Aggregation behaviour of a single-chain, phenylene-modified bolalipid and its miscibility with classical phospholipids

Aggregation behaviour of a single-chain, phenylene-modified bolalipid and its miscibility with classical phospholipids Simon Drescher*1, Vasil M. Garamus2, Christopher J. Garvey3, Annette Meister4,5 and Alfred Blume4 Full Research Paper Address: 1Institute of Pharmacy, Martin Luther University (MLU) Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany, 2Helmholtz-Zentrum Geesthacht (HZG), Centre for Materials and Costal Research, Max-Planck-Str. 1, 21502 Geesthacht, Germany, 3Australian Nuclear Science and Technology Organisation (ANSTO), Kirrawee DC, NSW, Australia, 4Institute of Chemistry, MLU Halle-Wittenberg, von-Danckelmann-Platz 4, 06120 Halle (Saale), Germany and 5Institute of Biochemistry and Biotechnology, MLU Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle (Saale), Germany Open Access Beilstein J. Org. Chem. 2017, 13, 995–1007. doi:10.3762/bjoc.13.99 Received: 15 March 2017 Accepted: 28 April 2017 Published: 23 May 2017 This article is part of the Thematic Series "Lipids: fatty acids and derivatives, polyketides and isoprenoids". Dedicated to Prof. Dr. Bodo Dobner on the occasion of his 65th birthday. Guest Editor: J. S. Dickschat Email: Simon Drescher* - © 2017 Drescher et al.; licensee Beilstein-Institut. License and terms: see end of document. * Corresponding author Keywords: aggregation behaviour; bolaamphiphiles; bolalipids; membrane lipids; mixing behaviour; nanofibres; self-assembly Abstract In the present work, we describe the synthesis of a single-chain, phenylene-modified bolalipid with two phosphocholine headgroups, PC-C18pPhC18-PC, using a Sonogashira cross-coupling reaction as a key step. The aggregation behaviour was studied as a function of temperature using transmission electron microscopy (TEM), differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, and small angle neutron scattering (SANS). We show that our new bolalipid self-assembles into nanofibres, which transform into flexible nanofibres at 27 °C and further to small elongated micelles at 45 °C. Furthermore, the miscibility of the bolalipid with bilayer-forming phosphatidylcholines (DMPC, DPPC, and DSPC) was investigated by means of DSC, TEM, FTIR, and small angle X-ray scattering (SAXS). We could show that the PC-C18pPhC18-PC is partially miscible with saturated phosphatidylcholines; however, closed lipid vesicles with an increased thermal stability were not found. Instead, bilayer fragments and disk-like aggregates are formed. Introduction Bolalipids are amphiphilic molecules consisting of two hydrophilic headgroups attached to both ends of a long hydrocarbon spacer [1]. The hydrophobic spacer is composed of either a single alkyl chain or two chains connected via a glycerol moiety. These bolalipids originate in membranes of some species of archaea, e.g., thermoacidophiles and these archaeal membranes can withstand extreme living conditions, such as high temperatures or low pH values [2-4]. Archaea are quite different from bacteria and eukaryotes [5-9], which is also reflected in the chemical structure of those archaeal membrane 995 Beilstein J. Org. Chem. 2017, 13, 995–1007. lipids: the alkyl chains are connected via ether linkages in the inverse sn-2,3 configuration to the glycerol, the alkyl chains sometimes contain a varying number of cyclopentane rings or several methyl branches [2,3], and some of the archaeal lipids consist of two transmembrane alkyl chains (caldarchaeol-type). Especially this type of bolalipids is of great interest for applications in material sciences, biotechnology, and pharmaceuticals [10-15]. Since these bolalipids are able to span the membrane of classical phospholipid bilayers, they can be used to stabilize liposomes for drug delivery purposes. The applicability of this approach was already tested for a large variety of natural and artificial bolalipids [12,16-22]. The isolation of archaeal bolalipids from natural sources is expensive and often leads to mixtures of bolalipids with different alkyl chain pattern. But also the synthesis of natural as well as artificial bolalipids is elaborate and time-consuming and, hence, present research tries to simplify the chemical structure of bolalipids by keeping up their membrane-stabilizing properties [23]. This simplification strategy led in our group to the synthesis of dotriacontane-1,32-diylbis[2-(trimethylammonio)ethylphosphate] (PC-C32-PC) [24,25], the simplest bola model lipid consisting of two phosphocholine (PC) headgroups connected by a long, unmodified C32 alkyl chain. If PC-C32PC is suspended in water, the formation of a dense network of nanofibres and, as a consequence, a clear and transparent hydrogel is observed [25]. The nanofibres have a thickness of about 6 nm, corresponding to the length of a PC-C32-PC molecule. Due to the bulky PC headgroup, the PC-C32-PC molecules are arranged side by side within the fibrous aggregate but slightly twisted relative to each other leading to a helical super structure of the fibres. This helicity was previously confirmed by cryo-transmission electron microscopy (cryo-TEM), high resolution atomic force microscopy (AFM) [26], and Monte Carlo simulations [27]. A temperature increase leads to a transformation of the nanofibres into small micelles and the gel character is lost. This reversible gel/sol transformation is accompanied by a cooperative endothermic transition at Tm = 48 °C, which can be followed by differential scanning calorimetry (DSC) [24]. However, the use of PC-C32-PC as “stabilizer” of phospholipid bilayers failed. In mixtures of PC-C32-PC with classical phospholipids, such as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), no significant insertion of the bolalipid into the bilayer was observed [28]. The reason for this behaviour is that packing problems due to the mismatch between the large space requirement of the PC headgroup of PC-C32-PC and the small cross-sectional area of its single alkyl chain arise. The insertion of a PC-C32-PC molecule in a stretched conformation into phospholipid bilayers is energetically unfavourable as it produces void volume, which can be filled by neither bolalipid nor phospholipid. Consequently, the self-assembly of PC-C32PC into nanofibres is preferred. To evade these packing problems, we expanded the crosssectional area of the alkyl chain of the bolalipid in order to fill the void volume. Besides heteroatoms [29,30], acetylene [31] or diacetylene groups [32], or methyl branches [31,33], also phenyl- or biphenyl rings were inserted into the long alkyl chain [34-37]. The insertion of a phenylene group led to PC-C16pPhC16-PC [36], which self-assembles at room temperature into small ellipsoidal micelles, and to PC-C17pPhC17-PC [35] that forms nanofibres with a significantly reduced thermal stability compared to PC-C32-PC. Unfortunately, the insertion of PC-C17pPhC17-PC into phospholipid bil (...truncated)