Chemistry and Physics of Lipids 183 (2014) 22–33

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Chemistry and Physics of Lipids

jou rn al h om epage : www.elsev ier .com/ locate /chemphys l ip

Stereoselective synthesis of perdeuterated phytanic acid, itsphospholipid derivatives and their formation into lipid modelmembranes for neutron reflectivity studies

Nageshwar R. Yepuria,∗, Stephen A. Holtb, Greta Moraesa, Peter J. Holdena,Khondker R. Hossainc, Stella M. Valenzuelac, Michael Jamesa,d,e, Tamim A. Darwisha

a National Deuteration Facility, Bragg Institute, Australian Nuclear Science and Technology Organisation, Locked Bag 2001, Kirrawee DC,NSW 2232, Australiab Bragg Institute, Australian Nuclear Science and Technology Organisation, Locked Bag 2001, Kirrawee DC, NSW 2232, Australiac School of Medical and Molecular Biosciences, University of Technology Sydney, Ultimo, NSW 2007, Australiad School of Chemistry, The University of New South Wales, Kensington, NSW 2052, Australiae Australian Synchrotron, 800 Blackburn Road, Clayton, Victoria 3168, Australia

a r t i c l e i n f o

Article history:Received 6 January 2014Received in revised form 1 April 2014Accepted 7 April 2014Available online 2 May 2014

Keywords:Perdeuterated phytanic acidTail-deuterated 1,2-di(3RS,7R,11R-phytanyl)-sn-glycero-3-phosphocholineTail-deuterated 1,2-di(3RS,7R,11R-phytanoyl)-sn-glycero-3-phosphocholineLangmuir monolayerTethered bilayer lipid membrane

a b s t r a c t

We describe a straightforward method, for synthesis of large scale (gram quantities) of highly deuteratedphytanic acid from commercially available phytol while preserving the stereochemistry around the chiralcentres. The subsequent synthesis of tail-deuterated analogues of the archeabacterial membrane lipids1,2-di(3RS,7R,11R-phytanyl)-sn-glycero-3-phosphocholine (DPEPC) and 1,2-di(3RS,7R,11R-phytanoyl)-sn-glycero-3-phosphocholine (DPhyPC) from perdeuterated phytanic acid is also described. Both lipidswere employed in construction of two different model membranes, namely Langmuir monolayers anda tethered bilayer membrane (TBM) on a solid substrate, characterised by pressure area isotherm andneutron reflectometry techniques. At 10 mN/m pressure the head-group thickness of both monolayerswas similar while the thickness of the tail region was significantly larger for tail-deuterated DPhyPC,which was evident from a smaller area per molecule. At 20 mN/m the thickness of the head and tailregions in both lipids was comparable, yet the area per molecule of tail-deuterated DPhyPC was 10%smaller than tail-deuterated DPEPC. In the TBM bilayer model membrane, the thickness of the lipid tailsin both inner and outer leaflets was 8.2 A, giving a total of 16.4 A. Deuteration enabled unambiguousdetermination of the relative proportion of the hydrogenous tether, phospholipid and subphase.

© 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Phytanic acid ((3RS,7R,11R)-3,7,11,15-tetramethylhexade-canoic acid; Fig. 1a) is a naturally occurring, saturated, branchedisoprenoid chain fatty acid of significant biological interest due toits association with diseases such as Refsum’s disease (Steinberg,1978; Herndon et al., 1969; Tsai et al., 1969), as well as prostate,breast, and colorectal cancers (Price et al., 2010; Allen et al.,2008). In nature phytanic acid occurs as a racemic mixture oftwo diastereomers, and humans can obtain this fatty acid via theconsumption of dairy products, certain fish and ruminant animal

∗ Corresponding author at: National Deuteration Facility, The Bragg Institute, Aus-tralian Nuclear Science and Technology Organisation (ANSTO), Locked Bag 2001,Kirrawee DC, NSW 2232, Australia. Tel.: +61 2 9717 6004; fax: +61 2 9717 3038.

E-mail addresses: raoy@ansto.gov.au, yepuri@hotmail.com (N.R. Yepuri).

fats, making it the most common branched fatty acid in the humandiet (Brown et al., 1993).

Phytanic acid-based lipids, such as 1,2-di(3RS,7R,11R-phytanyl)-sn-glycero-3-phosphocholine (DPEPC, Fig. 1b) and 1,2-di(3RS,7R,11R-phytanoyl)-sn-glycero-3-phosphocholine (DPhyPC,Fig. 1c) have been extensively used to develop membrane-basedbiosensors (Cornell et al., 1997; Römer and Steinem, 2004; Hanet al., 2007; Vogel et al., 2009; Hansen et al., 2009), and to aidthe study of the formation of pores in membranes (Lee et al.,2004; Huang et al., 2004; Arcisio-Miranda et al., 2008; Heller et al.,1997; Kresak et al., 2009) and structures formed by protein-basedion channels (Kelkar and Chattopadhyay, 2007; Busath et al.,1998; Duffin et al., 2003; Poulos et al., 2009; Naumann et al.,2003; Lundbaek et al., 2005, 2010). DPEPC and DPhyPC differonly in the mode of attachment between the phytanic acid chainand the glycerol backbone; ether or ester respectively. The keyreasons behind the utility of phytanic acid-based lipids are a

http://dx.doi.org/10.1016/j.chemphyslip.2014.04.0040009-3084/© 2014 Elsevier Ireland Ltd. All rights reserved.

N.R. Yepuri et al. / Chemistry and Physics of Lipids 183 (2014) 22–33 23

3711OH

O OO

OH

P O

O

O N

a b

OO

OH

PO

O

O N

O

O

c

Fig. 1. Chemical structures of (a) phytanic acid, (b) DPEPC and (c) DPhyPC.

combination of their excellent chemical and mechanical stabilityand their capacity to form highly insulating bilayer membranes,with electrical resistances typically observed in the G! range.

Phytanic acid-based lipids are also found in the cellular mem-branes of extremophile bacteria such as archaebacteria (van deVossenberg et al., 1998; Yamauchi et al., 1992, 1993), which cansurvive in harsh environments and are stable over a wide range oftemperatures. The cellular membranes of the extremely halophilic(salt-loving) organism Halobacterium cutirubrum for example con-tains phytanyl chains, a feature which taxonomically separatesthis unique class of microorganism from eubacteria and eukary-otes (Kates et al., 1965, 1966, 1967). There has been considerableactivity related to the investigation of the impact of phytanic acidon the stability and dynamics of phospholipid bilayers (Cushleyet al., 1979; Yue et al., 1988; Yue and Cushley, 1990), the formationand physiochemical properties of 1,2-di(3RS,7R,11R-phytanoyl)-sn-glycero-3-phosphocholine model membranes (Redwood et al.,1971; Janko and Benz, 1977; Lindsey et al., 1979; Gutknecht, 1988),as well as natural and synthetic archaebacterial membranes (Esserand Lanyi, 1973; Plachy et al., 1974; Gulik et al., 1985; De et al.,1986; Rodriguez-Valara, 1991; Moss and Fujita, 1990; Stewart et al.,1990).

While perdeuterated phytanic acid has not been previously pro-duced, partially deuterated phytanic acid with deuterium atoms atthe !- and "-positions (Brink et al., 1989; ten Brink et al., 1988,1992, 1993; Johnson and Poulos, 1988; Verhoeven et al., 1998)has been synthesised for GC–MS investigations of the pathwaysassociated with the metabolic breakdown of phytanic acid. Whenisotopically labelled compounds are used as internal standards inMS metabolic studies, the deuterium labelling has to be sufficientlylarge to achieve adequate mass separation from the mass distribu-tion resulting from the naturally occurring isotopes. For a relativelylarge molecular weight molecule such as phytanic acid and its lipidderivatives, the use of the perdeuterated form provides optimalperformance in such studies; although in some cases the isomeri-cally pure form is required.

Perdeuteration of phytanic acid and its phospholipid deriva-tives provides an excellent tool for neutron scattering and NMRstudies of model membranes. Deuteration of phospholipids is anessential first step in numerous 2H NMR (Gehman et al., 2011;Seelig and Macdonald, 1987) and neutron reflectometry (Fernandezet al., 2012, 2013; Shen et al., 2011; Stanglmaier et al., 2012;Wacklin, 2011; James et al., 2011; Le Brun et al., 2013) investiga-tions. Although deuterium (2H) and hydrogen (1H) behave similarlyin chemical reactions, the composition of their nuclei results invastly different NMR and neutron scattering responses.

DPhyPC in its protonated form has been recently used to formtethered bilayer model membranes (TBMs) for neutron reflec-tometry studies, and was shown to be effective at revealing themembrane structure against a solution interface comprised ofheavy water (D2O) (Valincius et al., 2006; McGillivray et al., 2007).

Membranes formed using protonated DPhyPC, however, are noteffective platforms for studies seeking to investigate the locationand structure of integral and trans-membrane proteins, as bothlipid and protein have similar neutron scattering length densities(nSLD). In these instances, tail-deuterated DPhyPC is essential tosupport such studies. On the other hand, DPEPC possesses phytanicacid tails attached to the glycerol backbone of the lipid through themore hydrophobic ether bonds, which are known to have superiorbond stability to the less hydrophobic ester bonds in DPhyPC.

In this paper, we describe a convenient method for the synthe-sis of perdeuterated phytanic acid in multi-gram scale quantitiesfrom commercially available phytol, as well as the synthesis oftail-deuterated phospholipids DPhyPC (94% D) and tail-deuteratedDPEPC (98% D). We demonstrate in these deuteration and syntheticprocesses that the stereochemistry of phytanic acid is preserved atpositions 7 and 11.

To demonstrate the utility of these deuterated lipids, two neu-tron scattering experiments were undertaken with different modelmembrane architectures. Firstly, the tail-deuterated DPhyPC andtail-deuterated DPEPC were used to make Langmuir monolayers atthe air/water interface. In this system, the neutrons reflect fromthe interfaces parallel to the water surface and provide a sub-nanometre sensitive depth profile of the structure perpendicularto the water surface (Fig. 2a). This also allows direct compar-ison between the two monolayer assemblies and the effect ofthe degree of hydrophobicity of ether versus ester bonds in thetwo lipids. Secondly, a tethered bilayer membrane (TBM, Fig. 2b)was constructed using the more stable tail-deuterated DPEPC lipid(commonly used in bio-sensing applications) where the neutronreflection provides information regarding the structure perpendic-ular to the gold/solution interface under conditions of completehydration. The long tether molecules (Phyt(EO4)2SSPh) penetrateinto the lower leaflet of the bilayer providing an anchor. The shorttether molecules provide for a water reservoir between the bilayerand the substrate which is essential for biosensor operation. In thiswork, the bilayer is formed by tail-deuterated DPEPC, and a full sur-face coverage as depicted prevents solute or ions from traversingfrom the reservoir region to the bulk solution. Damage to the bilayeror insertion of channels provides a ‘conduction’ path through thebilayer and the possibility of detecting an electrical signal.

2. Materials and methods

2.1. General

Chemicals and reagents of the highest grade were pur-chased from Sigma–Aldrich and were used without furtherpurification. Solvents were purchased from Sigma–Aldrich, Merckand Fronine Laboratory Supplies and were purified by liter-ature methods (Perrin et al., 1980). Phytol (97% purity) waspurchased from Sigma–Aldrich and consists of a mixture of

24 N.R. Yepuri et al. / Chemistry and Physics of Lipids 183 (2014) 22–33

Fig. 2. Model membrane architectures employed: (a) Langmuir monolayers where the lipids (tail-deuterated DPhtyPC, or tail-deuterated DPEPC) are confined to the air/waterinterface, (b) The TBM constructed using sulphur chemistry (Phyt(EO4)2SSPh and OH(EO4)SSPh) to bind the tether molecules to a gold surface, enabling biosensing applicationsusing tail deuterated DPEPC as the bilayer lipid.

isomers. Raney® 2800 nickel slurry in water was also purchasedfrom Sigma-Aldrich. When solvent mixtures were used as an elu-ent, the proportions are given by volume. NMR solvents werepurchased from Cambridge Isotope Laboratories Inc. and wereused without further purification. D2O (99.8%) was suppliedby AECL, Canada. Thin-layer chromatography (TLC) was per-formed on Fluka analytical silica gel aluminium sheets (25 F254).Davisil® silica gel (LC60 A 40–63 #m) was used for bench-top flashcolumn chromatography. The phytanyl bis-tetraethyleneglycolbenzyl disulphide (Phyt(EO4)2SSPh) and hydroxyl-terminatedtetra-ethyleneglycol benzyl disulphide (OH(EO4)SSPh) used in theTBM production were supplied by Surgical Diagnostics Pty Ltd. Thesilicon substrates were purchased from EL-CAT Pty Ltd.

Large scale (gram) quantities of phytanic acid from commer-cial sources were unavailable for this work; even small quantitieswere prohibitively (100 mg for ∼US $2000). The synthesis of 3RS-phytanic acid from the readily available phytol (4 kg for ∼US $2000),

was achieved in gram quantities via a modification of the methodof Burns et al. (1999).

2.2. Instrumental

Electrospray ionisation mass spectra (ESI-MS) were recorded ona 4000 QTrap AB SCIEX Mass Spectrometer. The overall percentdeuteration of the molecules was calculated by ER–MS (enhancedresolution – MS) using the isotope distribution analysis of the dif-ferent isotopologues. This was achieved by analysing the area undereach MS peak which corresponds to a defined number of deu-terium atoms. The carbon-13 (natural abundance) contribution tothe value of the area under each MS signal was subtracted basedon the relative amount of cabon-13 natural abundance found orestimated in the protonated version. 1H NMR (400 MHz), 13C NMR(100.6 MHz) and 2H NMR (61.4 MHz) spectra were recorded on aBruker 400 MHz spectrometer at 298 K. Chemical shifts, in ppm,

N.R. Yepuri et al. / Chemistry and Physics of Lipids 183 (2014) 22–33 25

OH1

Raney nickel

EtO H/H298%

OH2

CrO3 (CH3)2CO/H2OCH3COOH

R, S

OH

O

R, S3711

D2O/220 °C

NaOD/Pt/C75%

D3C CDCD2

D2C C

D2

CD C

D2

D2C C

D2

CD C

D2

D2C C

D2

CD C

D2OH

CD3 CD3 CD3 CD3 O

perdeuterat ed phyta nic acid34

84%

Scheme 1. Synthesis of perdeuterated phytanic acid (4) from phytol (1) and phytanol (2).

were referenced to the residual signal of the corresponding NMRsolvent. Deuterium NMR was performed using the probe’s lockchannel for direct observation.

2.3. Experimental

2.3.1. Synthesis of 3RS-phytanol (2) from phytol (1) (Scheme 1)The hydrogenation of the double bond of phytol (1) to give 3RS-

phytanol (2) is shown in Scheme 1. To a solution of phytol (1)(25.0 g, 84.3 mmol) in ethanol (100 mL) was added Raney nickelcatalyst as an aqueous slurry (2.5 g) and after bubbling nitrogenthrough the reaction mixture for 10 min, it was hydrogenated for24 h using a balloon filled with hydrogen. The reaction was mon-itored by 1H NMR, and the disappearance of the peak at 5.25 ppmindicated complete hydrogenation of 1. The catalyst was removedfrom the mixture by filtering through a 4 cm thick silica bed. Theethanol was evaporated on a rotary evaporator and the remainingaqueous portion was partitioned with DCM (100 mL × 4). The com-bined organic solvent layers were dried over MgSO4 and evaporatedto give a colourless viscous liquid, 3RS-phytanol (2) (Burns et al.,1999) (24.6 g, 82.4 mmol, 98% yield). 1H NMR (CDCl3): ı 0.88 (m,15H), 0.97–1.47 (m, 22H), 1.48–164 (m, 3H), 3.37 (m, 2H). 13C NMR(CDCl3): ı 9.6, 22.6, 22.7, 24.3, 24.4, 24.7, 27.9, 29.5, 32.7, 37.2, 37.3,39.3, 39.9, 40.0, 61.2. ESI-MS m/z: 321 (M+Na)+ (see supplementaryinformation for NMR, Figs. S1 and S2).

2.3.2. Synthesis of 3RS-phytanic acid (3) from 3RS-phytanol (2)The oxidation of 3RS-phytanol (2) to give 3RS-phytanic acid (3)

is shown in Scheme 1. To a cold solution (0 ◦C) of 3RS-phytanol(23.0 g, 77.04 mmol) in acetone (500 mL) and acetic acid (400 mL)was added a solution of CrO3 (18.4 g, 184.01 mmol) in water (20 mL)slowly using a dropping funnel. The mixture was slowly brought toroom temperature and was continuously stirred for 3 h. PowderedNa2S2O5 was added slowly until the colour was persistently pur-plish green and the solution filtered through a 4 cm thick silica bed.After washing the silica bed with acetone (100 mL × 2), the organicsolvent was evaporated to give an aqueous portion. The aqueousportion was extracted with diethyl ether (100 mL × 3); the com-bined organic layers were dried over MgSO4 and evaporated underreduced pressure to give colourless viscous liquid. This was fur-ther purified by flash chromatography using a solvent mixture of2:1 hexane:diethyl ether to give a colourless viscous liquid 3RS-phytanic acid (3) (Burns et al., 1999) (20.2 g, 64.74 mmol, 84% yield).1H NMR (CDCl3): ı 0.88–0.99 (m, 15H), 1.00–1.57 (m, 22H) 1.93 (m,1H), 2.11–2.17 (m, 1H), 2.32–2.39 (m, 1H). 13C NMR (CDCl3): ı 20.8,22.6, 22.7, 24.3, 24.4, 24.8, 27.9, 30.1, 30.2, 32.7, 32.8, 36.9, 37.0,37.05, 37.2, 37.4, 37.4, 39.3, 41.5, 41.63, 177.4, 179.6. ESI-MS m/z:311(M−1)− (See supplementary information for NMR, Figs. S3 andS4).

2.4. Deuteration of 3RS-phytanic acid (4)

The hydrothermal deuteration of 3RS-phytanic acid (3) is shownin Scheme 1. A mixture of 3RS-phytanic acid (3) (13 g, 41.66 mmol),10% Pt/C catalyst (1.62 g, 1.52 mmol) and 40% NaOD solution (1.70 g,41.66 mmol or 4.25 mL of 40% NaOD in D2O) in D2O (120 mL) wasstirred under hydrothermal conditions for 72 h at 220 ◦C in a MiniBenchtop 4560 Parr reactor (600 mL vessel capacity, 206 bar max-imum pressure, 350 ◦C maximum temperature) (Zimmermann,1989; Yepuri et al., 2013; Darwish et al., 2013). At 220 ◦C and withthese reagents, the Parr reactor reaches 25 bar in pressure. Aftercooling, the reaction mixture was diluted with dichloromethane(200 mL) and the mixture filtered through Celite to remove thecatalyst. The filtered catalyst was washed with water (100 mL × 3)and the filtrate acidified to pH 2 with dilute HCl. The productwas extracted with diethyl ether (100 mL × 3) and the combinedorganic phases were dried over Na2SO4, filtered and concentratedin vacuo to give a colourless gum (12.5 g). The product was thenrun through a second cycle in the Parr reactor with fresh Pt/C cata-lyst, NaOD and D2O under the same conditions as above. Followingextraction and isolation, the final product was further purified bycolumn chromatography using 25% diethyl ether in hexane to givea colourless viscous liquid (10.9 g, 31.05 mmol, 75% yield, deuter-ation level: 97%). Residual 1H NMR (CDCl3): ı 0.78–0.87 (m), 0.91(br, s), 1.01–1.31 (m), 1.46–1.56 (m), 1.90 (br s), 2.11 (br s), 2.32 (brs); 2H NMR (CDCl3): ı 0.80–1.44 (br m, 36D), 1.89 (m, 1D), 2.09 (m,1D), 2.30 (m, 1D). 13C NMR (CDCl3): ı 18.5 (m), 21.60 (m), 23.4 (m),26.7 (m), 29.24 (m), 31.6 (m), 36.1 (m), 37.9 (m), 40.8 (m), 179.7 (s).ESI-MS m/z: 350 (M−H)−, isotope distribution (97% D) 2.0% d35, 6.9d36, 21.0% d37, 37.5% d38, 32.6% d39 (See supplementary informationfor NMR and mass spectra, Figs. S5–S9).

2.5. Isolation of the diastereomers of perdeuterated 3RS-phytanicacid (Scheme 2)

The isolation of the 3R- and 3S-diastereomers of perdeuter-ated phytanic acid followed the method of Burns et al.(1999). In summary, the phytanic acid and chiral amine(R)-(+)-1-(1-naphthyl)ethylamine were coupled together usingN,N′-dicyclohexylcarbodiimide (DCC) reagent to give the 3R and 3Snaphthylethylamide diastereomers of phytanic acid, which weresubsequently isolated using flash chromatography. The individualdiastereomers were then converted to optically pure perdeuterated3R- and 3S-phytanic acid (see below).

2.5.1. Synthesis of (3R and 3S 7R,11R)-N-[1(R)-1-naphthylethyl]-3,7,11,1S-tetramethylhexadecanamide (5 and 6)

The conversion of 3RS-phytanic acid to its naphthylethy-lamide analogues is summarised in Scheme 2. A solution ofperdeuterated 3RS-phytanic acid (4) (0.35 g, 0.99 mmol) and

26 N.R. Yepuri et al. / Chemistry and Physics of Lipids 183 (2014) 22–33

D3CCDC

D2

D2C C

D2

CD C

D2

D2C C

D2

CD C

D2

D2C C

D2

CD C

D2OH

CD3 CD3 CD3 CD3 O

DCC/DMAP/CH2Cl2

90%NH2

D3C CDCD2

D2C C

D2

CD C

D2

D2C C

D2

CD C

D2

D2C C

D2

CD C

D2NH

CD3 CD3 CD3 CD3 O

(5) 3R,(6) 3S

4

D3C CDCD2

D2C C

D2

CD C

D2

D2C C

D2

CD C

D2

D2C C

D2

CD C

D2N

CD3 CD3 CD3 CD3 O

NO

D3C CDCD2

D2C C

D2

CD C

D2

D2C C

D2

CD C

D2

D2C C

D2

CDCD2

OH

CD3 CD3 CD3 OCD3

NaNO2acetic anhydride /acetic acid

THF/water LiOH/H2O2

(7) 3R = 35%(8) 3S = 30 %

Scheme 2. Conversion of perdeuterated 3RS-phytanic acid to their naphthylethy-lamide analogues and hydrolysis of the amides to gives isomerically pure 3R- and3S-phytanic acids.

N,N’-dicyclohexylcarbodiimide (0.20 g, 0.99 mmol) indichloromethane (15 mL) was stirred for 5 min. and, (R)-(+)-1-(1-naphthyl)ethylamine (0.17 g, 0.99 mmol) in dichloromethane(5 mL) added. The reaction mixture was stirred overnight at roomtemperature, filtered through a sintered funnel and then washedwith dichloromethane (50 mL × 3). The filtrate was evaporatedunder reduced pressure to give a pale yellow residue, which waspurified by RevelerisTM flash chromatography Grace Pty Ltd. on aprepacked silica cartridge using a gradient solvent system startingfrom 9:1 to 8:2 petroleum ether:ethyl acetate to give two colour-less compounds with slightly different Rf values. The 3R- productwith Rf 0.32 (5) eluted first followed by the 3S- product with Rf0.25 (6). The mixed fractions were combined and subjected tofurther flash chromatography to provide in total 0.45 g, 90% yield.This included 0.19 g of (5) 3R[˛]25

D +47.0◦ (c, 12.08 in CHCl3) (lit(Burns et al., 1999) [˛]27

D +40.75◦ in unlabelled version) and 0.26 gof (6) 3S[˛]25

D +54.0◦ (c, 12.14 in CHCl3) (lit (Burns et al., 1999) [˛]27D

43.39◦ of unlabelled version). 5 and 6 were subsequently analysedby 1H NMR, 2H NMR, and 13C NMR and MS (See supplementaryinformation for NMR, mass spectra and chromatogram, Figs.S10–S17 and S37).

2.5.1.1. Spectral data for (5) (3R,7R,11R)-N-[l(R)-l-naphthylethyl]-3,7,11,1S-tetramethylhexadecanamide. 1H NMR (CDCl3): ı0.86–0.94 (m, 2.05H), 0.96–1.42 (m, 3.2H), 1.45–1.58 (m, 0.29H),1.67 (d, J = 6.7 Hz, 3H, methyl group), 1.78–1.99 (m, 0.59H),2.11–2.19 (m, 0.13H), 5.68 (d, J = 8.1 Hz 1H), 5.92–5.99 (q, J = 7.59,and 6.86 Hz, 1H), 7.42–7.54 (m, 4H), 7.79 (d, J = 8.58 Hz, 1H), 7.86(d, J = 8.58 Hz, 1H), 8.11 (d, J = 8.15 Hz, 1H). 2H NMR (CDCl3): ı

0.89 (br s), 0.96–1.38 (m), 1.44 (br s), 1.88 (m), 2.11 (m). 13C NMR(CDCl3) {1H} and {2H} decoupled ı 18.4 (m), 20.5, 18.78 (m), 19.59(m), 20.5 (s), 21.3 (s), 21.4 (s), 21.7 (s), 21.8 (s), 22.5 (s), 22.6 (s),23.0 (m), 23.1 (m), 23.4 (m), 23.8 (m), 26.7 (s), 26.8 (s), 27.9 (s),31.5 (m), 35.7–36.2 (m), 36.5 (m), 37.1–37.4 (m), 37.9 (m), 38.0(s), 38.3 (s), 39.3 (s), 43.6 (s), 43.7 (s), 44.2 (s), aromatic carbons122.5, 123.5, 125.0, 125.8, 126.4, 128.2, 128.6, 131.1, 133.8, 138.3,171.4. ESI-MS m/z: 503 (M−1)− Isotope distribution (95% D) 3.1%d34, 6.1% d35, 15.2 d36, 24.0 d37, 30.7% d38, 20.9% d39.

2.5.1.2. Spectral data for (6) (3S 7R,11R)-N-[l(R)-l-naphthylethyl]-3,7,11,1S-tetramethylhexadecanamide. 1H and 13C NMR data areidentical to the corresponding NMR spectra of 3R (5) except thatthe amide proton appeared at 5.81 (d, J = 8.1 Hz, 1H) whereas in 3S(6) it appeared at 5.68 (d, J = 8.1 Hz, 1H).

2.5.2. Synthesis of perdeuterated 3R-phytanic acid (7)To a cold (0 ◦C) solution of (5) (0.154 g, 0.30 mmol) in a 2:1

mixture of acetic anhydride/acetic acid was added sodium nitrite(250 mg, 3.6 mmol). The reaction was allowed to come to roomtemperature with continuous stirring under nitrogen and allowedto react overnight. Ice (25 mL), followed by solid NaHCO3 wasadded slowly until no further reaction was observed. The aque-ous portion was extracted with dichloromethane (50 mL × 3). Thecombined organic layers were dried over MgSO4 and evaporatedunder reduced pressure to give a yellowish gum, which was usedwithout further purification. The gum was dissolved in THF (10 mL)and water (5 mL), chilled to 0 ◦C and lithium hydroxide (150 mg,6.26 mmol) was added, followed by H2O2 (2 mL, 30% solution).The reaction mixture was allowed to warm to room temperaturewith continuous stirring for 24 h. The reaction mixture was thencooled (0 ◦C) and solid Na2S2O5 (2 g, 10.52 mmol) was added. Water(25 mL) was added and the reaction mixture was stirred for 5 minat 0 ◦C, before extracting with dichloromethane (25 mL × 3). Thecombined organic extracts were washed with brine, dried overMgSO4 and evaporated under reduced pressure to give a residue.The residue was purified by flash chromatography using 10% ethylacetate in petroleum ether as the mobile phase to give a colourlessgum (7) (0.036 g, 35%): ([˛]25

D +4.0 (c, 1.8 in CHCl3) (lit (Burns et al.,1999) unlabelled version: [˛]27

D +4.5). 1H NMR and 13C NMR datawere identical to that of compound 4.

2.5.3. Synthesis of perdeuterated 3S-phytanic acid (8)This compound was prepared in a fashion identical to that used

for (7), with (6) using (0.15 g, 0.29 mmol) starting material to givea colourless gum (8) 3S (0.03 g, 30%): ([˛]25

D −3.79 (c, 1.2 in CHCl3)(lit (Burns et al., 1999) unlabelled form: [˛]27

D −4.45). 1H NMR and13C NMR data were identical to that of compound 4.

2.6. Synthesis of tail-deuterated DPEPC(1,2-di(3RS,7R,11R-phytanyl)-sn-glycero-3-phosphocholine) (13)

Tail-deuterated DPEPC was synthesised from the building blocksusing 3RS-phytanic acid according to Scheme 3.

2.6.1. Synthesis of perdeuterated phytanol (9)To a cold (0 ◦C) solution of perdeuterated phytanic acid 4

(10.0 g, 28.5 mmol) in THF (50 mL) was added slowly LiAlD4 (1.17 g,28.5 mmol) under nitrogen atmosphere. The reaction mixture wasbrought to RT then refluxed overnight. After cooling to 0 ◦C, thereaction mixture was quenched by pouring it slowly onto crushedice in a large beaker (Caution: addition must be done slowly to avoidexcessive generation of hydrogen). The reaction mixture was thenextracted with dichloromethane (75 mL × 3) dried over Na2SO4,and concentrated in vacuo. The product was purified by silica flash

N.R. Yepuri et al. / Chemistry and Physics of Lipids 183 (2014) 22–33 27

OO

O

CD2

D2CCD CD3

D2CCD2

D2CCD CD3

D2CCD2

D2CCD CD3

D2CCD2

D2CCD

D3CCD3CDD3C

D2CCD2

D3C CDCD2

D2CCD2

D3C CDCD2

D2CCD2

D3C CDCD2

D2CCD2

CD3

PO

O

O

N

D3C CD2

CDCD2

D2C

CD2

CDCD2

D2C

CD2

CDCD2

D2C

CD2

CDCD3 CD3 CD3 CD3 OH

O

AlLi D4/TH F

reflux/overnight D3C CD2

CDCD2

D2C C

D2

CDCD2

D2C C

D2

CDCD2

D2C C

D2

CDCD3 CD3 CD3 CD3

CD2

OH

80%

N OOBr

PPh3/CH2Cl284%

HO

HO

O

Ph

KOH/TBAB/THF/48 hr

D3C CD2

CDCD2

D2C C

D2

CDCD2

D2C C

D2

CDCD2

D2C C

D2

CDCD3 CD3 CD3 CD3

CD2

Br

10%w/w Pd /CEtOH7hrs

95%

40%

1.

PO Cl

O O

C6H6

2. N(CH3)3/ACN 43%

9

1011

12

13

4

O

OHO

CD2

D2C

CDCD3

D2C

CD2

D2C CD

CD3

D2C

CD2

D2C

CDCD3

D2C

CD2

D2C

CD CD3

CD3

CDCD3D2

C CD2

CD3CD

CD2

D2C C

D2

CD3CD

CD2

D2C C

D2

CD3CD

CD2

D2C C

D2CD3

O

OO

CD2

D2C

CDCD3

D2C

CD2

D2C

CDCD3

D2C

CD2

D2C

CDCD3

D2C

CD2

D2C

CD CD3

CD3

CDCD3D2

CCD2

CD3CD

CD2

D2C C

D2

CD3CD

CD2

D2C C

D2

CD3CD

CD2

D2C C

D2CD3Bn

Scheme 3. Total synthesis of tail-deuterated 1,2-di(3RS,7R,11R-phytanyl)-sn-glycero-3-phosphocholine DPEPC (13).

chromatography to give colourless liquid (9) (7.7 g, 22.67 mmol,yield 80%) (Scheme 3). 1H NMR (CDCl3): ı residual protons 0.89 (m,0.67H), 1.00–1.98 (m, 2.5H). 2H NMR (CDCl3): ı 0.89 (m, 14.42D),1.0–1.87 (m, 23.74D), 3.65 (s b, 2D). 13C NMR (CDCl3): protondecoupled spectra values ı 18.4 (m), 20.1 (m), 23.2 (m), 26.4 (m),27.9 (m), 31.1 (m), 35.7 (m), 38.2 (m), 59.9 (m) (See supplementaryinformation for NMR spectra, Figs. S18 and S19).

2.6.2. Synthesis of perdeuterated phytanyl bromide (10)To a cold (0 ◦C) solution of perdeuterated phytanol (9) (7.0 g,

20.61 mmol) and triphenyl phosphine (6.48 g, 24.73 mmol) indichlormethane (100 mL) was added slowly N-bromosuccinimide(3.6 g, 20.61 mmol). The reaction mixture was brought to roomtemperature and stirring was continued for 45 min, then washedwith sodium thiosulfate saturated solution (150 mL). The organiclayer was evaporated to give a solid. The solid was taken intoa mortar triturated with hexane (200 mL × 5), and the combinedhexane portions evaporated. The residue was purified by flash chro-matography on silica column using 100% petroleum ether to givea colourless liquid (10) (6.93 g, 17.20 mmol, 84%) (Scheme 3). 1HNMR (CDCl3): ı residual protons 0.77–0.89 (m), 0.99–1.33 (m), 3.44(m). 2H NMR (CDCl3): ı 0.88 (m, 14.42D), 0.93–1.36 (m,18.69D),1.41–1.88 (m, 4.26D), 3.40 (m, 2D). 2H NMR (CDCl3): ı 0.87 (br s,14.86D), 1.00–1.55 (m, 19.29D), 1.61 (m, 3.36), 1.81 (m, 1.14D), 3.2(m, 2D). 13C NMR (CDCl3): proton decoupled spectra values ı 18.5(m), 21.61 (m), 23.4 (m), 26.4 (m), 30.5 (m), 31.6 (m), 36.0 (m), 38.3(m), 59.0 (m) (See supplementary information for NMR and massspectra, Figs. S20–S22).

2.6.3. Synthesis of tail-deuterated1,2-di(3RS,7R,11R-phytanyl)-sn-glycero-3-benzyl (11)

A solution of phytanyl bromide 10 (6.0 g, 14.96 mmol, 4mol eq.), 1-O-benzyl-sn-glycerol (0.68 g, 3.54 mmol), KOH (4.18 g74.8 mmol) (pellets) and tetrabutylammonium bromide (0.48 g,1.4 mmol) in THF (60 mL) was stirred for 48 h. The reaction wasdiluted with diethyl ether (200 mL) and washed with 1 N HCl(50 mL × 2) and water (50 mL × 2). The organic layer was dried overMgSO4 and evaporated to give a residue which was purified by flashchromatography. Initial elution with 100% hexane gave unreactedperdeuterated phytanylbromide, and further elution with 10% ethylacetate in hexane gave the desired product, tail-deuterated 1,2-di(3RS,7R,11R-phytanyl)-sn-glycero-3-benzyl (11) as a colourlessgum (2.0 g, 2.42 mmol, 40%) (Scheme 3). 1H NMR (CDCl3): ı resid-ual protons 0.76–0.96 (m, 2.52H), 0.99–1.37 (m, 2.26H), protonatedhead-group 3.47–3.63 (m, 5H), 4.57 (s, 2H), 7.32 (m, 5H). 2H NMR(CDCl3): ı 0.88 (s b, 30D), 0.91–1.63 (m, 46D), 3.51 (m, 4D). 13C NMR(CDCl3): proton decoupled spectra values ı 18.8 (m), 21.61 (m), 23.4(m), 26.7 (m), 28.7 (m), 31.7 (m), 36.1 (m), 37.8 (m), 50.4 proton-ated head-group peaks (m), 70.3, 70.7, 70.8, 73.3, 77.8, 127.4, 127.5,128.3, 138.4 (See supplementary information for NMR spectra, Figs.S23–S25).

2.6.4. Synthesis of 1,2-di(3RS,7R,11R-phytanyl)-sn-glycerol(tail-deuterated GDPE) (12)

A solution of 1,2-di(3RS,7R,11R-phytanyl)-sn-glycero-3-benzyl(11) (2.0 g, 2.42 mmol) was mixed with Pd/C (0.2 g, 10% w/w ofPd/C 10%) in ethanol (25 mL). After bubbling nitrogen and thenhydrogen through the solution, the reaction was left for 7 h with

28 N.R. Yepuri et al. / Chemistry and Physics of Lipids 183 (2014) 22–33

stirring at RT, under a hydrogen atmosphere. The catalyst was thenfiltered through Celite and the filtrate evaporated to give an oilysubstance which was purified by flash chromatography. Elutionwith 10% ethyl acetate in hexane gave a colourless liquid (12) (1.7 g,2.31 mmol, 95%) (Scheme 3). 1H NMR (CDCl3): ı residual protons0.80 (m, 1.2H), 0.99–1.33 (m, 1.23H), 1.55 (m, 0.46H), protonatedhead-group 3.44–3.56 (m, 3H), 3.61 (m, 2H), 3.71 (m, 2H). 2H NMR(CDCl3): ı 0.80 (s b, 28.4D), 0.92–1.59 (m, 44D), 3.51 (m, 4D). 13CNMR (CDCl3): proton decoupled spectra values ı 18.5 (m), 21.5 (m),23.3 (m), 26.7 (m), 28.6 (m), 31.7 (m), 36.0 (m), 37.9 (m), proton-ated head-group peaks 63.0 (s), 67.8 (m), 69.3 (m), 70.9 (s), 78.2(s). ESI-MS m/z: 758 (M+Na)+ (See supplementary information forNMR spectra, Figs. S26 and S27).

2.6.5. Synthesis of 1,2-di(3RS,7R,11R-phytanyl)-sn-glycero-3-(1,3,2-dioxaphospholane)

To a solution of 1,2-di(3RS,7R,11R-phytanyl)-sn-glycerol (12)(1.6 g, 2.17 mmol) in benzene (20 mL) was added triethylamine(0.26 g, 2.6 mmol). The reaction mixture was cooled to 0 ◦C andto this was added in one lot, a solution of 2-chloro-2-oxo-1,3,2-phospholane (0.31 g, 2.17 mmol) in benzene (10 mL). The reactionwas left stirring at room temperature for 16 h and then filteredthrough a sintered funnel to remove precipitated (Et)3N–HCl. Thefiltrate was concentrated to give a residue, which was used for thenext reaction without further purification.

2.6.6. Synthesis of1,2-di(3RS,7R,11R-phytanyl)-sn-glycero-3-phosphocholine (13)(tail-deuterated DPEPC)

A solution of the above compound (1.8 g, 2.14 mmol) in dry ace-tonitrile (30 mL) was transferred to a pressure flask. It was cooledin a liquid nitrogen and acetone freezing mixture bath (−78 ◦C)and approximately 2 mL of anhydrous trimethylamine (condensed)added. The bottle was sealed and then heated at 68 ◦C for 24 h. Thepressure bottle was cooled to 0 ◦C and the solvent was evaporatedto give a residue, which was purified by flash chromatography usinga solvent system 70:30:0.4 CHCl3:MeOH:H2O to give a colour-less gum (13) (0.7 g, 43%) (Scheme 3). 1H NMR (CDCl3): ı residualprotons 0.80 (m, 1.2H), 0.99–1.33 (m, 1.23H), 1.55 (m, 0.46H), pro-tonated head-group 3.44–3.56 (m, 3H), 3.61 (m, 2H), 3.71 (m, 2H).2H NMR (CDCl3): ı 0.80 (s b, 28.4D), 0.92–1.59 (m, 44D), 3.51 (m,4D). 13C NMR (CDCl3): proton decoupled spectra values ı 18.5 (m),21.5 (m), 23.3 (m), 26.7 (m), 28.6 (m), 31.7 (m), 36.0 (m), 37.9 (m),protonated head-group peaks 63.0 (s), 67.8 (m), 69.3 (m), 70.9 (s),78.2 (s). ESI-MS m/z: 758 (M+Na)+ (See supplementary informationfor NMR and MS spectra, Figs. S28–S32).

2.7. Synthesis of tail-deuterated DPhyPC1,2-di(3RS,7R,11R-phytanoyl)-sn-glycero-3-phosphocholine (14)

DPhyPC with deuterated phytanyl chains was synthesisedaccording to a modifi cation of Singhs method (Singh, 1990)(Scheme 4). Deuterated phytanic acid (1.59 g, 4.53 mmol) dissolvedin 25 mL alcohol free chloroform (anhydrous) was added to a50 mL single-necked round-bottomed flask containing sn-glycero-phosphocholine–CdCl2 complex (0.5 g, 1.13 mmol). The resultingsuspension was vigorously stirred and 4-dimethylamino pyridine(0.27 g, 2.26 mmol) added followed by dicyclohexylcarbodiimide(0.93 g, 4.53 mmol). The contents of the flask were then degassedwith N2, stoppered, protected from light, and sonicated in a son-ication bath for 5 h at room temperature. The solution was stirredfor 3 days at room temperature. The progress of the reaction wasmonitored by TLC on silica gel CHCl3:CH3OH:H2O 65:25:4. Whenthe reaction was complete chloroform was added and the mixturewas filtered through a Celite pad, which was then washed with afurther 50 mL of chloroform. The chloroform was removed under

D3CCDC

D2

D2C C

D2

CD C

D2

D2C C

D2

CD C

D2

D2C C

D2

CD C

D2OH

CD3 CD3 CD3 CD3 O

DCC/D MAP65%

OH O

OH

PO O

ON

CdCl2

O

O

OPO

OO

N

CD2

CD C

D2

D2C C

D2

CD C

D2

D2C C

D2

CD C

D2

D2C C

D2

CDCD3

D2C D

CD2C C

D2

D2C D

CD2C C

D2

D2C D

CD2C C

D2

D2C D

C CD3

O

O

CD3

CD3 CD3

CD3

CD3

CD3

CD3

CD3

4

14

Scheme 4. Synthesis of tail-deuterated 1,2-di(3RS,7R,11R-phytanoyl)-sn-glycero-3-phosphocholine.

reduced pressure at room temperature, and the residue was dis-solved in 5 mL of CHCl3:CH3OH 1:1 and passed through a silicacolumn which was pre-conditioned with CHCl3. The column waseluted with MeOH:CHCl3 (40:60) which eluted product directlyafter eluting the DMAP. The fractions containing the product werecombined and the solvent was removed in vacuo at 25 ◦C. The tail-deuterated DPhyPC (14) phospholipid thus obtained (0.65 g, 65%)gave a single 31P NMR resonance. 1H NMR (CDCl3): ı residual pro-tons 0.78 (m, 0.58H), 1.01 (m, 0.39H), 1.15–1.33 (m, 0.81H), 1.46 (m,0.22H), protonated head-group 3.36 (s, 9H), 3.40–3.61 (m, 3.8H),3.73–3.83 (m, 4.5H), 4.99 (m, 1H), 4.30 (m, 2H). 2H NMR (CDCl3):ı 0.78 (s b, 29.6D), 0.90–1.69 (m, 41.95D), 3.48 (m, 4D). 13C NMR(CDCl3): proton decoupled spectra values ı 18.6 (m), 21.4 (m), 23.4(m), 26.8 (m), 28.9 (m), 31.7 (m), 36.2 (m), 38.2 (m), protonatedhead-group peaks 54.5 (s), 59.1 (s), 65.0 (s), 66.5 (m), 70.9 (s), 78.2(q). ESIMS m/z: 901 (M+1)+. 94% D isotope distribution 2.1% d73, 4.3%d74, 11.1% d75, 27.1% d76, 37.0% d77, 18.4% d78 (See supplementaryinformation for NMR and mass spectra, Figs. S33–S36).

2.8. Monolayer production for neutron reflection measurements

The tail-deuterated DPyPC and tail-deuterated DPEPC were eachdissolved in chloroform at ca 1 mg/mL. 50 #L of this solution wasadded dropwise to the surface of HEPES buffer (pH 7.2) in a Lang-muir trough (Nima Scientific, Coventry, UK). The trough area was600 cm2 and compressions were carried out at a barrier speed of10 cm2/min. The isotherms were collected from zero surface pres-sure up to ca 30 mN/m. Brewster Angle Microscopy (BAM) imageswere collected at 5 mN/m steps on a Nanofilm EPI2000.

2.9. Tethered bilayer production

TBMs production was based upon the methods outlined byCornell et al. (1997). The bilayers here were produced on sili-con (1 0 0) disks, 100 mm in diameter × 10 mm thick, sequentiallycoated in the same vacuum chamber with ca 4 nm chromiumfollowed by ca 15 nm gold by electron beam deposition (Aus-tralian National Fabrication Facility, Canberra node). A 20:80mixture (300 #M in ethanol) of phytanyl bis-tetraethyleneglycolbenzyl disulphide (Phyt(EO4)2SSPh) and hydroxyl-terminatedtetraethyleneglycol benzyl disulphide (OH(EO4)SSPh), was imme-diately incubated on the freshly coated surface for 180 s to form thetether base layer. The surface was then washed with dry ethanol

N.R. Yepuri et al. / Chemistry and Physics of Lipids 183 (2014) 22–33 29

and stored under ethanol until the bilayer assembly was completedby the following procedure. The tether coated silicon substrate wasplaced into a solid/liquid neutron reflectometry (NR) cell, having asurface area of ca 50 cm2 exposed to solution. The cell was assem-bled with a thin Teflon gasket that restricted the depth of solutionover the surface to <100 #m. The cell reservoir was then filled withtail-deuterated DPEPC (3 nM in ethanol), and incubated for 2 minprior to washing with Hepes buffer flowing through the cell at5 mL/min. The TBM was then ready for NR measurements.

2.10. Neutron Reflectometry

Specular neutron reflectometry (NR) involves impinging a beamof neutrons at a glancing angle onto the interface of interest andmeasuring the reflected intensity relative to the incident intensitywhen the exit angle is equal to the incident angle. Under these con-ditions NR probes the variation in neutron scattering length density(nSLD) perpendicular to the interface in question. The nSLD (Å−2)for a material can be determined from the scattering length (b), Å,of each atom and the molecular volume (V), Å3 according to Eq. (1)

nSLD =!

ibi

V(1)

Data is typically presented as a function of momentum transfer(Q) defined as

Q = 4" sin #$

(2)

where # is the angle of incidence (and exit) of the neutron beam,and $ is the neutron wavelength.

NR data from the monolayer films at the air/liquid interface wascollected on the PLATYPUS reflectometer (James et al., 2011) at theOPAL Research Reactor, Australian Nuclear Science and TechnologyOrganisation, Sydney, Australia. PLATYPUS was operated with theincident neutron wavelength ranging from 2.8 to 18.0 A with inci-dent angles of 1.0◦ and 4.8◦ (Q range of 0.013–0.31 A−1). Data wasre-binned to a Q resolution of 5%. The TBM samples were exam-ined on the OffSPEC neutron reflectometer (Target Station 2, ISISspallation neutron source, Oxfordshire, UK), with incident neutronwavelengths of 1.2–14.5 A, and angles of 0.55◦ and 2.3◦, resultingin a Q range of 0.0076–0.42 A−1. Data reduction followed the sameprocedure for both instruments. A ‘straight-through’ beam run wascollected for each instrument configuration. Each reflection datasetwas then divided through by the appropriate straight-through run.Datasets at different angles were then stitched together by compar-ing the data in the Q range where the incident angles overlappedto produce a single reduced dataset.

For soft matter studies a major advantage of neutron scat-tering is the large difference in neutron scattering length (b)between hydrogen and its isotope deuterium. Hydrogen has anegative neutron scattering length (b = −3.7406 fm), while deu-terium is positive (b = 6.671 fm), and one may for example exchangehydrogen and deuterium in a sample without altering the struc-ture but having a large impact on the neutron scattering lengthdensity. As the systems of interest here are, fully hydrated incontact with water, this also allows for the possibility of mixingH2O (nSLD = −0.56 × 10−6 A−2) and D2O (nSLD = 6.33 × 10−6 A−2) tomake buffers of different contrast that can be made to match dif-ferent components of the system. For example a H2O:D2O mixture(92% H2O by volume) is contrast matched to air, and thus for a tail-deuterated DPEPC and tail-deuterated DPhyDC monolayer on aircontrast matched water (ACMW), the scattering signal results fromthe monolayer only with the aqueous sub-phase invisible to theneutron beam. Langmuir monolayers were measured against D2Oand ACMW sub-phases; while the TBMs were measured againstD2O and H2O buffers.

The neutron reflectometry data fitting was carried out usingthe Motofit software (Nelson, 2006) in the Igor Pro environment(Wavemetrics). Briefly the interface is partitioned into a series ofslabs, each described by three parameters, the nSLD, thickness andGaussian roughness with the adjacent slab. Motofit calculates thereflectivity from a model defined starting point, this is comparedwith the real data and the least squared residuals minimised via agenetic algorithm. For the TBM the two solution contrast datasetswere fitted simultaneously with the thickness and roughness ofeach layer constrained. However the nSLD values were allowed tovary between datasets appropriately.

Data fits and errors were assessed via a Monte Carlo resam-pling method (Holt et al., 2009; Heinrich et al., 2009; Valinciuset al., 2008). After the initial fit was completed at least one thou-sand datasets were synthesised from the original dataset by MonteCarlo resampling. Each dataset was then fitted following the sameprocedure as the real data, resulting in a distribution of values foreach parameter, the midpoint of a Gaussian fit to this distributionis the value presented in the text with the width of the distributionquoted as the error.

3. Results and discussion

3.1. Synthesis and deuteration outcomes

3.1.1. Impact on stereochemistryPerdeuterated branched hydrocarbons can be prepared readily

by gas-phase exchange with deuterium gas (Dixon and Marr, 1963)or using mild metal catalysed H/D exchange reactions (Atkinsonet al., 1967). However these methods are either susceptible tostructural isomerisation with high boiling hydrocarbons (>200 ◦C),or have been reported to affect epimerisation of asymmetric ortertiary carbon atoms in cyclic hydrocarbons (Atkinson et al.,1967). Essentially, studies detailing the stereoselective deuteriumlabelling of branched alkyl carbon chains are absent from the lit-erature. Therefore in this study, in order to investigate the fateof the chiral centres of the phytanyl alkyl chain following thehydrothermal deuteration step, the different diastereoisomers ofperdeuterated phytanic acid were separated using a chiral auxil-iary agent. This was undertaken to characterise and compare theoptical purity of the deuterated isomers of phytanic acid with liter-ature values of their protonated analogues. Although little is knownon how the value of optical rotation will be affected when a hydro-gen atom is substituted by a deuterium atom at a chiral centre,it is assumed that the optical rotation values should not signifi-cantly change. In fact, a search in the literature for optical rotationvalues of chiral molecules showed similar values between proton-ated compounds and their deuterated analogues, e.g. d-leucine-2-dand d-leucine; d-glucose-1,2,3,4,5,6,6-d7 and d-glucose; l-alanine,l-alanine-2-d, l-alanine-2,3,3,3-d4 (Sigma–Aldrich website). Inaddition, studies using (−)-(R)-3-methyl-1-indanone and (−)-(R)-3-methyl-1-indanone-1-d reported very similar optical rotationvalues of both compounds (Almy and Cram, 1969). Therefore, itis in accordance with commonly accepted practice to compare theoptical rotation values of the deuterated phytanic acid isomers withthe optical rotation of protonated analogues found in literature tocheck the retention of chirality for the phytanic acid’s chiral centres.

Fatty acids are known to undergo H/D exchange when treatedwith alkaline D2O in the presence of metal catalyst (Pt or Pd) athigh temperature (180–250 ◦C) (Zimmermann, 1989; Yepuri et al.,2013; Darwish et al., 2013). Phytanic acid 3 was deuterated to giveperdeuterated phytanic acid 4 in good yield using a hydrothermalH/D exchange reaction with heterogeneous platinum metalcatalyst. Under these conditions a high degree of deuteration wasachieved (D > 95%) as determined by mass spectrometric analysis

30 N.R. Yepuri et al. / Chemistry and Physics of Lipids 183 (2014) 22–33

D3C CDCD2

D2C C

D2

CD C

D2

D2C C

D2

CD C

D2

D2C C

D2

CD C

D2NH

CD3 CD3 CD3 OCD3

3R

D3C CDCD2

D2C C

D2

CD C

D2

D2C C

D2

CD C

D2

D2C C

D2

CD C

D2NH

CD3 CD3 CD3 O3SCD3

Fig. 3. Chemical structures of amide (5) 3R and (6) 3S.

and 1H NMR analysis using an internal standard (supplementaryinformation Figs. S3–S9). 13C NMR spectra of 4 (supplementaryinformation Figs. S5 and S6) shows that all of the carbon signalsare depleted due to deuterium coupling, including the hinderedsites at the ! position with respect to the methyl groups.

A key element of this study was to investigate whetherhydrothermal deuteration of (3RS,7R,11R)-3,7,11,15-tetramethylhexadecanoic acid (perdeuterated phytanic acid)using Pt catalyst leads to racemisation at the 7R- and 11R- centres,or whether these centres retain their original stereochemistry.

In the current study, since a mixture of protonated 3RS-phytanicacid diastereomers (3) was used for the deuteration step, it wasanticipated that this mixture would also be reproduced in thedeuterated product. If any isomers were formed during deutera-tion at positions 7 and 11, it would be difficult to separate them bystandard chromatography techniques and they would not be iden-tified by NMR spectral analysis. Therefore, the procedure of Burnset al. (1999) was used to isolate the diastereomers based on position3.

The method follows the separation of diastereomeric mixture ofperdeuterated 3RS-phytanic acid (4) by coupling with a chiral aux-iliary agent (R)-(+)-(1-napthyl)ethylamine, which created a subtledifference in chromatographic behaviour. Upon analysis, it wasfound that 3R- (5) and 3S- (6) naphthylethylamide diastereomers ofperdeuterated phytanic acid were in a ∼4:6 ratio respectively withan overall yield of 90% (Scheme 2). These two amides had differ-ent Rf values on thin layer chromatography (TLC) using 10% ethylacetate in hexane with 3R, Rf = 0.32 (5) and 3S, Rf = 0.25 (6). Thesame solvent system was used to separate both these compoundsusing medium pressure flash chromatography (pre packed silicacartridges RevelerisTM). The optical rotation data and 13C {1H, 2H}NMR spectra of these compounds (5) and (6) (Fig. 3) are compa-rable to that reported by Burns et al. (1999) (for spectra refer tosupplementary information Figs S10–S17).

All the subsequent hydrolysis reactions were performed on theseparated pure diastereomers 3R-(5) and 3S-(6), and all the sub-sequent reaction products were assumed to be enantiomericallypure. Hydrolysis of amides 3R-(5) and 3S-(6) to obtain the enan-tiomerically pure perdeuterated phytanic acids 3R-(7) and 3S-(8)was carried out following the procedures published by Burns et al.with overall yield of 35% (Burns et al., 1999). The optical rota-tion values of 7 and 8 obtained in this study were comparable tothose reported in literature for their respective unlabelled versions(Burns et al., 1999). Since the optical rotation values of the isolatedperdeuterated phytanic acid isomers and their amides derivativesare comparable with their protonated versions reported in litera-ture, it was assumed that no significant racemisation had occurredduring the deuteration exchange reaction.

3.2. Neutron reflection

3.2.1. Neutron reflection from Langmuir monolayersFig. 4 shows the Langmuir isotherm for tail-deuterated DPhyPC

(14) and tail-deuterated DPEPC (13) monolayers. Both monolayerfilms demonstrated a steady increase in surface pressure as thetrough area was reduced. Phytanyl lipids are expected to be in thefluid phase at room temperature, and this was borne out by thelack of phase transitions observed as a function of surface pressure.

Fig. 4. Pressure area isotherm of tail-deuterated DPEPC and tail-deuterated DPhyPCmonolayers on HEPES buffer.

The small breaks seen in the tail-deuterated DPhyPC isotherm arewhere the barrier was paused for approximately 2 h while NR datawas collected. The BAM images (data not shown) were essentiallyfeatureless at all surface pressures confirming the fluid nature ofboth monolayers. The deuterated molecules displayed the samemonolayer characteristics that would be expected of the hydroge-nous analogues.

The real space density profile from the fit to neutron reflec-tion data from the tail-deuterated DPhyPC is shown in Fig. 5. Asimilar set of density profiles was observed for the tail-deuteratedDPEPC. Table 1 summarises the fitting parameters for each surface

Fig. 5. Real space nSLD profile resulting from fitting the NR data from a Langmuirmonolayer of tail-deuterated DPhyPC at 10 (green trace) and 20 (red trace) mN/msurface pressure. To simplify the figure the density profile is only shown for themonolayer on the ACMW subphase. The vertical dashed lines represent the interfacepositions for each layer for the 20 mN/m dataset. A similar set of density profiles isobserved for the tail-deuterated DPEPC. (For interpretation of reference to colour inthis figure legend, the reader is referred to the web version of this article.).

N.R. Yepuri et al. / Chemistry and Physics of Lipids 183 (2014) 22–33 31

Table 1Area per molecule and monolayer thicknesses for tail-deuterated DPhyPC and tail-deuterated DPEPC as a function of surface pressure.

Lipid Area per molecule (Å2) Head-group thickness (Å) Tail thickness (Å) Monolayer thickness (Å)

DPEPC 10 mN/m 105.6 ± 1.3 5.7 ± 1.6 5.3 ± 1.0 11.0 ± 1.9DPEPC 20 mN/m 91.2 ± 1.2 6.5 ± 1.0 9.6 ± 1.4 16.1 ± 1.7DPhyPC 10 mN/m 90.6 ± 1.2 5.7 ± 1.3 8.3 ± 1.2 14.0 ± 1.8DPhyPC 20 mN/m 82.5 ± 1.2 6.0 ± 1.4 10.8 ± 1.2 16.0 ± 1.8

pressure examined. The thickness of the monolayers is compa-rable but the tail-deuterated DPEPC had an increased area permolecule by ∼10–15%, when compared to the tail-deuteratedDPhyPC molecule at the same pressure. The head-group thicknessis independent of phospholipid type and surface pressure. The areaper molecule ranges from ca 105 (for DPEPC at 10 mN/m) downto 83 A2 (for DPhyPC at 20 mN/m). This compares with a crosssectional area of 76 A2 determined by Wu et al. (1995) from well-ordered multibilayer stacks of DPhyPC and ∼75 A2 by Heinrich et al.(2009) from tethered bilayers. A single monolayer at the air/liquidinterface would be expected to be less ordered, and the area permolecule that we observe was consistent with a monolayer wellbelow the collapse pressure.

Both lipids are essentially of the same length with same num-ber of bonds, they only differ by the way of attachment to theglycerol backbone. However, the consistency in observing lowerarea per molecule values for the tail-deuterated DPhyPC in com-parison with tail-deuterated DPEPC is likely to be due to the morehydrophilic nature of the ester containing lipids which rendersthe head-group more embedded in the water sub-phase result-ing in more compact packing of the alkyl chains and lower areaper molecule values (Fig. 2a). On the other hand, in the case ofthe more hydrophobic ether bonds containing lipid (tail-deuteratedDPEPC), the water layer pushes the hydrophobic tails away whichthen protrude further from the water sub-phase resulting withpoorer packing assembly and thus larger APM in comparison withthe tail-deuterated DPhyPC.

Fig. 6 shows observed NR data and the associated fit for thetwo solution contrasts (H2O and D2O) from the TBM formed usingtail-deuterated DPEPC, with associated refined structural param-eters listed in Table 2. Following analysis, it is evident from thedata fitting that the bilayer deposition parameters require opti-misation for these large substrates. None-the-less an isotopicallyasymmetric bilayer has been produced, i.e. the inner leaflet of thebilayer consists of 41% protonated phytanyl tails (due to the teth-ered layer Phyt(EO4)2SSPh) and 12% tail-deuterated DPEPC. Thelarge water content indicates that the assembly method did not effi-ciently deliver tail-deuterated DPEPC to the inner leaflet. The outerleaflet consists of tail-deuterated DPEPC (29%) and subphase (71%).The thickness of the lipid tails, 8.2 A is about half that determinedby Heinrich et al. (2009) for TBMs on different tethers completed

Table 2Summary of primary fit parameters from the tail-deuterated DPEPC TBM.

Parameter Value

Cr thickness (Å) 46.7 ± 1.2Cr Density (% of theoretical) 98.7 ± 1.4Au thickness (Å) 181.7 ± 1.0Au density (% of theoretical) 91.3 ± 0.4Tether (Å) 15.9 ± 2.6Water content of reservoir (%) 67.0 ± 4.0HG (Å) 5.7 ± 0.9Inner tail (Å) 8.2 ± 0.6Inner tails volume fractions (h-lipid, d-lipid, solvent) 0.41, 0.12, 0.47 ± 0.03Outer tail (Å) 8.2 ± 0.6Outer tails volume fractions (d-lipid, solvent)a 0.29, 0.71 ± 0.03

a Note outer tails are deuterated DPEPC only.

by DPhyPC. The low surface coverage observed in our sample isevidence of high disorder in the bilayer and inefficient lipid packing.

This structural characterisation of the Phyt(EO4)2SSPh-basedTBM is a precursor to a wider study elucidating the auto-insertionproperties of the metamorphic chloride ion channel protein intomodel membranes, an area in which we have an active researchprogramme (Valenzuela et al., 2013). The use of tail-deuteratedDPEPC will enable the disentanglement of signal from lipids,cholesterol, protein and solution to a far greater extent than

Fig. 6. (a) Data (symbols) and fit (line) from a TBM of Phyt(EO4)2SSPh/tail-deuterated DPEPC, ⃝ – D2O and * – H2O dataset. (b) Real space SLD profile fromthe fit in (a).

32 N.R. Yepuri et al. / Chemistry and Physics of Lipids 183 (2014) 22–33

previously possible using NR with protonated lipids or using elec-trochemical methods.

4. Conclusions

We have demonstrated a straightforward and effective methodfor the production of highly deuterated phytanic acid in multi-gram quantities while preserving the stereochemical chiralityof positions 7 and 11. The perdeuterated phytanic acid wasused for the preparation of tail-deuterated 1,2-di(3RS,7R,11R-phytanyl)-sn-glycerol-3-phosphocholine (tail-deuterated DPEPC)and 1,2-di(3RS,7R,11R-phytanoyl)-sn-glycerol-3-phosphocholine(tail-deuterated DPhyPC). The tail-deuterated DPEPC and tail-deuterated DPhyPC were employed to construct two differentmodel membranes, namely Langmuir monolayers at the air–waterinterface and tail-deuterated DPEPC was used in the productionof tethered bilayer membrane on a solid substrate. Pressure areaisotherm and neutron reflectrometry techniques were used tocharacterise the two model membranes and the structures com-pared with those from hydrogenous versions of the lipids (whereavailable) reported in the literature. The area per molecules deter-mined using neutron reflectometry ranged from ca. 105 downto 82.5 A2 at high monolayer compression. This compares with across sectional area of 76 A2 determined by Wu et al. (1995) fromwell-ordered multilayer stacks of DPhyPC. Results demonstratedthat the head-group thickness is independent of phospholipid typeand surface pressure. The area per molecule of tail-deuteratedDPhyPC was ∼10–15% smaller than tail-deuterated DPEPC. Thiswas attributed to the more hydrophilic nature of the ester con-taining lipids which attracts the lipid head-groups more into thewater sub-phase resulting with better packing of the alkyl chainsabove the water layer. On the other hand, in the case of the morehydrophobic ether bonds containing lipid (tail-deuterated DPEPC),the water layer pushes the hydrophobic tails away which pro-trude more from the water sub-phase resulting in poorer packing.In the TBM composed of tail-deuterated DPEPC the bilayer thick-ness of the disordered lipid tail regions was 16.4 A. The availabilityof such deuterated phytanyl-based lipid membrane models willhave substantial impact on neutron studies investigating proteinion channel systems such as the auto-insertion of the metamorphicchloride ion channel protein into membranes which has been previ-ously restricted by the limited scattering length density differencebetween the different components of the systems.

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgment

The authors acknowledge the National Deuteration Facilityat the Australian Nuclear Science and Technology Organisation(ANSTO); the operation of which was partially funded by theNational Collaborative Research Infrastructure Strategy (NCRIS).Mrs Marie Gillon is gratefully acknowledged for her assistance inpreparing the deuterated fatty acid for this work and Ms AnwenKrause-Heuer for proof reading the manuscript.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.chemphyslip.2014.04.004.

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