Design and synthesis of novel dimeric lipids and amphiphiles and characterization of their vesicular and micellar properties

Abstract

The thesis titled “Design and Synthesis of Novel Dimeric Lipids and Amphiphiles and Characterization of Their Vesicular and Micellar Properties” describes the biophysical and morphological aspects of various supramolecular organizations generated from newly developed dimeric lipids and amphiphiles that are covalently connected by a spacer chain at the level of the headgroup. The thesis has been divided into four chapters. Chapter 1 gives an overview of the general area of aggregate chemistry, especially in the context of dimeric or gemini lipids and amphiphiles. It provides a comprehensive account of research by various groups working in membrane?mimetic chemistry toward the development of novel vesicle? and micelle?forming systems.

Chapter 2 — Vesicle Formation from Ion?Paired Systems Chapter 2 deals with vesicle formation from different ion?paired systems and is divided into three sections. Section A This section describes a series of vesicle?forming ion?paired bis(hexadecyl dimethylammonium)alkane dipalmitate amphiphiles where the two headgroup charges are separated by a flexible polymethylene chain [–(CH?)?–], such that the conformation of the spacer chain determines the intra?“monomer” headgroup separation. Electron microscopy indicated that each of these forms bilayer membranes upon dispersion in aqueous media. Membrane properties were examined by differential scanning calorimetry (DSC) and temperature?dependent fluorescence anisotropy. Interestingly, the T? values for vesicular 1a–1h decreased with an increase in spacer chain length (m). Thus, while the apparent T? of 1a with m = 2 is 74.1?°C, the corresponding value for 1h with m = 12 is as low as 38.9?°C. Entrapment of a fluorophoric dye (e.g., riboflavin) and its response to an imposed transmembrane pH gradient demonstrated that these bilayers form enclosed vesicles. X?ray diffraction of cast films was performed to understand the nature and thickness of these membrane organizations, which were in the range of 30–51?Å. To rationalize the observations, molecular modeling studies were also carried out. 1a–1h, m = 2, 3, 4, 5, 6, 8, 10, 12

Section B This section focuses on the synthesis of another class of ion?paired amphiphiles, bis(hexadecyl dimethylammonium)alkanebis(hexadecyl phosphate) amphiphiles, 2a–2d, in which the –NMe??···–OOC headgroups (as in Section A) are replaced by –NMe??···O–P(=O)OR–O–(CH?)?–O–P(=O)OR–O···?O– headgroups (a phosphatidylcholine mimic), with the aim of studying headgroup dynamics indirectly governed by the spacer using ³¹P NMR spectroscopy. All of these form vesicles, as evidenced by transmission electron microscopy and dye entrapment. The thermal phase transition temperatures also respond strongly to spacer chain variation. The influence of the spacer on vesicular properties was examined in detail by calorimetry, fluorescence depolarization, ¹H NMR, and ³¹P NMR. Paramagnetic relaxation experiments were performed to probe transbilayer lipid asymmetry. X?ray diffraction studies explored possible polymorphism in these bilayers. A rationale for the spacer effects was provided by energy?minimized structures for each amphiphile. 2a–2d, m = 2, 4, 6, 10

Section C This section highlights the syntheses of three new hybrid (bolaphile–amphiphile) ion pairs: bis(hexadecyl trimethylammonium)phenyl?1,2?, 1,3? and 1,4?di(oxyundecanoate), 3, 4, and 5, where the bolaamphiphilic counterions originate from different isomeric positions on the central phenyl ring; and the corresponding (monopolar) anion/cation pairs cetyltrimethylammonium (CTA) 11?phenoxyundecanoate (6) and CTA palmitate (7). Electron microscopy indicated that each forms bilayer membranes with different morphologies upon dispersion in aqueous media. Membrane properties were examined by DSC, microcalorimetry, temperature?dependent fluorescence anisotropy, and UV–Vis spectroscopy. The T? values for vesicular 3, 4, 5, 6, and 7 were 38, 12, 85, 31.3, and 41.6?°C, respectively. Interestingly, T? for 3 and 5 depended on concentration. Entrapment of small solutes and permeability studies further demonstrated enclosed vesicles. X?ray diffraction of cast films revealed membrane widths in the range of 33–47?Å. Molecular modeling supported these observations. Thus, membrane properties can be modulated by simple structural changes at the amphiphile level; judicious incorporation of centrally disubstituted aromatic units as structural anchors in bolaphiles allows modulation of vesicle properties.

Chapter 3 — Gemini Bis?Cationic Pseudoglyceryl Lipids (Vesicles) Chapter 3 describes the synthesis of eight new gemini bis?cationic pseudoglyceryl lipids 8a–8h, where two dimethylammonium headgroups are separated by a variable number of polymethylene units [–(CH?)?–]. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) of aqueous dispersions confirmed formation of vesicular aggregates. Vesicle sizes and morphologies depended strongly on m, the preparation method, and thermal history. Thermotropic properties were obtained from microcalorimetry and temperature?dependent fluorescence anisotropy. Strikingly, T? values for vesicular 8a–8h showed a nonlinear dependence on m. These vesicles entrapped small water?soluble solutes and sustained transmembrane pH gradients. Reflection X?ray diffraction of cast films elucidated membrane nature and thickness, revealing three distinct membrane architectures depending on m. The ESR spin?probe method (doxyl stearic acids 5NS, 12NS, 16NS) established chain?flexibility gradients and bilayer homogeneity. Fusion characteristics of these bipolar tetraether lipids in the presence of sodium sulfate were examined using FRET fusion assays. Molecular modeling helped assign structural reasons for these unusual membrane?level findings. Interactions with cholesterol and DPPC vesicles depended strongly on m. This demonstrates another gemini lipid system in which membrane properties can be fine?tuned by simple monomer?level structural changes. R = n?C??H??; m = 3, 4, 5, 6, 12, 16, 20, 22 (8a–8h)

Chapter 4 — Dimeric Micellar Aggregates (SANS Studies) Chapter 4 focuses on the physical and morphological behavior of dimeric micellar aggregates and is divided into two sections. Section A — Hydrocarbon Spacers (9a–9g) Micelles of Br? · n?C??H??NMe??–(CH?)?–N?Me?–n?C??H?? · Br? (where m = 3, 4, 5, 6, 8, 10, 12), 9a–9g, adopt different morphologies and internal packing arrangements in aqueous media depending on spacer length. Detailed small?angle neutron scattering (SANS) measurements of these bis?cationic dimeric micelles in D?O were analyzed using the Hayter–Penfold model for macro?ion solutions to compute the interparticle structure factor S(Q), accounting for screened Coulomb interactions. SANS analysis clearly indicated that aggregate growth and micelle shape depend primarily on spacer length. For m < 4, micellar growth is particularly pronounced. Effects of varying surfactant concentration (m = 5 and 10) and temperature (m = 10) were also examined. Critical micelle concentrations (CMC) and microviscosities (from the probe 1,6?diphenyl?1,3,5?hexatriene) were determined. Changes in CMC and microviscosity versus m were explained by conformational variations and progressive looping of the spacer in the micellar core with increasing m. Similar studies on mixed micelles composed of CTAB and 9a, 9c, 9f in D?O indicated that aggregate growth and micelle shape can be modulated by the amount of dimeric surfactant present. 9a–9g, m = 3, 4, 5, 6, 8, 10, 12

Section B — Oxyethylene Spacers (10a–10c) This section discusses the synthesis and SANS studies of three new gemini surfactants 10a–10c containing mono?, di?, and tri?oxyethylene spacer chains. Data were analyzed as in Section A. SANS showed that micellar morphology depends on both the nature and length of the spacer. Detailed analysis indicates that introducing an oxyethylene spacer is not sufficient to prevent spacer looping; the average separation between dimethylammonium headgroups is considerably lower than that expected from fully extended spacer conformations. Micelles were further characterized by CMC, microviscosity, and micropolarity measurements, which indicated little difference in micellar properties across 10a–10c.