ag((O₂CCHC(CH₃)₂))(PPh₃)₂] (3b) shows that the senecioato ligand is chelated with silver atom and generate, a distorted tetrahedron.     

Reactions of the complexes [Re2(CO)9(η-P-P)] (P-P=Ph2P(CH2)nPPh2, n=1-6) with Me3NO: formation of close-bridged complexes [Re2(CO)8(μ-P-P)] and phosphine oxide complexes [Re2(CO)9{P-P(O)}]

Reactions of [Re₂(CO)₁₀] with Me₃NO and diphosphines [Ph₂P(CH₂)_nPPh₂, n=1-6] yield mixtures of the monodentate-coordinated diphosphine complexes [Re₂(CO)₉(η¹-P-P)] (P-P=Ph₂P(CH₂)_nPPh₂, n=1-6) (yields 5-40%) and bridged dimers [{Re₂(CO)₉}₂(μ-P-P)] (5-50%). These complexes were isolated as either equatorial or axial isomers, or a mixture of two isomers. Reactions of the monodentate complexes with Me₃NO yield close-bridged complexes [Re₂(CO)₈(μ-P-P)] and phosphine oxide complexes [Re₂(CO)₉{P-P(O)}]. The structures of the close-bridged complexes 1 (n=3) and 2 (n=4), were determined by X-ray crystallography. The Re-Re bond in the close-bridged complex with the longest phosphine chain (n=6) is readily cleaved in CDCl₃ to give the complex [{cis-ReCl(CO)₄}₂(μ-dpph)] (3) as the product, the structure of which was also determined by X-ray crystallography.     

Effects of β-Cyclodextrin on the Structure of Sphingomyelin/Cholesterol Model Membranes

The interaction of β-cyclodextrin (β-CD) with mixed bilayers composed of sphingomylein and cholesterol (Chol) above and below the accepted stable complexation ratio (67:33) was investigated. Membranes with the same (symmetric) and different (asymmetric) compositions in their inner and outer leaflets were deposited at surface pressures of 20, 30, and 40 mN/m at the solid-liquid interface. Using neutron reflectometry, membranes of various global molar ratios (defined as the sum of the molar ratios of the inner and outer leaflets), were characterized before and after β-CD was added to the subphase. The structure of bilayers with global molar ratios at or above the stable complexation ratio was unchanged by β-CD, indicating that β-CD is unable to remove sphingomyelin or complexed Chol. However, β-CD removed all uncomplexed Chol from bilayers composed of global molar ratios below the stable complexation ratio. The removal of Chol by β-CD was independent of the initial structure of the membranes as deposited, suggesting that asymmetric membranes homogenize by the exchange of molecules between leaflets. The interaction of β-CD with the aforementioned membranes was independent of the deposition surface pressure except for a symmetric 50:50 membrane deposited at 40 mN/m. The scattering from 50:50 bilayers with higher packing densities (deposited at 40 mN/m) was unaffected by β-CD, suggesting that the removal of Chol can depend on both the composition and packing density of the membrane.     

The arylation of [Pt2(μ-S)2(PPh3)4]

Routes to the synthesis of the mixed sulfide-phenylthiolate complex [Pt₂(μ-S)(μ-SPh)(PPh₃)₄]⁺ have been explored; reaction of [Pt₂(μ-S)₂(PPh₃)₄] with excess Ph₂IBr proceeds readily to selectively produce this complex, which was structurally characterised as its PF₆⁻ salt. Reactions of [Pt₂(μ-S)₂(PPh₃)₄] with other potent arylating reagents (1-chloro-2,4-dinitrobenzene and 1,5-difluoro-2,4-dinitrobenzene) also produce the corresponding nitroaryl-thiolate complexes [Pt₂(μ-S){μ-SC₆H₂(NO₂)₂X}(PPh₃)₄]⁺ (X = H, F). The complex [Pt₂(μ-S)(μ-SPh)(PPh₃)₄]⁺ reacts with Me₂SO₄ to produce the mixed alkyl/aryl bis-thiolate complex [Pt₂(μ-SMe)(μ-SPh)(PPh₃)₄]²⁺, but corresponding reactions with the nitroaryl-thiolate complexes are plagued by elimination of the nitroaryl group and formation of [Pt₂(μ-SMe)₂(PPh₃)₄]²⁺. [Pt₂(μ-S)(μ-SPh)(PPh₃)₄]⁺ also reacts with Ph₃PAuCl to give [Pt₂(μ-SAuPPh₃)(μ-SPh)(PPh₃)₄]²⁺.     

Synthesis of some pentanuclear platinum clusters [Pt5(CO)6(L)4]. The X-ray structure of [Pt5(μ-CO)5(CO)(PCy3)4]

[Pt₅(μ-CO)₅(CO)L₄] (L = PPh₃1, PPh₂Bz 2, AsPh₃3, PEt₃4, PCy₃5) have been synthesized by reacting [Pt₃(μ-CO)₃(PR₃)₃] with H₂O₂ (1 and 2), by reduction of cis-[PtCl₂(CO)(PEt₃)] with Zn dust (4), and by the Zn reduction of [Pt₃(μ-CO)₃(PCy₃)₃] in the presence of [PtCl₂(CH₃CN)₂] (5). Complex 5 has not been observed previously and has been characterized by X-ray crystallography. Oxidation of the phosphine ligands with H₂O₂ is a new way to synthesize 1 and 2. The first complete NMR characterization of these complexes has also been achieved, and showed that these pentanuclear cluster complexes exhibit similar stereochemistries in solution and in the solid state. The observed ¹J_Pt-Pt values do not have any correlation with the corresponding bond lengths, again pointing out the irregular behaviour of such parameter in Pt complexes.