Aggregation Behavior Of ?-Ketooctanoic Acid And Sodium ?-Ketooctanoate In Water

Amphiphile molecules in aqueous solutions can self-assemble to form various aggregate structures such as micelles,vesicles and lamellar phases,in which vesicles have been extensively studied recently.Vesicle assemblies have played an important role in the fields of bio-membrane simulation,drug delivery and material synthesis,especially that they are commonly used as model systems for exploring the origin of life.Generally,primitive cellular membranes are presumed to have originated on prebiotic Earth by the self-assembly of simple amphiphilic molecules,thus fatty acid vesicles are widely used as model systems of protocell.However,the hypothesis favoring primordial vesicle formation by fatty acids faces severe challenges,such as the fact that fatty acids only form vesicles in a narrow pH range(cal.7-9),the instability of the vesicles in the presence of divalent cations,and the relatively high vesicle formation concentration(>50 mM for C8-C10 fatty acid systems).Single-tailed ?-ketooctanoic acid(KOCOOH)is a derivative of fatty acids.Recent studies have found that,under simulated sunlight irradiation,KOCOOH in water can form vesicles at low concentration(?6 mM),arising from the formation of double-tailed chemically bonded dimer,which presents a credible model for primitive cellular membranes.Thus,an interesting question raised is how KOCOOH itself assembles in water,since it may be related to the formation of primitive cells.So far,there have been no reports on it.In this dissertation,the aggregation behavior of KOCOOH and sodium ?-ketooctanoate(KOCOONa)in aqueous solution was studied,and further compared with octanoic acid and sodium octanoate,to improve the understand the self-assembly behavior of single-tailed keto-acid amphiphiles and to provide information for exploring the origin of life and the basis for the application of the aggregates.The main contents and conclusions of the doctoral dissertation are listed as follows:(1)The basic properties of KOCOOH and KOCOONa,such as spectral characteristics,light irradiation stability,melting point and solubility were investigated.In addition,the apparent acid dissociation constant(Ka)were measured,and the effect of concentration(C)on pKa was investigated and compared with octanoic acid and sodium octanoate.The results show that the chemical shifts(?)of the protons of KOCOOH and KOCOONa move to the downfield significantly compared with the octanoic acid and sodium octanoate;KOCOOH and KOCOONa are stable in aqueous solution under the laboratory routine light(no photoinitiated reaction occurred for at least 2 weeks);and the solubility of KOCOOH in water is obviously higher than that of octanoic acid(?4.3 mM),but the solubility of KOCOONa(-300 mM)is significantly lower than that of sodium octanoate(?3.69 M).With increasing C,the pKa of KOCOONa increases arising from the molecular aggregation.At given C,the pKa of KOCOONa is obviously lower than that of sodium octanoate.The obvious difference in the properties of KOCOOH or KOCOONa with octanoic acid or sodium octanoate can be attributed to the hydrophilicity and electron-withdrawing ability of the ketone group.These results improve the understanding of the effect of ketone carbonyl substitution on the properties of fatty acids.(2)The aggregation behavior of KOCOOH in aqueous solution was studied.The morphology,structure and size of the aggregates were characterized by negative staining(NS-)and cryogenic(cryo-)transmission electron microscopy(TEM),atomic force microscopy(AFM),conductivity and dynamic light scattering(DLS).It's found that KOCOOH itself can form vesicles at a very low critical vesicle concentration(CVC,ca.1.4 mM),obviously lower than that of octanoic acid/sodium salt(ca.120-200 mM).Uni-and multilamellar vesicle structures were observed,which coexist in the KOCOOH solution;the hydrodynamic diameter was about 92 nm and the thickness of the KOCOOH vesicle membranes is about 2.1 nm.Importantly,the vesicles exhibit remarkable stability within a wide pH range(ca.2-10),upon long-term storage(at least 10 months at room temperature),and even in artificial seawater(with a salinity of 36.5 g/L and containing 33.8 mM of equimolar Mg2+ and Ca2+).In addition,the formation mechanism of vesicles is discussed.The "acid-soap" structure formed by the hydrogen bonding between the carboxyl groups plays a key role.Furthermore,it is possible that a "water-bridge" is formed between two adjacent keto groups through hydrogen bonding,also contributing to the high stability of vesicles.Owing to the fact that KOCOOH vesicles can form at a very low concentration,within a wide pH range and exhibit remarkable stability in the presence of divalent cations,KOCOOH vesicles may be a good model system for protocell-like vesicles.In addition,they may have potential applications,such as in drug delivery and material synthesis.(3)The aggregation behavior of KOCOONa in aqueous solution was studied.The morphology,structure and size of the aggregates were characterized by NS-TEM,cryo-TEM,freeze-fracture(FF-)TEM,AFM,confocal laser scanning microscopy(CLSM),conductivity and DLS.Similar to KOCOOH,it is found that KOCOONa itself can also form vesicles in aqueous solution,and its CVC is about 15 mM,obviously lower than that of sodium octanoate(?120-200 mM),but higher than that of KOCOOH;the hydrodynamic diameter of the vesicles was about 70-150 nm and the thickness of the vesicle membranes is about 2.0 nm.Uni-and multilamellar vesicle structures were observed,which coexist in the KOCOONa solution.With increasing C,a vesicle-to-micelle transition can occur,showing a critical micelle concentration(CMC)of?80 mM.The mechanism of vesicle formation and structural transformation was discussed.In addition,the membrane permeability of the KOCOONa vesicles was examined using small-size calcein and large-size FITC-BSA as fluorescence probes,showing a size-selective permeability,similar to biological cell membranes.(4)The microviscosity,encapsulation,permeability and thermal stability of KOCOOH vesicle membranes were investigated by steady-state and time-resolved fluorescence techniques,using 1,6-diphenyl-1,3,5-hexatriene(DPH),riboflavin,calcein and Nile red as fluorescent probes.The results show that the microviscosity of KOCOOH membranes is about 30-90 mPa s,which is related to the pH value.With increasing pH from 3-10,the microviscosity initially increasse and then decreases,with a maximum appearing at pH?7.In addition,KOCOOH vesicles have the ability to encapsulate hydrophilic and hydrophobic guests;keep stable below 50 ? and rupture above 50 ?,and are permeable to small ions.The permeability of OH-across KOCOOH membranes can be well described by the first-order kinetic model.Thus,KOCOOH vesicles possess some of the necessary functional properties of the early cell membrane,which further validates that KOCOOH vesicles may be an alternative primitive membrane model.