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Design, Synthesis, Aggregation And Gene Transfection Properties Of Novel Gemini Cationic Lipids And LipopolymersBajaj, Avinash 12 1900 (has links)
The thesis entitled “Design, Synthesis, Aggregation and Gene Transfection Properties of Novel Gemini Cationic Lipids and Lipopolymers” elucidates the design, synthesis, aggregation and gene transfection properties of novel gemini cationic lipids based on pseudoglyceryl, aromatic and cholesterol/thiocholesterol backbone, and PEI-cholesterol based lipopolymers . The work has been divided into five chapters.
Chapter 1: Introduction to Gene Delivery
This chapter presents an overview of the general area of gene delivery and also gives a comprehensive account of the research towards the development of novel cationic lipids and PEI derived polymers. Utilization of these non-viral vectors for gene delivery and their aggregation studies has also been reviewed.
Chapter 2 deals with the Design, Synthesis, Membrane-Forming and Gene Transfection Properties of Pseudoglyceryl Gemini Lipids and has been divided into four parts.
Part 2A: Synthesis of Pseudoglyceryl Gemini Lipids Possessing Polymethylene and Oxyethylene Spacers
We have synthesized pseudoglyceryl gemini cationic lipids possessing polymethylene [-(CH2)m-] or oxyethylene [-CH2-(CH2-O-CH2)m-CH2-] spacers between the cationic ammonium headgroups. We have varied the length and nature of the spacer between the headgroups, from hydrophobic polymethylene [-(CH2)m-] to hydrophilic oxyethylene [-CH2-(CH2-O-CH2)m-CH2-] units (Figure 1). In these two series, we have also varied the hydrocarbon chain lengths from tetradecyl (n-C14H29) to hexadecyl (nC16H33) chains. Ether functionality has been introduced between the pseudoglyceryl backbone and the hydrocarbon chains.
Figure 1(Refer PDF File)
Part 2B: Thermotropic and Hydration Studies of Membranes Formed from Pseudoglyceryl Gemini Lipids Possessing Polymethylene spacers
In this part, the aggregation, thermotropic and hydration properties of pseudoglyceryl gemini lipids possessing polymethylene [-(CH2)m-] spacers (Figure 1) have been discussed using transmission electron microscopy (TEM), high sensitivity differential scanning calorimetry (DSC) and Paldan fluorescence studies. Electron microscopic studies revealed the vesicular nature of all the lipid aggregates. Thermotropic studies showed that the incorporation of a -(CH2)3- (lipid (16)2-3-(16)2) spacer between cationic ammonium headgroups dramatically increased the phase transition temperature (Tm) for gemini lipid aggregates irrespective of the hydrocarbon chain lengths. Further increase in the number of polymethylene units brought about decreases in the Tm. Hydration studies indicate that gemini lipid aggregates bearing hexadecyl (n-C16H33) chains sense greater hydration at membrane interfaces and among them, aggregates of lipid (16)2-12-(16)2 were found to be most hydrated in the gel state.
Part 2C: Membrane-Forming Properties of Pseudoglyceryl Gemini Lipids Possessing Oxyethylene Spacers
Here, we report the membrane-forming properties of glycerol backbone based gemini cationic lipids with two pairs of hexadecyl (n-C16H33) chains and with a hydrophilic, flexible oxyethylene [-CH2-(CH2-O-CH2)m-CH2-] spacer of variable length and hydration properties between headgroups (Figure 1). Their membrane-forming properties have been studied by transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurements, X-Ray diffraction (XRD), differential scanning calorimetry (DSC), Paldan fluorescence studies. The aggregates of lipid (16)2-1ox-(16)2 possess the highest phase transition temperature (Tm), lowest zeta potential and are highly hydrated, whereas that of gemini lipid (16)2-5ox-(16)2 aggregates are smallest in size, have highest zeta potential and greater bilayer width in the series examined, but possess comparable Tm as that of monomeric lipid (16)2.
Part 2D: Gene Transfection Properties of Pseudoglyceryl Gemini Lipids Possessing Polymethylene and Oxyethylene Spacers
We undertook a chemical-biology investigation on gene delivery efficacies of pseudoglyceryl gemini lipids (Figure 1). These gemini lipid formulations showed a significant enhancement in the gene transfection activities as compared to that of Lipofectin, which is a monomeric, structurally related to the present set of gemini lipids and commercially available reagent based on 1:1(w/w) ratio of DOTMA:DOPE formulation. The transfection efficacies depend on the hydrocarbon chains lengths and the spacer between the cationic ammonium headgroups as shown in Figure 2. The present set of gemini lipids were found to be serum compatible and even the presence of serum caused enhancement of the gene transfection activities of some of the lipid formulations. Lipid (16)2-3ox-(16)2/DOPE formulation was able to transfect nearly 35% of the cells in 50% FBS conditions. The simplicity of the use of pseudoglyceryl backbone, their high chemostability and shelf-life make these formulations particularly attractive.
Figure 2(Refer PDF File)
Chapter 3 deals with Design, Synthesis, Membrane-Forming and Gene Transfection Properties of Cationic Gemini Lipids based on Aromatic Backbone and have been divided into four parts.
Part 3A: Synthesis of Gemini Lipids Possessing Aromatic backbone between the Hydrocarbon chains and the Cationic Headgroup
In this chapter, we report the synthesis of new gemini cationic lipids based on an aromatic backbone that differ in the hydrocarbon chain lengths. We have also varied the length and nature of the spacer segment from hydrophobic polymethylene [-(CH2)m-] to hydrophilic oxyethylene [-CH2-(CH2-O-CH2)m-CH2-] units between the cationic headgroups .(Fig3)
Figure 3(Refer PDF FILE)
Part 3B: Membrane-Forming Properties of Aromatic derived Gemini Lipids Possessing Polymethylene Spacers
The membrane-forming properties of lipids (12)2Bz and (12)2Bz-(CH2)m-Bz(12)2 (Figure 3) have been studied in detail by transmission electron microscopy (TEM), dynamic light scattering (DLS), X-ray diffraction (XRD), high sensitivity differential scanning calorimetry (DSC), Paldan fluorescence studies and UV-vis absorption spectroscopy. The vesicle sizes, morphologies and thermotropic phase transition properties of the lipid aggregates depend on the length of the spacer chain. Paldan fluorescence studies indicate that the gemini lipid aggregates are less hydrated as compared to that of their monomeric counterpart in their solid-gel state. In contrast in their fluid liquid-crystalline phase, the hydration was found to depend strongly on the length of the spacer. UV-vis absorption studies suggest an H-type aggregate formation in the gemini lipid membranes in the gel states. In fluid state of the lipid membranes, H-aggregate formation was found to be enhanced depending on the length of the spacer.
Part 3C: Gene Transfection Properties of Aromatic derived Gemini Lipids Possessing Polymethylene Spacers
Gene transfection properties of novel aromatic derived gemini possessing polymethylene [-(CH2)m-] spacers and three monomeric cationic lipids (Figure 3) that differ in the hydrocarbon chain lengths have been reported in this chapter. We investigated their gene transfection properties in detail in HeLa cells in the absence and presence of serum conditions. The lipids bearing n-C14H29 hydrocarbon chain lengths have been found to be the best transfecting agents as compared to their analogues with n-C12H25 and n-C16H33 hydrocarbon chains (Figure 4). Formulation of lipid (14)2Bz-5-Bz(14)2, possessing tetradecyl hydrocarbon chains and pentamethylene [-(CH2)5-] spacer showed highest gene transfection efficacy in this series. Lipid (14)2Bz-5-Bz(14)2 formulation is also able to deliver genes in the presence of high percentages of serum.
Figure 4(Refer PDF File)
Part 3D: Gene Transfection Properties of Aromatic derived Gemini Lipids Possessing Oxyethylene Spacers
In this part, the transfection properties of six novel gemini cationic lipids based on aromatic backbone possessing n-C14H29 or n-C16H33 hydrocarbon chains (Figure 3) have been reported. We have varied the length of oxyethylene type spacers [(-CH2-CH2-O-CH2-CH2-)m] between the headgroups, where m varies from 1 to 3. Transfection studies showed that among lipids bearing n-C14H29 chains, transfection efficacies decrease with increase in the length of the spacer, whereas in case of lipids bearing n-C16H33 chains, transfection efficacies increase with increase in the length of the spacer. Lipid ((16)2Bz-3ox-Bz(16)2) bearing n-C16H33 hydrocarbon chains with [-(CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2)-] spacer was found to be highly serum compatible even in the presence of 50% serum conditions.
Chapter 4 deals with the Design, Synthesis and Gene Transfection Properties of Gemini Cationic Lipids based on Cholesterol/Thiocholesterol backbone and have been divided into three parts.
Part 4A: Design, Synthesis and Gene Transfection Properties of Cholesterol based Gemini Cationic Lipids Possessing Polymethylene Spacers
Here we represent the synthesis and gene transfection properties of five cholesterol based gemini cationic lipids, which differ in the length of the polymethylene [-(CH2)m-] spacer between cationic ammonium headgroups (Figure 5).
Transfection studies showed that with the increase in spacer chain length from propanediyl [-(CH2)3-] to pentanediyl [-(CH2)5-], transfection efficiency increased both in the absence and presence of serum (Figure 6). However, with further increase in the length from pentanediyl [-(CH2)5-] to dodecanediyl [-(CH2)12-] spacer transfection efficiency decreases. Transfection efficiencies of all the gemini lipids except lipid chol-3-chol were maintained even when the serum was present during the transfection conditions as compared to the monomeric lipid M, with which a dramatic decrease in transfection efficiency was observed(figure6)
Figure 5 and 6(Refer PDF File) .
Part 4B: Synthesis and Gene Transfection Properties of Cholesterol based Gemini Cationic Lipids Possessing Oxyethylene type Spacers
Four novel cholesterol based gemini cationic lipids differing in the length of oxyethylene [(-CH2-CH2-O-CH2-CH2-)m] type spacers between each ammonium headgroups have been synthesized (Figure 7) and studied for gene transfection properties.
All the cholesterol based gemini lipids induced better transfection activity than their monomeric counterpart M. Major characteristic feature of these oxyethylene spacer based cholesterol gemini lipids was that 10% serum conditions does not inhibit the transfection activity of these gemini lipids, whereas the transfection activity of their monomeric counterpart decreased drastically in the presence of serum. One of cholesterol based gemini lipid chol-1ox-chol possessing -CH2-CH2-O-CH2-CH2- spacer showed highest transfection activity.
Figure 7(Refer PDF File)
Part 4C: Effect of the Nature of the Spacer on Gene Transfection Properties of Novel Thiocholesterol derived Gemini Cationic Lipids
In this chapter, we present the synthesis and gene transfection properties of three thiocholesterol derived gemini cationic lipids possessing biodegradable disulfide linkages between the cationic ammonium headgroup and thiocholesterol backbone (Figure 8).
We varied the nature of the spacer between cationic headgroups from hydrophobic flexible -(CH2)5- (Lipid TC-5) to hydrophobic rigid (-C6H4-) (Lipid TC-px) to hydrophilic flexible (-CH2-CH2-O-CH2-CH2-) (Lipid TC-1-ox) spacer, to examine the effect of the nature of the spacer on gene transfection properties in different cell lines. Gene transfection properties of these gemini lipids were found to depend upon the nature of the spacer and the cell line. Cytotoxic studies confirmed the nontoxic nature of these lipid:DNA complexes at different N/P ratios used for transfection studies.
Figure 8(Refer PDF File)
Chapter 5 deals with the Synthesis and Gene Transfection Properties of PEI-Cholesterol based Lipopolymers, and Their Interactions with L-α-dipalmitoyl phosphatidylcholine (DPPC) membranes and has been divided into two parts
Part 5A: Synthesis and Gene Transfection Properties of PEI-Cholesterol based Lipopolymers
Nine lipopolymers based on low molecular weight Polyethyleneimines (PEI) and cholesterol via an ether linkage between the polymer amine and the cholesterol backbone have been synthesized (Figure 9). Different percentage of cholesterol moieties had been grafted on three types of PEI of molecular weights 800 (Mw), 1200 (Mn), 2000 (Mw). These lipopolymers were studied for gene transfection activities in HeLa cells. All lipopolymer formulations are better transfecting agents and highly serum compatible than commercially available PEI-25KDa. Transfection efficacies and serum compatibility of lipopolymer formulations depend upon the M.W. of PEI used for lipopolymers’ synthesis and percentage of cholesterol grafting on lipopolymers. Cell viability assay showed that PEI-25KDa is highly toxic as compared to all the lipopolymers.
Figure 9(Refer PDF File)
Part 5B: Thermotropic and Fluorescence studies of the Interactions of PEI-Cholesterol based Lipopolymers with L-α-dipalmitoyl phosphatidylcholine (DPPC) membranes
The interactions of PEI-cholesterol based lipopolymers (Figure 9) with L-α-dipalmitoyl phosphatidylcholine (DPPC) membranes had been examined using fluorescence anisotropy and differential scanning calorimetry (DSC). These lipopolymers were found to quench the chain motion of the acyl chains of DPPC, when incorporated in membranes. Detailed analysis of the fluorescence anisotropy and DSC data indicates that the nature of perturbation induced by lipopolymers is dependent upon the molecular weight of the PEI used and the % of cholesterol grafting on PEI.
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Evolution of New Lipids and Molecular Gelators : Syntheses, Aggregation Properties and ApplicationsMaiti, Bappa January 2015 (has links) (PDF)
The thesis entitled “Evolution of New Lipids and Molecular Gelators: Syntheses,
Aggregation Properties and Applications” elucidates the design, synthesis, aggregation properties and application of new lipids based on α-tocopheryl backbone and also with triazacyclononane (TACN) moiety. This thesis also elucidates the synthesis and aggregation properties of molecular gelators based on pyrene-pentapeptide and naphthalene diimide (NDI) moiety. The work has been divided into five chapters.
Chapter 1: Introduction: Self-assembled Molecular Aggregates and their Potential
Applications
This chapter describes the importance of different self-assemble mainly lipids and molecular gelator. Lipids mediated gene delivery, drug delivery and metal ion induced interaction are discussed. For liposomal gene delivery here we mainly describe example of cationic gemini lipids. This chapter also gives a comprehensive account of the research towards the development of novel liposomal drug delivery containing tocopheryl backbone. It also includes the utilization of liposome which could coordinate with metal ions and their interaction with different biological analyte. Here we also discuss a wide range of molecular gelator mainly based on NDI and amino acid or peptide.
Chapter 2A: Physicochemical Characterization of Bilayer Membranes Derived from (±) α-Tocopherol Based Gemini Lipids and their Interaction with plasmid-DNA and Phosphatidylcholine Bilayers In this sub-chapter we discuss the membrane formation and aggregation properties of a series of (±) α-tocopherol based cationic gemini lipids (Figure 1) varying polymethylene spacer length (TnS; n = 3, 4, 5, 6, 8 and 12) are studied extensively while comparing with corresponding properties of monomeric counterpart (TM). Liposomal suspensions of all cationic lipids are characterized by atomic force microscopy (AFM), transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurements and small angle x-ray diffraction studies. Aggregation properties of the gemini lipids are highly dependent on the spacer length and were significantly distinct from that of monomeric lipid (TM).
Figure 1. Molecular structures of (±) α-tocopherol based cationic monomeric and six gemini lipids that differ in polymethylene spacer length.
Stable monolayer formation at air water interface formation of each amphiphile is studied by Langmuir film balance technique. Interaction of liposome with plasmid
DNA is studied by ethidium bromide (EB) intercalation assay. Micellar sodium dodecyl sulphate (SDS) mediated release of the plasmid DNA from various pre-formed lipoplex is also studied. Structural transformation of pDNA upon complexation with liposome is characterized by circular dichroism (CD) spectroscopy. Interaction of all tocopheryl lipids with a model phospholipid, L-α-dipalmitoyl phosphatidylcholine (DPPC) derived vesicles is thoroughly examined by differential scanning calorimetry (DSC) and DPH fluorescence anisotropy measurements. Succinctly, we perform a detailed physicochemical characterization on cationic monomeric and gemini lipids bearing tocopherol as their hydrophobic backbone.
Chapter 2B: Physicochemical Characterization of Bilayer Membranes Derived from (±) α-Tocopherol Based Gemini Lipids Containing Hydroxyethyl Functionality in the Headgroups and their Interaction with plasmid-DNA and Phosphatidylcholine Bilayers
This sub-chapter describes the synthesis and aggregation properties of series of tocopheryl-based monomeric and gemini cationic lipids with hydroxyethyl functionality (Figure 2) in the headgroup region. Gemini lipids of this given series differ in their polymethylene spacer -(CH2)n- chain lengths between cationic headgroups.
All monomeric and gemini lipids are found to generate stable suspensions in aqueous media. Average hydrodynamic diameter and surface charge of liposome are characterized by DLS and zeta potential measurements. Atomic force microscopy and transmission electron microscopic studies show that all lipids form vesicular
Figure 2. Molecular structures of (±) α-tocopherol based cationic monomeric and five new lipids with hydroxyethyl functionality in the headgroups that differ in polymethylene spacer length aggregates in aqueous media. XRD studies with the cast films of lipids reveal interdigitated bilayer arrangement of liposome.
pDNA binding and release studies show that the interactions between gemini lipids and DNA depend upon the nature of head group as well as the length of the spacer between cationic head groups. Circular Dichroism (CD) spectra of lipoplex are measured to characterize structural transformation of pDNA upon complexation with liposome. DPH anisotropy and DSC studies of the DPPC-cationic lipid co-aggregates show that ~20 mol-% of of the tocopheryl gemini lipids is enough to abolish phase transition of DPPC membranes whereas more than 20 mol-% is required in case of their monomeric counterparts. Furthermore thermotropic properties of co-aggregates depend upon the length of the spacer of gemini lipid included in the mixture.
Chapter 2C: Transfection Efficacies of α-Tocopherol Based Cationic Gemini Lipids with Hydroxyethyl Containing Headgroups.
In this sub-chapter, we demonstrate transfection efficiency of five α-tocopheryl gemini lipid with hydroxyethyl containing headgroups (Figure 3). Co-liposomal formulations with helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) form highly stable formulations in water. Co-liposomal formulations with high molar ratio of DOPE (1.5:1 and 2:1) show higher transfection efficiency than liposome with low DOPE content liposome. Co-liposome of gemini lipids with longer spacer (n = 8 and 12) have higher level of luciferase expression in HepG2 cell line. In A549 and MCF-7 cell lines also co-liposomes of TH8S (2:1) are proved to be better than other co-liposome. N/P ratios of highest transfection are 1-1.5. These formulations are more potent than L2K in all three cancer cell line. The comparison with gemini lipid (T8T) without
Figure 3. Molecular structures of (±) α-tocopherol based cationic gemini lipids that differ in polymethylene spacer length and helper lipid DOPE.
hydroxylethyl group also proves the importance of hydroxyethyl functionalities. High serum stability of DOPE-gemini lipid formulation is attributed to tocopherol backbone and also hydroxyethyl functionalities. Circular dichroism data also show that lipoplex of DOPE-TH8S (2:1) have different conformation than the other. Relatively moderate binding efficiency and easy release of pDNA is also observed with DOPE-TH8S (2:1) in the EB-displacement assay which could be plausible reason for high transfection efficiency.
Chapter 2D: Reduction Responsive Nanoliposomes of α-Tocopheryl-Lipoic Acid
Conjugate for Efficacious Drug Delivery to Sensitive and Resistant Cancer Cells
In this sub-chapter, we present lipid conjugates derived from biologically relevant molecules, i.e., tocopherol and lipoic acid (Figure 4). These conjugates (TL1 and TL2) are able to form stable nanoliposomes (~100 nm) that respond to the reducing environment of cells as shown by the treatments of 1,4-Dithiothreitol (DTT) and Glutathione (GSH).
Figure 4. Molecular structures of tocopheryl-lipoic acid conjugates, TL1 and TL2
Nanoliposomes could efficiently load the drug (DOX) molecules and release them in response to the stimulus. Nanoliposomes are stable enough in the presence of serum and could deliver DOX inside drug sensitive and drug resistant cells in an efficient manner that is even better than the drug alone treatments as shown by means of flow cytometry and confocal microscopy analysis. DOX loaded nanoliposomal formulations show relatively less cell viability counts than those drug alone treatments.
Chapter 3A: Interaction of Nickel (II) and mida ole it
Triazacyclononane Modified Chelator Amphiphiles: A Potential Substrate for Immobilization of His-tag Protein on Hydrophilic Surface
This sub-chapter describes two chelator amphiphiles based on 1, 4, 7-traiazaclonone (TACN) (Figure 5). These compounds could bind efficiently Ni2+ ion. Self-assemble of these amphiphiles form vesicular aggregates. Their packing properties of these amphiphiles are influence by Ni2+ and imidazole. Also influence of Ni2+ and imidazole in Langmuir monolayer isotherm of these amphiphiles at air-water interface are also studied.
Figure 5. Molecular structures of TACNA chelator amphiphiles.
These studies show the newly synthesized amphiphiles could immobilize histidine tagged protein on both bilayer and monolayer surface. One of these compounds with Ni2+ (C16TACNA-Ni2+) is used to transfer a His-tagged protein nucleolin on hydrophilicobic glass surface by Langmuir-Blodgett transfer technique. So, these compounds with Ni2+ could be very useful to attach different His-tagged protein or polypeptide of interest on the bilayer (liposome) or monolayer surface.
Chapter 3B: Supramolecular Hosts for Enhancing the Selectivity of TACN Based Probes towards Copper (II): Differential Output Signals for Cysteine and Histidine
In this sub-chapter, we have developed a new amphipathic probe compound 1 having TACN as the binding site and dansyl as signaling moiety (Figure 6). As TACN is known for its’ unspecific interaction with multiple ions, the probe shows response with most of the transition metal ions. However, incorporation into different supramolecular hosts (like micelle and vesicles) drastically improves the selectivity of compound 1 towards Cu2+ (diminution of bright green fluorescence) in water. Then we
Figure 6. Molecular structures of dansylated TACN chelator amphiphiles.
have also employed the Cu2+ complex of compound 1 for selective estimation of amino acids. Addition of cysteine regains the green emission of compound while histidine exhibits blue intense emission upon formation of ternary conjugate.
Chapter 4: Transforming a β-Sheet Pyrenylated-VPGKG Sequence into pH Tolerent, Thixotropic Hydrogel by Arene-Perfluoroarene Interactions and Visualized Sensing of Calcium (II) Ion
In this chapter we discuss self-assembly studies of a novel thermoresponsive, lipidated, pyrene-appended peptide, PyP (Figure 7). Size of the vesicular aggregates of the β-sheet forming peptide, PyP, strongly depends on the temperature of the solution in water. Further pyrene-octafluoronaphthalene (OFN) pair has been used as supramolecular synthon to induce hydrogelation of PyP in presence of equimolar amount of OFN via complementary quadrupole-quadrupole interactions. The gel shows excellent pH tolerant as well as thixotropic behavior. Detailed studies suggested the lamellar packing of the gelator in a right-handed helical fashion yielded vesicular aggregates. The sticky vesicles form gel via inter-
Figure 7. Molecular structure of the Pyrenylated-VPGKG peptide (PyP) and octafluoronapthalene (OFN).
Ca2+ ion reinforces the mechanical strength and also reduces the critical gelator concentration of the native gel through coordination with the free -COO- group of the gelator. Therefore, this present system could be used as a visualized sensor of Ca2+ ion.
Chapter 5: First Report of Naphthalenediimide Based Metallo(organo)gel
In this chapter, we have demonstrated synthesis of a novel asymmetric bolaamphiphilic (Figure 8). NDI derivative is capable of self assemble into stable gel in EtOH. Detailed studies reveal the gelator molecule of 1 adopt a parallel alignment in the lamellae during self-aggregation as nanoscopic spherical assemblies. In addition, dried gel of 1 shows nematic liquid crystalline phase. Further, we synthesize a novel metal-ligand discrete complex 2 in a nearly quantitative yield by reacting equimolar amount of 1 and PdCl2(PhCN)2.
Figure 8. NDI derivative, 1, and its discrete metal complex 2.
Complex 2 has been found to yield stable gel in dichloromethane (DCM) or chloroform (CHCl3) through the formation of high aspect ratio fibers. ROESY NMR experiment of
Complex 2 has been found to yield stable gel in dichloromethane (DCM) or chloroform
(CHCl3) through the formation of high aspect ratio fibers. ROESY NMR experiment of
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