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Azide-modified Membrane Lipids: Synthesis, Properties, and Reactivity Sindy Lindner, Kai Gruhle, Rico Schmidt, Vasil M Garamus, Daniel Ramsbeck, Gerd Hause, Annette Meister, Andrea Sinz, and Simon Drescher Langmuir, Just Accepted Manuscript • Publication Date (Web): 30 Apr 2017 Downloaded from http://pubs.acs.org on May 2, 2017

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Azide-modified Membrane Lipids: Synthesis, Properties, and Reactivity Sindy Lindner,†,‡ Kai Gruhle,‡ Rico Schmidt,† Vasil M. Garamus,# Daniel Ramsbeck,⊥ Gerd Hause,¶ Annette Meister,§ Andrea Sinz,† Simon Drescher*,‡ †

Institute of Pharmacy – Pharmaceutical Chemistry and Bioanalytics, Martin Luther University ‡

(MLU) Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, 06120 Halle (Saale), Germany;

Institute of Pharmacy – Biophysical Pharmacy, MLU Halle-Wittenberg, Wolfgang-LangenbeckStrasse 4, 06120 Halle (Saale), Germany; # Helmholtz-Zentrum Geesthacht: Zentrum für Materi⊥

al- und Küstenforschung GmbH (HZG), Max-Planck-Strasse 1, 21502 Geesthacht, Germany;

Fraunhofer Institute for Cell Therapy and Immunology IZI, Weinbergweg 22, 06120 Halle (Saale), Germany;

¶

Biocenter, MLU Halle-Wittenberg, Weinbergweg 22, 06120 Halle (Saale),

Germany; § Institute of Chemistry and Institute of Biochemistry and Biotechnology, MLU HalleWittenberg, von-Danckelmann-Platz 4, 06120 Halle (Saale), Germany. KEYWORDS Membrane Lipids, Azide-modified Phospholipids, Aggregation Behavior, Interdigitation, PhotoCross-Linking, Peptide/Lipid Interactions, WAL-Peptide

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ABSTRACT In the present work, we describe the synthesis and the temperature-dependent behavior of photoreactive membrane lipids as well as their capability to study peptide/lipid interactions. The modified phospholipids contain an azide group either in the middle part or at the end of an alkyl chain and also differ in the linkage (ester vs. ether) of the second alkyl chain. The temperaturedependent aggregation behavior of the azidolipids was studied using differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, and small angle X-ray scattering (SAXS). Aggregate structures were visualized by stain and cryo transmission electron microscopy (TEM) and were further characterized by dynamic light scattering (DLS). We show that the position of the azide group and the type of linkage of the alkyl chain at the sn-2 position of the glycerol influences the type of aggregates formed as well as their long-term stability: P10AzSPC and r12AzSHPC show the formation of extrudable liposomes, which are stable in size during storage. In contrast, azidolipids that carry a terminal azido moiety either form extrudable liposomes, which show time-dependent vesicle fusion (P15AzPdPC), or self-assemble in large sheet-like, non-extrudable aggregates (r15AzPdHPC) where the lipid molecules are arranged in an interdigitated orientation at temperatures below Tm (LβI phase). Finally, a P10AzSPC:DMPC mixture was used for photochemically induced cross-linking experiments with a transmembrane peptide (WAL-peptide) to demonstrate the applicability of the azidolipids for the analysis of peptide/lipid interactions. The efficiency of photo-cross-linking was monitored by attenuated total reflection infrared (ATR-IR) spectroscopy and mass spectrometry (MS).


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INTRODUCTION

Proteins embedded in biomembranes or peripheric proteins et cetera are referred to as membrane proteins. They are ubiquitously present in various organisms as receptors, transporters, and ion channels and represent an important group of drug targets, with 40 % of all commercially available drugs acting on membrane proteins.1 Changes in membrane proteins may cause or promote a number of diseases.2 Despite the physiological and pathophysiological significance of membrane proteins and the fact that 30% of the naturally occurring proteins are embedded in biological membranes,3 high resolution structures of membrane proteins are currently underrepresented. This is due to different aspects; probably one of the most important reasons is that integral membrane proteins are difficult to solubilize caused by their lipophilic substructure that lead to aggregation or precipitation in aqueous solution.4,5 Moreover, membrane proteins tend to lose their activity when removed from their physiological environment.6 The methods available for a structural characterization of proteins are manifold, but not all of them can be used for membrane proteins since these proteins should be embedded in their native environment, i.e. membrane lipids. Even a tiny fraction of membrane lipids stabilizing membrane proteins to maintain their functions, can adversely affect X-ray and NMR analysis.7 On the contrary, if membrane lipids are completely removed, membrane proteins are usually no longer stable and lose their tertiary structure. Besides X-ray, NMR, and high resolution EM, chemical cross-linking combined with mass spectrometry (MS) has matured into an alternative method to study protein/protein interactions.8 Using the cross-linking/MS approach, information on the three-dimensional structure of proteins as well as on interactions between proteins can be obtained.9,10 For further details, we refer to Arlt et al.10 and the references cited therein. For photocross-linking, different cross-linkers are currently employed, such as benzophenones, phenyl-


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azides, and diazirines.11 They are activated by UV-irradiation leading to the formation of highly reactive carbene or nitrene species, which will react non-specifically with amino acids residues in peptides and proteins. The major advantage of the cross-linking/MS approach is that also membrane proteins can be investigated in their native environment, i.e. in the presence of membrane lipids. For this purpose, a variety of membrane models are available, among the most common ones are amphipols,12 bicelles, nanodiscs,6,13 and liposomes.14 Beside the modification of isolated peptides and proteins with different cross-linkers, membrane lipids, with membrane peptides/proteins embedded, might be modified. The idea of inserting photochemical cross-linkers, namely azides, diazirines, diazirinophenoxy groups as well as their fluorinated analogs—either in the hydrophobic alkyl chain or in the hydrophilic headgroup of the membrane phospholipid—is not entirely new.11,15,17 Pioneering work had been performed by Khorana et al. on the synthesis of phospholipids containing photo-reactive precursors for studying protein/lipid interactions.18-25 Also, a membrane-spanning lipid (bolalipid) bearing a photo-reactive group in the middle part of the alkyl chain has been designed.26 In case the photoreactive group is inserted in the alkyl chain of the membrane lipid at different positions, further information about the insertion mode of membrane proteins and peptides will be obtained (Figure 1).


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Figure 1. Left: schematic representation of an integral membrane protein/peptide embedded in a phospholipid bilayer including photo-reactive membrane lipids. Right: chemical structure of an azide-modified membrane lipid bearing an azide group in the sn-2 acyl chain of the glycerol. Previous studies on phospholipids containing photo-reactive groups, as noted above, have in common that either a comparable physicochemical behavior of modified and classical membrane lipids is assumed or that both types of membrane lipids show a perfect miscibility. In-depth physicochemical investigations regarding the aggregation behavior of pure membrane lipids bearing photo-reactive groups as well as the mixing behavior of them with classical membrane lipids are missing so far. In the present study, we perform a systematic evaluation of the aggregation behavior of photoreactive membrane lipids. Therefore, we synthesized four membrane lipids containing an azide group at different positions of the alkyl chain (Scheme 1). For our initial study, we focus on azides since the advantages of this group combine its facile incorporation into the fatty acid chain, its enhanced stability during the experiments, and the fact that potential perturbations of alkyl chain packing are minimized due to its small size. The latter should result in miscibility with classical phospholipids—a prerequisite for future peptide/lipid and protein/lipid interaction studies. In addition, we prepared classes of membrane lipids, containing either an ester or an ether bond at the sn-2 position of the glycerol. By this, we would like to study the effects of these alternative linkages on the aggregation behavior and the stability of the different azidolipids. The temperature-dependent aggregation behavior of the pure azidolipids in aqueous suspension was investigated by means of transmission electron microscopy (TEM), dynamic light scattering (DLS), differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, and small angle X-ray scattering (SAXS). We show that several of the azidolipids are capable to form closed lipid vesicles (liposomes), while other azidolipids self-assemble in


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large sheet-like aggregates where the lipid molecules are arranged in an antiparallel (interdigitated) orientation in the gel phase. Finally, a transmembrane model peptide related to the WAL-peptide27,28 family was embedded in a phospholipid bilayer composed of an azidolipid/phospholipid mixture (P10AzSPC:DMPC). Photochemically induced cross-linking demonstrates the applicability of our azidolipid to analyze peptide/lipid interactions. The efficiency of photo-cross-linking was monitored by attenuated total reflection infrared (ATR-IR) spectroscopy and MS.

EXPERIMENTAL SECTION Chemicals. Chemicals for the synthesis were purchased from Sigma Aldrich Co. (Steinheim, Germany) and were used without further purification. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were purchased from Lipoid KG (Ludwigshafen, Germany)and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was obtained from Avanti Polar Lipids (Alabaster, USA). Syntheses. Azidolipids. The synthetic procedures and the analytical data of azide-modified membrane lipids are described in detail in the Supporting Information (SI). WAL-peptide. The peptide Ac-GKKLALALALALALALALALWWA-NH2 (referred to as KLAW23) was synthesized using fast Fmoc-SPPS29 on a Tetras Peptide Synthesizer (Advanced Chemtec, Louisville, KY, USA) using a standard protocol. The procedure is described in the SI in detail. The purity and identity of the purified peptide was confirmed by HPLC (Figure S1) and MALDI-TOF-MS (Figure S2, Figure S3).


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Methods. Sample preparation. The pure azidolipid was suspended in H2O (MilliQ Millipore water with a specific resistance ρ = 18.2 MΩ cm). Homogeneous suspensions were obtained by heating to 90 °C and vortexing. Liposomes were prepared by extrusion (17 times) of the lipid suspension through a polycarbonate membrane (100 nm) at a temperature approximately 10 K above the transition temperature observed in the DSC experiments. Transmission Electron Microscopy (TEM). The stained samples were prepared by spreading 5 µL of the lipid suspension (c = 0.05 mg mL–1) onto a copper grid coated with a Formvar film. After 1 min, excess liquid was blotted off with filter paper and 5 µL of 1% aqueous uranyl acetate solution were placed onto the grid, drained off after 1 min, and the samples were dried at room temperature for at least 24 h. All specimens were examined with a Zeiss EM 900 transmission electron microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). Cryo-TEM. Vitrified specimens for cryo-TEM were prepared by a blotting procedure, performed in a chamber with controlled temperature and humidity using an EM GP grid plunger (Leica, Wetzlar, Germany). A drop of the sample solution (c = 1 mg mL–1) was placed onto an EM grid coated with a holey carbon film (C-flatTM, Protochip Inc., Raleigh, NC, USA). Excess solution was then removed with a filter paper, leaving a thin film of the solution spanning the holes of the carbon film on the EM grid. Vitrification of the thin film was achieved by rapid plunging of the grid into liquid ethane held just above its freezing point. The vitrified specimens were kept below 108 K during storage, transfers to the microscope, and investigation. Specimens were examined with a Libra 120 Plus TEM (Carl Zeiss Microscopy GmbH, Jena, Germany), operating at 120 kV. The microscope is equipped with a Gatan 626 cryotransfer system and with a BM-2k-120 Dual-Speed on axis SSCCD-camera (TRS, Moorenweis, Germany).


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Dynamic Light Scattering (DLS). DLS experiments were carried out with a Litesizer 500 (Anton Paar GmbH, Graz, Austria) or an ALV-NIBS-HPPS particle sizer (ALV-Laser Vertriebsgesellschaft m.b.H., Langen, Germany). A 3 mW laser with a wavelength λ = 632.8 nm and a scattering angle of 173° was used. All samples (c = 1 mg mL–1) were filled into cuvettes (path length 10 mm). Before starting the measurement, each sample was equilibrated for at least 10 min at 25 °C. Three individual measurements were performed for each sample to test the reproducibility with one measurement consisting of 3 runs of 120 s each. The experimental data were analyzed with the aid of the ALV-correlator software (version 3.0) assuming a viscosity of η = 0.8872 mPa s, a refractive index of 1.33 and using an exponential regularized fit. Differential Scanning Calorimetry (DSC). DSC measurements were performed using a MicroCal VP-DSC differential scanning calorimeter (MicroCal Inc. Northampton, MA, USA). Before the measurements, the sample suspension and the water reference were degassed under vacuum while stirring. A heating rate of 60 K h–1 was used and the measurements were performed in the temperature interval from 5–75 °C. To check the reproducibility, three consecutive scans were recorded for each sample. The water-water baseline was subtracted from the thermogram of the sample, and the DSC scans were evaluated using MicroCal Origin 8.0 software. Fourier-transform Infrared Spectroscopy (FTIR). Infrared spectra were collected on a Bruker Vector 22 Fourier transform spectrometer with DTGS detector operating at 2 cm-1 resolution. The sample suspension (c = 100 mg mL–1 in H2O) was placed between two CaF2 windows, separated by a 6 µm spacer. IR spectra were recorded in steps of 2 K in the temperature range from 9 °C to 61 °C. The temperature was adjusted with a Haake F6 thermostat (C25, Thermo Electron Corporation, Karlsruhe, Germany) and controlled with Delphi-based home-written software. After an equilibration time of 8 min, 128 scans were recorded and accumulated. The


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corresponding spectra of the solvent (H2O) were subtracted from the sample spectra using the OPUS software supplied by Bruker. Small angle X-ray scattering (SAXS). The SAXS measurements were performed with a laboratory SAXS instrument (Nanostar, Bruker AXS GmbH, Karlsruhe, Germany). The instrument includes an IµS micro-focus X-ray source with a power of 30 W using the wavelength of the Cu Kα line. As the detector, a VÅNTEC-2000 detector (14 × 14 cm2 and 2048 × 2048 pixel) was used. The sample to detector distance was 108.3 cm and the accessible q range was from 0.01 to 0.23 Å–1. Samples were filled into glass capillaries of 2 mm diameter with temperature control (∆T = 0.1 K). The raw scattering data were corrected for the background from the solvent measured in a capillary with the same diameter and then converted to absolute units using the scattering of pure water measured at 20 °C (program SuperSAXS, Prof. C. L. P. Oliveira and Prof. J. S. Pedersen). UV-irradiation. For reactivity studies we used a solution of pure azidolipid (c = 5 mM), an azidolipid:DMPC mixture (c = 10 mM; 1:4, n:n; premixed in CHCl3/MeOH), or an azidolipid:DMPC:KLAW23 mixture (c = 10 mM; 10:40:1, n:n:n; premixed in CHCl3/MeOH). For mixed samples, the organic solvent was removed in a stream of N2 and the resulting film was kept in an evacuated flask for 48 h to remove residual traces of solvent. Liposomes were prepared by mixing with MilliQ water and extrusion through a polycarbonate membrane of 100 nm pore size. Afterwards, the solution was filled into quartz cuvettes (Quartz SUPRASIL, path length 10 mm; Hellma Analytics, Müllheim, Germany) and gently stirred during the experiment. The cross-linking was performed using a low-pressure mercury lamp (λ = 254 nm, P = 15 W). At pre-defined time points, 50 µL-samples were removed for ATR-FTIR and MS studies. Attenuated Total Reflection Fourier-transform Infrared (ATR-FTIR) spectroscopy. Samples


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of cross-linked liposomes were analyzed using a Bruker BioATR2 unit with a Bruker Tensor27 FTIR spectrometer (Bruker Optics GmbH, Ettlingen, Germany). Single-channel IR spectra (128 scans) were recorded between 900 and 5000 cm–1 using unpolarized IR light with 2 cm–1 resolution. The temperature (T = 25 °C) was adjusted with a Haake Pheonix II thermostat (C25P, Thermo Electron Corporation, Karlsruhe, Germany) and controlled with Delphi-based homewritten software. As reference, single-channel spectrum (128 scans) taken from the pure water at 25 °C was used and this spectrum was subtracted from the corresponding sample spectra using the OPUS software supplied by Bruker. Afterwards, the wavelength range of sample spectra were set to 1900–3100 cm–1, all spectra were normalized with respect to the intensity of the symmetric methylene stretching vibrational band (νsCH2), and the intensity of the antisymmetric azide stretching vibrational band (νasN3) was analyzed. Mass spectrometry (MS). MALDI-TOF-MS was performed on an Ultraflex III MALDITOF/TOF mass spectrometer (Bruker Daltonik, Bremen, Germany) equipped with a 355 nm Smart Beam ® Laser. All measurements were performed in positive ionization and reflector mode. Eight hundred single laser shots were added to each mass spectrum. 2,5-Dihydroxybenzoic acid (DHB) (c = 40 mg mL–1 in methanol (50% v/v) and distilled water (40% v/v) containing 0.1% (v/v) trifluoroacetic acid) was used as matrix. All spectra were analyzed with the FlexAnalysis (3.3) software (Bruker Daltonik, Bremen, Germany). LC/MS. Samples of irradiated azidolipid:DMPC:KLAW23 mixtures were separated on an Agilent 1200 HPLC System equipped with Jupiter C4 column (Phenomenex, 150 × 2 mm, 5 µm, 300 Å). Analytes were eluted with a 45-min linear gradient from 10% to 90% B at 0.1 mL min–1 and 50 °C; with A: 40% aqueous acetonitrile, 0.1% formic acid; and B: 10% acetonitrile, 90% isopropanol, 0.1% formic acid. Analytes were detected with a QExactive Plus mass spectrometer


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(Thermo Fisher Scientific, Bremen, Germany) with Nanospray Flex Ion Source coupled online to the HPLC system. MS detection was carried out in the orbitrap mass analyzer (R = 140,000 at m/z 200) and for every MS scan the 20 most intense signals were selected for higher-energy collision induced dissociation (HCD) at 29% normalized collision energy. Fragment ions were analyzed in the orbitrap mass analyzer (R = 17,500 at m/z 200). Precursors were excluded for further fragmentation within 60 s after MS/MS acquisition. The HPLC system and the mass spectrometer were operated with HyStar (Version: 3.2, Bruker, Bremen, Germany) and XCalibur (Version: 4.0, Thermo Fisher Scientific, Bremen, Germany). XCalibur and Progenesis QI (Version: 2.1, nonlinear Dynamics, Newcastle Upon Tyne, UK) were used for data evaluation. Quantitation results obtained from Progenesis QI were normalized using total ion abundances of each LC/MS analysis.

RESULTS AND DISCUSSION Synthesis of azide-modified membrane lipids. For the preparation of monopolar, azide-modified membrane lipids two different strategies are conceivable: On the one hand, one can apply a partial synthetic approach using classical phospholipids, such as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cleave the fatty acid chain in sn-2 position of the glycerol with the help of phospholipase A2 (PLA2 received from honey bee venom or porcine pancreas), and finally perform a reacylation reaction with the fatty acid of interest and, for example, the Steglich esterification.30 This approach is straightforward and allows an easy preparation of various modified phospholipids including two ester bonds. One the other hand, and if one would like to insert an ether instead of an ester bond, a total synthetic approach is the method of choice. For this, Arnold et al.31 and later Nuhn et al.32 described


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the use of a selectively blocked and alkylated glycerol derivative, namely rac-1-O-benzyl-2-Ohexadecyl-sn-glycero-3-phosphocholine (2), which could easily be synthesized in four steps from 1,3-O-dibenzylidene glycerol. After cleavage of the benzyl moiety one can insert any desired fatty acid using for example the Steglich esterification reaction. In this study, we use both the partial and the total synthetic approach to prepare two azidemodified phospholipids (P10AzSPC and P15AzPdPC) including two ester bonds in the glycerol backbone and two azidolipids (r12AzSHPC and r15AzPdHPC) that bear an ether bond in the 2 position of the glycerol (Scheme 1). The latter ones are used to study a possible effect on the stability of phospholipids, since it is well-known that the acyl chain in sn-2 position is amenable to cleavage. As azide-modified fatty acids we use either (10RS)-10-azidostearic acid33 (1a), (12RS)-12-azidostearic acid18,19,33,34 (1b), or 15-azidopentadecanoic acid35,36 (1c), which were synthesized according to the literature (see Scheme 1, top).

Scheme 1. Synthetic Pathway for the Preparation of Azide-Modified Membrane Lipidsa


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Azido-modified fatty acids (10RS)-10-azidostearic acid

(12RS)-12-azidostearic acid

COOH 3

N3 1a[33]

15-azidopentadecanoic acid

COOH 2

4

COOH

N3

5

7

N3 1b[18,19,33,34]

1c[35,36]

Azido-modified phospholipids Method A: partial synthesis

O O

sn-1 sn-2 sn-3

PC

*

O i >95%

O

O 7

O

7

PC

O

O

DPPC

7

ii 47-54%

OH

O

N

O

O 7

O P O O

PC:

PC

O

R O

N3

P10AzSPC: R =

lyso-PPC

4

3

N3

P15AzPdPC: R = 7

Method B: total synthesis

O

Bn

O

O

OH iii

iv or v 52-57%

PC

O

O

O O

7 [24,25]

2

PC

O

7

3

R

PC

O

7

N3

r12AzSHPC: R = 5

2

N3

r15AzPdHPC: R = 7

a

Reactions and conditions: (i) PLA2 from Apis mellifera, Et2O/MeOH, r.t.; then H2O, EtOH. (ii) MNBA, DMAP, 1a or 1c, CH2Cl2, r.t. (iii) EtOH, Pd/C, H2, 3–4 atm, r.t. (iv) 1b, DCC, CCl4; then 3, toluene, DMAP, r.t. (v) 1c, DCC, DMAP, CH2Cl2, r.t. The partial synthetic approach starts from classical monopolar membrane lipids, DPPC in our

case. The corresponding lyso-derivative 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (lyso-PPC) was prepared according to a method described by Keough and Davis:37 Incubation of an ethereal (+ 5% MeOH) solution of DPPC with PLA2 from honey bee venom (Apis mellifera), dispersed in 5 mM CaCl2, resulted in the formation of lyso-PPC after 2 h of gently shaking in nearly quantitative yield. For the subsequent reacylation reaction, we firstly tried to use the method by Steglich30 with a 1.5fold excess of the corresponding azide-modified fatty acid (1a or 1c), 1.65 equivalents of dicyclohexylcarbodiimide (DCC), and 10 mol% of 4-(dimethylamino)-


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pyridine (DMAP). But, after 48 h, we did not observe any noteworthy conversion and the addition of further DCC and DMAP did not result in an improvement, too. Thus, we changed the activating reagent to 2-methyl-6-nitrobenzoic anhydride (MNBA, 5 equivalents), following the method described by Yasuda et al.38 Together with a high excess of DMAP (10 equiv. instead of 0.1 equiv.) we were able to prepare two azide-modified phospholipids bearing an ester bond in the sn-2 position of the glycerol, namely 1-palmitoyl-2-[(10RS)-10-azidostearoyl]-sn-glycero-3phosphocholine (P10AzSPC) and 1-palmitoyl-2-(15-azidopentadecanoyl)-sn-glycero-3-phosphocholine (P15AzPdPC, see Scheme 1, middle) in good over-all yields (47-54%). The total synthetic approach starts from rac-1-O-benzyl-2-O-hexadecyl-sn-glycero-3-phosphocholine (2). This precursor, which was synthesized according to Nuhn et al.,32 already contained an alkyl chain in the 2 position of the glycerol. The benzyl protecting group was removed using a hydrogenation reaction under increased pressure and palladium on carbon as catalyst, yielding the rac-1-hydroxy-2-O-hexadecyl-sn-glycero-3-phosphocholine (3). For the reacylation reaction, the corresponding fatty acid (1b, 5fold excess) was pre-activated in dry CCl4 using the DCC. The subsequent coupling step was conducted in dry toluene with catalytic amounts of DMAP—a modification to the method described by Steglich.30 Alternatively, one can apply a ‘one-pot’ synthesis using a mixture of compound 3, 1.25 equivalents of the fatty acid 1c, DCC (3 equivalents), and DMAP (10 equivalents) in dichloromethane. However, in both cases, the repeated addition of DCC after certain time of reaction is advantageous to activate the free fatty acid again and, hence, to increase the yields obtained. Using both ways, two azide-modified phospholipids, namely rac-1-[(12RS)-12-azidostearoyl]-2-O-hexadecyl-sn-glycero-3-phosphocholine (r12AzSHPC) and rac-1-(15-azidopentadecanoyl)-2-O-hexadecyl-sn-glycero-3-phosphocholine (r15AzPdHPC), could be prepared in good over-all yields (52-57%). It has to be


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noted that, in contrast to both lipids described above, r12AzSHPC and r15AzPdHPC are racemic phospholipids due to the preparation steps used in the synthesis of precursor 2. Should it become obvious that the stereospecificity of the glycerol has an impact on, e.g., the physicochemical behavior of the azide-modified membrane lipids, the synthesis of optically pure derivatives with an alkyl chain in the sn-2 position is also possible using (S)-1,2-O-isopropylideneglycerol as staring compound and the valuable protecting group strategy previously reported by Markowski et al.39-41 The synthetic strategies presented herein allow the preparation of azide-modified phospholipids with a variable substitution pattern on the glycerol moiety. Moreover, it can be easily adapted to the synthesis of other phospholipids including for example other photo-reactive fatty acids. Properties: The temperature-dependent aggregation behavior in aqueous suspension. At first, we investigated aqueous suspensions of azide-modified phospholipids in pure manner to get insights into the temperature-dependent aggregation behavior of these lipids and to detect possible perturbations caused by the azide group. TEM and DLS. When suspended in water, all azidolipids (c = 1 mg mL–1) form a more or less turbid (opalescent) suspension after several cycles of heating and vortexing. This fact is a first indication for the formation of larger aggregate structures, such as multilamellar vesicles or sheet-like assemblies, since the formation of very small aggregates, like micelles, should result in a virtually clear and transparent suspension. Afterwards, all suspensions were extruded through a polycarbonate membrane of 100 nm pore size in order to possibly produce unilamellar liposomes. To visualize the structures of aggregates formed in the aqueous suspensions at 20 °C, stained samples were prepared before and right after extrusion (Figure 2 and Figure S4 in the SI). In addition, DLS measurements were performed from the aqueous suspensions of the azidolipids


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before extrusion, after extrusion, and also one and seven days of storage in the refrigerator (at 4 °C) in order to get information about the stability of the liposomes. The correlation functions obtained were analyzed using an exponential regularized fit and the corresponding results are shown in Figure S7 and Table S1 (SI) given as particle sizes of mass-weight size distribution. Before extrusion, all azidolipids form different mostly large aggregates when suspended in water: TEM images (see Figure S4) of stained samples show the existence of a variety of morphologies: very small aggregates (10–30 nm) and vesicles of about 100 nm in diameter for P10AzSPC, large vesicles (several hundreds of nanometer in diameter) for P15AzPdPC, and also sheet-like structures of about 500 nm in diameter for r12AzSHPC. For r15AzPdHPC only small fragments of sheet-like aggregates are visible in the TEM image (see Figure S4E,F); but, the corresponding DLS measurements show the presence of very large aggregates instead (d > 1 µm; see Figure S7 and Table S1). That means, TEM images of stained samples of the azidolipid suspensions, and other phospholipids in general, that are not extruded should be taken with caution because such large aggregates are blotted away during EM sample preparation in some cases. The situation changes after extrusion of the azidolipid suspensions through a 100 nm polycarbonate membrane. P10AzSPC shows the formation of round shaped aggregates of two different sizes (Figure 2A): smaller ones with a diameter of around 50 nm (white arrows) and larger ones with diameters ranging from 150 to 200 nm. This observation is confirmed by DLS measurements showing two populations of aggregates with particle sizes that are in a comparable range as described before: d1 = 32 nm and d2 = 146 nm (see Table S1). The reason for the formation of two different size populations remains unclear at this time. P15AzPdPC and also r12AzSHPC show the formation of liposomes with sizes of 100 to 180 nm in diameter (Figure 2B,C). The


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appearance of crashed vesicles, e.g. in Figure 2B, is typical for TEM images of stained lipid samples since liposomes usually collapse during the drying process of the EM sample preparation. The corresponding DLS measurements reveal consistent results: d = 138 nm for P15AzPdPC and d = 108 nm for r12AzSHPC. Another situation is found for r15AzPdHPC. Here, TEM image of stained sample shows the existence of very small possibly disk-like aggregates (Figure 2D). But, DLS measurements display again the presence of very large aggregates so that the TEM image of this sample and, hence, the exclusive existence of small objects should be taken with caution.

A

200 nm

B

200 nm

C

D

200 nm

200 nm

Figure 2. TEM images of aqueous suspensions (c = 0.05 mg mL–1) of (A) P10AzSPC, (B) P15AzPdPC, (C) r12AzSHPC, and (D) r15AzPdHPC after extrusion through a polycarbonate membrane of 100 nm pore size. The samples were prepared at 20 °C and stained with uranyl acetate. The white arrows point to small aggregates of P10AzSPC. To further clarify the structure of aggregates formed of r15AzPdHPC, additional cryo-EM


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investigations were performed and the corresponding cryo-TEM images are shown in Figure 3. One can clearly see that r15AzPdHPC forms large sheet-like aggregates of several micrometers and also very large vesicular structures. It is conceivable that the existence of both the terminal azido moiety and the hexadecyl chain in 2 position of the glycerol, i.e. an ether bond instead of a classical ester bond, are responsible for self-assembly of r15AzPdHPC in large sheet-like structures. This assumption becomes even more obvious since the counterparts that exhibit only one of the characteristics mentioned above, P15AzPdPC and r12AzSHPC, show liposome formation (see above).

A

200 nm

B

200 nm

Figure 3. Cryo-TEM images of an aqueous suspension of r15AzPdHPC (c = 1 mg mL–1) quenched from room temperature. To get information about the stability of the extruded liposomes, additional DLS measurements were performed after storage of liposomes at 4 °C for several days. The results show that


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liposomes prepared from azidolipids bearing the azide group in the middle part of the alkyl chain (P10AzSPC and r12AzSHPC) are stable in size for at least one week (see Figure S7 and Table S1). It is reasonable that the azido group prevents the lipid molecules from dense packing and, as a consequence, these two azidolipids might be in the liquid-crystalline phase (Lα) when stored at 4 °C (see below). In contrast, liposomes of P15AzPdPC increase in size during storage from d = 138 ± 1 nm at day 0 to d = 357 ± 2 nm at day 1 and further to d = 771 ± 2 nm at day 7. It is conceivable that the terminal azide group of P15AzPdPC causes a destabilization of the lipid layer, possibly due to the additional polarity derived from the azido group, which might lead to fusion of small liposomes into larger ones or even into large lamellar sheets comparable to aggregates found in P15AzPdPC suspensions prior extrusion (Figure S4B). Another reason could be that P15AzPdPC is in the gel phase (Lβ) during storage (see below), which might also trigger vesicle aggregation and fusion.42-45 To clarify this phenomenon, additional stability measurements of liposomes prepared from classical phospholipids were performed. Due to similar alkyl chain length, we used DPPC for the comparison with P15AzPdPC and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) for the comparison with P10AzSPC. The results of DLS and TEM investigations are shown in Figures S5, S7 as well as Table S2 (SI). The findings confirm our assumption that the (size)stability of extruded liposomes is mainly affected by the phase state of the phospholipid during storage: If the phospholipid is in the liquid-crystalline (Lα) state, the liposomes are stable in size for at least one week, which is the case for P10AzSPC, r12AzSHPC, and POPC. Contrary, if the lipid is in the gel phase, the corresponding liposomes are unstable and show aggregation and vesicle fusion over time. DSC. To investigate the temperature-dependent aggregation behavior of the azidolipids in


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aqueous suspension, DSC and FTIR measurements were carried out. The results are shown in Figure 4, Figure 5, and Figure S8-Figure S11. 132 128 60

-1

50 -1

Cp / kJ mol K

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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40 DPPC

30

DMPC

20

r15AzPdHPC r12AzSHPC

10

P15AzPdPC P10AzSPC

0 10

20

30 40 50 Temperature / °C

60

Figure 4. DSC heating curves of aqueous suspensions (c = 1 mg mL–1) of azidolipids as well as DMPC and DPPC shown for comparison. The heating rate was 60 K h–1. The curves are shifted vertically for clarity. Unmodified phospholipids, for example DMPC or DPPC, show two well-known endothermic transitions in the DSC heating scan: the so-called pretransition (Tp) from the lamellar gel phase (Lβ’) to the ripple phase (Pβ’) at Tp = 15.6 °C and Tp = 36.1 °C for DMPC and DPPC, respectively, and the main transition (Tm) from Pβ’ to the lamellar liquid-crystalline phase (Lα) at Tm = 24.5 °C and Tm = 41.7 °C for DMPC and DPPC (Figure 4). In the corresponding cooling scan the main transitions appear at slight lower temperatures with a small hysteresis of ∆T = 0.8 K (Figure S8) for both classical phospholipids. The DSC heating curve of P10AzSPC and r12AzSHPC shows no transition between 5–75 °C assuming that both azidolipids, which bear the azide group in the middle part of the alkyl chain, are in the Lα phase (Figure 4). The azido group perturbs a dense packing of the alkyl chains and,


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hence, Tm is shifted to lower temperatures (< 5 °C). This perturbation is comparable to the insertion of a double bond into one alkyl chain of conventional phospholipids, e.g., 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC) shows a transition from Lβ’ to Lα at Tm = −2.5 °C.46 The situation changes if the azide group is moved to the end of the alkyl chain: The DSC heating curve of P15AzPdPC reveals a broad transition at Tm = 36.3 °C (FWHM = 2.9 K) with an enthalpy change of ∆H = 36.8 kJ mol–1. This transition is possibly accompanied by a transition from a gel to a liquid-crystalline phase, without any additional pretransition (Figure 4). Compared to DPPC (∆H = 34.3 kJ mol–1),46 this transition appears 5.4 K lower, which might be due to the slightly shorter alkyl chain length (pentadecanoyl instead of palmitoyl) and/or the presence of the terminal azide group that, in turn, perturb the alkyl chain packing to some extent. However, it might also be conceivable that P15AzPdPC forms a lamellar gel phase with interdigitated lipid molecules (LβI) due to the existence of an azide group, which carries an affinity for polar environment. Such a spontaneous alkyl chain interdigitation in a lamellar gel phase is observed for phospholipids carrying, e.g., a single fluorine atom at the end of one alkyl chain: F-DPPC (1palmitoyl-2-(16-fluoropalmitoyl)-sn-glycero-3-phosphocholine) shows a transition at Tm = 52.0 °C (∆H = 40.9 kJ mol–1) from an LβI to Lα phase.47 But, the existence of an Lβ I phase for P15AzPdPC below Tm cannot be deduced from DSC experiments alone. The corresponding DSC cooling scan of P15AzPdPC shows the same transition with a small hysteresis at Tm = 33.5 °C (∆T = 2.8 K). For r15AzPdHPC we observed a very cooperative transition at Tm = 41.9 °C (FWHM = 0.5 K, ∆H = 35.2 kJ mol–1) in the DSC heating curve and, again, without any additional pretransition. However, since r15AzPdHPC carries a hexadecyl chain (ether bond) in the sn-2 position of the glycerol, a comparison with 1-palmitoyl-2-O-hexadecyl-sn-glycero-3-phosphocholine


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(PHPC) seems to be more reasonable. This unmodified acyl-alkyl-phosphocholine shows a transition at Tm = 44.4 °C (∆H = 36.0 kJ mol–1) from a lamellar chain-interdigitated gel phase (LβI) to an Lα phase and likewise without a pretransition.48 The slightly lower Tm value of r15AzPdHPC compared to PHPC might be attributed to the azide group, perturbing the alkyl chain packing, and/or to the slightly shorter alkyl chain. The corresponding DSC cooling curve reveals a transition peak at Tm = 37.6 °C including a small shoulder at 39.7 °C. Lewis et al. have also found such a “biphasic cooling exotherm at temperatures near the freezing point of its [PHPC] lipid hydrocarbon chains”,48 i.e., the phase behavior of r15AzPdHPC and PHPC are comparable to some extent. However, the existence of an alkyl chain interdigitation for r15AzPdHPC aggregates below Tm as found for PHPC and also other ether lipids, such as 1-Ohexadecyl-2-palmitoyl-sn-glycero-3-phosphocholine48

(HPPC)

or

1,2-di-O-hexadecyl-sn-

glycero-3-phosphocholine49,50 (DHPC), cannot be deduced from DSC measurement alone, although the comparable DSC behavior of r15AzPdHPC and PHPC gives a strong evidence therefor. Interestingly, r15AzPdHPC shows a much larger hysteresis (∆T = 4.3 K) compared to PHPC (∆T = 0.4 K) that is possibly due to the azide group at the end of the alkyl chain acting again as a perturbation and preventing a fast reorganization of the lipid molecules in the low temperature phase. FTIR. To obtain more information on the nature of the thermotropic transition observed in the DSC scans of P15AzPdPC and r15AzPdHPC, respectively, and to get evidence about the phase state of P10AzSPC and r12AzSHPC at ambient temperature, we performed FTIR experiments on suspensions of all azidolipids. The wavenumbers of the symmetric (νsCH2) and antisymmetric (νasCH2) methylene stretching vibrational band provide information about the conformational order of the alkyl chains.51,52


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For those two azidolipids showing no DSC transition, P10AzSPC and r12AzSHPC, the frequencies of the bands at ambient temperature are at 2924 cm–1 for νasCH2 and 2853 cm–1 for

νsCH2 (Figure S11). These frequency values indicate that the alkyl chains of both azidolipids are in “fluid” state, i.e. both lipids are in the Lα phase at room temperature, a fact already supposed by DSC and DLS measurements described above. The other two azidolipids bearing the azide group at the end of the alkyl chain show a different behavior. For P15AzPdPC, the frequencies of the bands at low temperature are at 2849.9 cm–1 for νsCH2 (Figure 5A) and 2916.8 cm–1 for νasCH2 (Figure S9), indicating that the alkyl chains of P15AzPdPC are in an all-trans conformation and well-ordered at this temperature, i.e. the azidolipid is in a lamellar gel phase. With increasing temperature the wavenumber of both bands remain nearly constant up to T ≈ 36 °C. Above this temperature, which correlates to Tm found in the DSC measurement, the wavenumber of the νsCH2 band jumps to 2852.8 cm–1 and increases further to 2853.6 cm–1 at 61 °C whereas the wavenumber of the νasCH2 band jumps to 2923.0 cm–1 and increases further to 2924.1 cm–1. These values indicate that the alkyl chains are now in the “fluid” state (Lα phase); and the “melting” of the chains occur at Tm of P15AzPdPC as can be seen by the stepwise increase in the wavenumbers at this temperature. The corresponding cooling curves of the frequencies of both CH2 stretching vibrational bands reveal exactly the same pattern without any indication of a hysteresis. This is kind of remarkable since we observed a small hysteresis of ∆T = 2.8 K in the DCS measurements (see above).


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B

A 2853.5

δ(CH2)

2102

ν(N3)

2101

1467.6

2852.0 2851.5 2851.0 2850.5

heating cooling

2850.0

-1

2100 1467.4

Wavenumber / cm

2852.5

Wavenumber / cm-1

-1

Wavenumber / cm

C 1467.8

νs(CH2)

2853.0

1467.2 1467.0 1466.8

2098 2097 2096

heating cooling

1466.6

2099

heating cooling

2095 10

20

30

40

50

60

10

20

Temperature / °C

E

2853.5

40

50

60

10

ν s(CH2)

F

1468.4

δ(CH2)

2850.5 2850.0

heating cooling

Wavenumber / cm

2851.0

1467.8 1467.6 1467.4 1467.2 heating cooling

1467.0

40

50

60

ν(N3)

2101 2100 2099 2098 heating cooling

2096

1466.8 30

Temperature / °C

60

2104

2097

2849.5 20

50

2102

-1

Wavenumber / cm

-1

2851.5

40

2103

1468.0

2852.0

30

Temperature / °C

1468.2

2852.5

10

20

-1

2853.0

30

Temperature / °C

D

Wavenumber / cm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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10

20

30

40

50

60

Temperature / °C

10

20

30

40

50

60

Temperature / °C

Figure 5. Wavenumber (heating, red; cooling, blue) of the symmetric methylene stretching vibrational band (A, D), the methylene scissoring vibrational band (B, E), and the azido stretching vibrational band (C, F) as a function of temperature for suspensions of P15AzPdPC (A–C) and r15AzPdHPC (D–F; c = 100 mg mL–1 in H2O). For r15AzPdHPC, the phase behavior is quite comparable to P15AzPdPC: The frequencies of both methylene stretching vibrational bands are at 2849.8 cm–1 for νsCH2 (Figure 5D) and at 2916.3 for νasCH2 (Figure S10), respectively, at low temperature indicating again an all-trans conformation of the alkyl chains (gel phase). With increasing temperature, the wavenumbers of both bands present a distinct jump at T ≈ 41 °C, which fits quite nicely to Tm found in DSC measurements. Above this temperature, values for νsCH2 at 2852.9 cm–1 and νasCH2 at 2923.1 cm–1, respectively, are observed that indicate again an increasing number of gauche-conformers in the alkyl chains of r15AzPdHPC and, hence, the presence of an Lα phase above Tm. A difference to P15AzPdPC is that in the corresponding cooling curves a distinctive hysteresis can be found (Figure 5D, Figure S10). This hysteresis is even more pronounced than the hysteresis found in the DSC measurement (∆TIR = 9.0 K compared ∆TDSC = 4.3 K), which is obviously due to the hundredfold higher concentration of the azidolipid used in the FTIR experiment compared


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to the DSC measurement, as the kinetics of reformation is, of course, concentration dependent. This hysteresis might be attributed to a pronounced hindrance of the reorganization of r15AzPdHPC lipid molecules within the low temperature phase. However, the fact that such a hysteresis in FTIR measurements has not been observed for P15AzPdPC supports the conclusion that the concomitance of a hexadecyl chain in sn-2 position and a terminal azide group is crucial for this hysteresis behavior. For a closer look at the nature of the transitions, the methylene scissoring vibrational band (δCH2) was analyzed. The position of this band as well as an occurrence of a characteristic splitting of this band are indicative for chain packing geometry.52 The temperature-dependent position of the δCH2 vibrational band for P15AzPdPC is depicted in Figure 5B. At low temperatures, this band is located at around 1467.7 cm–1. This indicates that the alkyl chains of P15AzPdPC show a hexagonal chain packing.52 Upon heating, the wavenumber of δCH2 vibrational band decreases with a jump at Tm (≈ 36 °C), indicating the chain melting transition. The corresponding cooling curve follows the same pattern. In contrast, the position of the δCH2 vibrational band for r15AzPdHPC scatters between 1468.4 and 1467.0 cm–1 within the temperature range from 9–61 °C (Figure 5E) and shows no clear temperature-dependent behavior. However, from the position of this band we assume again a hexagonal packing of the alkyl chains. Moreover, we analyzed the temperature-dependent position of the antisymmetric stretching vibrational band of the azide group (νasN3) of our azidolipids. It is known that the position of

νasN3 gives information about the hydration state of azides; e.g., Owrutsky et al. show that the νasN3 vibrational band of azide ions in different DMSO/water mixtures shifts to higher wavenumbers with increasing mole fraction of water.53 For P15AzPdPC, the frequency of the νasN3 band at low temperature is at 2101.7 cm–1. With


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increasing temperature the wavenumber of this band decreases slightly at the beginning, drops down between T = 32–35 °C, and finally stays nearly constant at 2096.3 cm–1 at temperatures above 36 °C (Figure 5C). The corresponding cooling curve reveals exact the same pattern without any hysteresis. A comparable curve progression was observed for the temperature-dependent position of the νasN3 band of r15AzPdHPC: Starting at a value of 2103.9 cm–1, the wavenumber initially stays constant with increasing temperature up to T ≈ 41 °C. At this temperature, a distinct drop to 2096.2 cm–1 can be found (Figure 5F) and above 41 °C the position of the νasN3 band stays constant. In contrast to P15AzPdPC, r15AzPdHPC shows a pronounced hysteresis in the cooling curve, which is stretched over a wide temperature range from 34 °C to 20 °C. From the temperature-dependent behavior of the νasN3 band and the reference cited above we came to the assumption that the azide group of both azidolipids is more hydrated in the low temperature phase than in the high-temperature, liquid-crystalline (Lα) phase. At first glance this might be implausible since the hydration should increase with increasing temperature.53 But, if we assume an interdigitated gel phase (LβI) at temperatures below Tm for both azidolipids, then the terminal azide groups of P15AzPdPC and r15AzPdHPC are arranged in the headgroup region within the lipid aggregates and, hence, well-hydrated. Upon heating above Tm, the proposed interdigitated gel phase (LβI) transforms into a non-interdigitated liquid-crystalline (Lα) phase and the azide groups are now placed in the middle, more hydrophobic part of the lipid bilayer and, consequently, less hydrated. To further prove this assumption, we analyzed the position of the νasN3 band of the other two azidolipids bearing the azide group in the middle of the alkyl chain. At ambient temperature, both lipids are in the Lα phase and the frequency of the

νasN3 band is at 2097.2 cm–1 and 2098.2 cm–1 for P10AzSPC and r12AzSHPC (see Figure S11), which corresponds to frequency values of the νasN3 band of P15AzPdPC and r15AzPdHPC at


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temperatures above Tm. That means the position of the νasN3 band gives a first indication whether the azidolipid forms an interdigitated gel phase or not. SAXS. To gain information about the structural organization of lipid molecules within the bilayer, whether an interdigitated alkyl chain packing occurs or not, aqueous suspensions of the azidolipids were investigated using small angle X-ray diffraction (Figure 6, Figure S15, and Figure S16). The SAXS curve of an aqueous suspension of P10AzSPC (c = 40 mg mL–1) depicts two distinct reflections with equidistant spacing at q1 = 0.094 Å–1 and q2 = 0.188 Å–1 at 20 °C (Figure 6A, filled green squares), which corresponds to a lamellar repeat distance (membrane thickness plus interlamellar water layer thickness) of d = 66.8 Å. This value is in the range of typical phospholipid bilayers in the Lα phase, e.g., DPPC reveals a repeat distance of d = 67.2 Å in the fully hydrated Lα phase including an interlamellar water layer of dw’ = 20.5 Å.54,55 The other azidolipid that bears the azido function in the middle part of the alkyl chain (r12AzSHPC) shows a broad halo in the SAXS region with a single reflection at q = 0.108 Å–1 at 20 °C (Figure 6A, open blue circles), Although we cannot define the arrangement of r12AzSHPC molecules within the aggregates from one single Bragg peak alone, we assume the existence of a lamellar phase due to our EM investigations (see above) and, hence, determine a repeat distance of d = 58.2 Å. The smaller d-value of r12AzSHPC compared to P10AzSPC could be caused by a thinner interlamellar water layer between the azidolipid bilayers possibly due to existence of only one carbonyl group (ester bond) in the glycerol backbone; i.e. less water seems to be necessary for complete hydration of the r12AzSHPC headgroups. The broad halo in the diffractogram of r12AzSHPC could originate from smaller aggregates, e.g., bilayer fragments.


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A P10AzSPC at 20 °C r12AzSHPC at 20 °C

Intensity / cm

-1

0.3

0.2

0.1

0.0 0.05

0.10

0.15 -1 q/A

0.20

0.25

B r15AzPdHPC at 20 °C r15AzPdHPC at 50 °C

-1

0.03

Intensity / cm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.02

0.01

0.00 0.05

0.10

0.15 -1 q/A

0.20

0.25

Figure 6. SAXS diffractograms of aqueous suspensions of various azidolipids at different temperatures: (A) P10AzSPC (filled green squares, c = 40 mg mL–1) and r12AzSHPC (open blue circles, c = 40 mg mL–1) at 20 °C; (B) r15AzPdHPC (c = 10 mg mL–1) at 20 °C (filled black squares) and at 50 °C (open red circles) after an equilibration of the sample at 4 °C for one week. The situation changes for the azidolipids with a terminal azide group. First of all, both P15AzPdPC and r15AzPdHPC show very low scattering due to the lower concentration used in the SAXS experiment (10 mg mL–1 instead of 40 mg mL–1) and the fact that the lamellar structures formed are poorly ordered in the z-direction. Unfortunately, we were not able to achieve a suitable SAXS diffractogram from P15AzPdPC using different sample preparations (Figure S15). The SAXS diffractogram of an aqueous suspension of r15AzPdHPC (c = 10 mg mL–1) shows a single reflection at q = 0.132 Å–1 at 20 °C (Figure 6B, filled black squares). Since we found large sheet-like aggregates for this azidolipid in aqueous suspension (see Figure 3), we


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assume a lamellar phase at temperatures below Tm and, hence, determine a repeat distance of d = 47.6 Å. This d-value, which combines the membrane thickness and the interlamellar water layer thickness, is very small in comparison to DPPC with d = 63.5 Å in the Lβ’ phase.54 In the case of DPPC, the alkyl chains are tilted by 32° with respect to the membrane normal.54 In the case of r15AzPdHPC, neither a larger tilt of the alkyl chains nor a much smaller water layer between the bilayer can explain the observed small d-value and, hence, the presence of an interdigitated gel phase (LβI) is obvious—a fact, already supposed by DSC and FTIR measurements. This dvalue of 47.6 Å is also comparable to the repeat distance of other phospholipid layer showing alkyl chain interdigitation in the gel phase: for example, F-DPPC reveals a repeat distance of d = 50.2 Å,47 DHPC depicts a value of d = 48 Å,50 1,3-dipalmitoyl-sn-glycero-2-phosphocholine (1,3-DPPC) shows a d-value of 47 Å,56,57 and 1,3-dipalmitamidopropan-2-phosphocholine (PadPC-Pad) shows a repeat distance of 46.8 Å.58 The question whether a gel phase with interdigitated alkyl chains is generally formed or not depends on several facts. At first, the polarity of the lipid headgroup plays a role: While DHPC, which includes two ether bonds and is therefore less polar, self-assembles in lamellar sheets with interdigitated lipid molecules, DPPC forms a classical bilayer. Secondly, a terminal modification of the alkyl chain that carries a certain polarity can trigger interdigitation as found for fluorine atoms in F-DPPC and, as in the present case, an azide moiety. If we heat the sample above Tm, we observe two reflections in the SAXS pattern with equidistant spacing at q1 = 0.095 Å–1 and q2 = 0.190 Å–1 (Figure 6B, open red circles). These Bragg reflexes correlate to a repeat distance of d = 66.1 Å indicating a lamellar Lα phase, which is comparable to P10AzSPC (see above) or DPPC Lα phases. It is noteworthy that the single reflection shown in Figure 6B and Figure S16 (filled black


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squares), indicating an LβI phase of r15AzPdHPC at 20 °C, was only obtained after storage of the sample for one week at 4 °C. If we store the same sample only for one day at 4 °C, the corresponding SAXS pattern reveals two reflections: the first at qA = 0.105 Å–1 and the second at qB = 0.132 Å–1 (Figure S16, open blue circles). If we assume two sets of different lamellar phases, we determine a repeat distance of dA = 59.8 Å and dB = 47.6 Å, respectively. The latter one corresponds to an interdigitated gel phase (LβI), whereas the first one indicates a non-interdigitated gel phase (Lβ). It is conceivable that r15AzPdHPC forms a non-interdigitated gel phase immediately after preparation, which gradually transforms into an interdigitated gel phase during storage, and we observe a coexistence of an LβI and an Lβ phase after one day of storage. Reactivity: The impact of UV-irradiation. The photochemistry of alkyl azides has been described thoroughly.59,60 UV-irradiation at λ = 254 nm generates nitrene intermediates after the loss of nitrogen. The nitrene will react nonspecifically with various amino acid residues of peptides and proteins embedded in a membrane bilayer or with adjacent acyl chains of other membrane lipids. However, nitrenes can also rearrange to imines after 1,2-migrations, i.e., insertion of nitrogen in the alkyl chain and hydrogen shift, respectively.61 These imines will further be hydrolyzed to aldehydes/ketones and corresponding amines (Scheme 2). Scheme 2. Possible reactions of azide-modified lipids after UV-irradiation at 254 nma


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P10AzSPC O

Cross-Linking

O

7

N

N

N

N

UV - N2

O PC

O

3

O

H

non-specific reaction with amino acid residues or other alkyl chains

O H 3 O nitrene intermediate insertion

3

H-shift

3

NH O

O O

3

3

N

O

3

O

3

N

3

O

3

O O O P10OSPC

NH2

O 3

3

O P9AmC9PC

3

O O P10OC10PC

3

O

a

Abbreviations: P10OSPC, 1-palmitoyl-2-(10-oxostearoyl)-sn-glycero-3-phosphocholine; P9AmC9PC, 1-palmitoyl-2-(9-aminononanoyl)-sn-glycero-3-phosphocholine; P10OC10PC, 1palmitoyl-2-(10-oxodecanoyl)-sn-glycero-3-phosphocholine. The applicability of azide-modified lipids to study interactions between membrane proteins and phospholipids was previously shown by several groups using N-([125I]iodo-4-azidosalicylamidyl)-1,2-dilauryl-sn-glycero-3-phosphoethanolamine [125I]-ASA-DLPE.62-66 A non-radioactive analog of this probe (ASA-DLPE) as well as a corresponding phosphatidylcholine derivative were used by the group of Gubbens et al. to analyze the proteome of Saccharomyces cerevisiae interacting with the lipid headgroups.16,67 Although in these studies, the photo-reactive group was located in the headgroup region of the phospholipid and is hence easily accessible, other groups have also reported successful cross-linking of membrane proteins with phospholipids bearing the photo-reactive group (diazirine) in the hydrophobic alkyl chain.17,68 To evaluate the impact of UV-irradiation at 254 nm on our novel azide-modified membrane lipids, P10AzSPC was exemplarily investigated. Besides the pure azidolipid, we also examined a mixture of P10AzSPC and DMPC (1:4, n:n) as an excess of photo-reactive lipids is not favorable regarding future protein/lipid interaction studies. The miscibility of both types of phospholipids was clarified by DSC studies (see below); detailed investigations regarding the mixing


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behavior of azide-modified lipids with classical phospholipids are part of ongoing research. In order to prove the reactivity of the azidolipid with membrane peptides, we used a membrane model peptide of the WAL-peptide family.27,28 These peptides consist of repeating alanineleucine sequences of different length, flanked with tryptophan residues at both termini. They form membrane-spanning α-helices that can incorporate into phospholipid bilayers and, hence, mimic protein transmembrane domains.69-73 Since the previously used peptide WALP23 (sequence: Ac-GWW(LA)8LWWA-NHEtOH) is highly hydrophobic and its purification proved rather challenging, we decided to replace the N-terminal tryptophan residues with lysines and synthesized the modified KLAW23 (sequence: Ac-GKK(LA)8LWWA-NH2) membrane model peptide. At first, empty vesicles, i.e., vesicles without a membrane peptide embedded, of P10AzSPC (c = 5 mM) and a P10AzSPC:DMPC mixture (1:4, n:n; c = 10 mM) were irradiated using a lowpressure mercury lamp (λ = 254 nm, P = 15 W). The pure azidolipid was used to get a first indication whether the azide group of P10AzSPC will react or not. At pre-defined time points, samples were removed to check the intensity of the antisymmetric azide stretching vibrational (νasN3) band and to collect MS data. The ATR-FTIR data of P10AzSPC and P10AzSPC:DMPC (1:4, n:n) liposomes are shown in Figure S12. In both cases, the intensity of the νasN3 band decreases with increasing time of UV-irradiation and after 3 hours the signal intensity of the νasN3 band has disappeared. The loss of P10AzSPC after irradiation was also confirmed by MS (Figure S17). Here, the signal at m/z 803.2 for the protonated azidolipid [P10AzSPC+H]+ disappeared and signals for decomposition products appeared at m/z 776.2 for the corresponding ketone derivative [P10OSPC+H]+ and m/z 651.1 for the truncated amine species [P9AmC9PC+H]+, which is generated upon nitrogen insertion and subsequent hydrolysis


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(Scheme 2, middle pathway). In the higher mass range we identified two new signals in the irradiated sample (Figure S18): the signal at m/z 1453.6 was tentatively assigned to a noncovalent dimer of P10OSPC and DMPC, while the signal at m/z 1328.4 might correspond to covalent dimer. However, the chemical structure of this dimer has not been clarified yet. In addition, we performed DSC measurements of the mixed phospholipid sample before and after UV-irradiation. The corresponding heating scans are shown in Figure 7. Prior to irradiation, the P10AzSPC:DMPC mixture (1:4, n:n) revealed a broad endothermic transition at Tm = 18.5 °C (red solid line), which is below Tm of pure DMPC. Although the pure P10AzSPC showed no transition in the temperature range investigated, we assume a miscibility of both phospholipid components due to the downshifted DSC peak. If one would assume that both lipid components were not miscible in the gel phase, a DSC peak close to Tm of pure DMPC would have been expected for the mixture. As mentioned before, detailed studies with respect to the miscibility of both types of phospholipids are under way. The DSC transition peak of the P10AzSPC:DMPC mixture is shifted to Tm = 37.2 °C after UV-irradiation (red dashed line)—even above Tm of pure DMPC (24.5 °C). This could be an indication for structural rearrangement of lipids within the bilayer membrane due to novel phospholipid species, probably dimers, generated by UVirradiation. After the addition of 2 mol% of the transmembrane model peptide KLAW23, the DSC transition peak of the mixture prior UV-irradiation is slightly shifted to lower temperatures (Tm = 16.6 °C) due to perturbations caused by the incorporation of the peptide into the membrane (Figure 7, green solid line). The existence of the α-helical structure of KLAW23 was proven by ATR-IR measurement: the frequencies of the amide I and amide II bands are at 1657.1 cm–1 and 1548.1 cm–1, respectively (Figure S14), which are indicative for an α-helical arrangement of


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KLAW23.74 The frequencies of the antisymmetric and symmetric methylene stretching vibrational band are at 2922.2 cm–1 and 2852.4 cm–1 indicating that the alkyl chains of the phospholipids are in the “fluid” state (Lα phase; Figure S14) at T = 25 °C. Additionally, the shape of aggregates was visualized by TEM of stained samples before (Figure S6A) and after (Figure S6B) extrusion.

-1

44 42 40

-1

Cp / kJ mol K

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- UV + UV

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2

DMPC

P10AzSPC:DMPC 1:4 + KLAW23

P10AzSPC:DMPC 1:4 - KLAW23

P10AzSPC

10

20

30 40 50 60 Temperature / °C

70

Figure 7. DSC heating curves of aqueous suspensions a P10AzSPC:DMPC (1:4, n:n, c = 1–3 mM) mixture without (red) and with (green) the membrane peptide KLAW23 (2 mol%) embedded, each prior (solid lines) and after UV-irradiation (dashed lines). The heating curves of pure P10AzSPC (black) and DMPC (blue; c = 1 mg mL–1) are shown for comparison. The heating rate was 60 K h–1. The curves are shifted vertically for clarity. After UV-irradiation of the lipid sample including KLAW23, several effects were observed. The DSC transition peak is again shifted to higher temperatures (Tm = 21.0 °C, Figure 7, green dashed line). If we compare the not irradiated with the irradiated sample, the increase in Tm for the sample containing KLAW23 is not as pronounced as for the empty vesicles, which might be


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due to different irradiation times used in the experiments. The ATR-IR experiments showed that the intensity of the νasN3 band decreases again with increasing irradiation time (Figure S13), the frequencies of both methylene stretching vibrations and, hence, the fluidity of the alkyl chains slightly increase (Figure S14A), and the α-helix of KLAW23 is still intact (Figure S14B). Additionally, the shape of the aggregates did not change after 30 min of irradiation, which was confirmed by TEM images of stained samples (Figure S6C,D). The UV-irradiation of the lipid sample including KLAW23 also gave rise to several new species detectable in LC/MS analyses. Besides hydrolyzed decomposition products of the formed nitrene (see above), also triply charged precursors were observed. Two isobaric products that are absent in the samples without irradiation (Figure 8A,B) and increase in abundance during the first five minutes of irradiation, exhibit a mass corresponding to the KLAW23 peptide plus azidolipid with N2 loss and an additional mass shift of −2 amu (m/z 1059.035 for [M+3H]3+, Figure 8C). Fragmentation of this species reveals an intense signal at m/z 184.073 (Figure 8D), characteristic for the phosphocholine group. Further signals in the fragment ion mass spectrum match the fragmentation pattern of the unmodified peptide, indicating either an instable modification under higher-energy collisional dissociation (HCD) conditions, or a modification on the Cterminus, that is not covered by the b-type ion series. As both, the unmodified peptide (tR = 44.7 min) and the P10AzSPC (tR = 51.4 min) are well separated from the resulting products, a gas phase adduct can be excluded and a covalent cross-link between azidolipid and membrane peptide is likely. However, further studies are required not only to improve the yield of peptide/lipid cross-links, but also to elucidate the structure of cross-linked species.


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Figure 8. (A) Extracted ion chromatograms of m/z 1059.035 without (red) and after (black) 5 min of UV-irradiation (λ = 254 nm): (B) Time course of [M+3H]3+ (m/z 1059.035) with tR = 55.1 min after different time points of UV-irradiation. (C) Mass spectrum (MS) and (D) fragment ion mass spectrum (HCD-MS/MS) of the cross-linked product at tR = 55.1 min.


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SUMMARY The investigation of interactions between membrane peptides and proteins with surrounding phospholipids using photo-reactive membrane lipids and the cross-linking/MS approach requires an in-depth understanding of the physicochemical properties of modified membrane lipids. Although there are numerous assays on the use of photo-reactive lipids in peptide/lipid and protein/lipid interaction studies, there is still a lack of a comprehensive examination regarding the aggregation behavior of pure photo-reactive lipids and their mixing behavior with classical phospholipids. To close this gap, four novel azide-modified membrane lipids have been synthesized using either a partial or a total synthetic approach. Both straightforward synthetic routes result in comparable over-all yields and they can also be used for the preparation of other modified membrane phospholipids including variable substitution pattern on the glycerol moiety. All azidolipids show the formation of layered structures in aqueous suspension. In the case of P10AzSPC, r12AzSHPC, and P15AzPdPC, these aggregates can be extruded to produce mostly uniform liposomes. These liposomes are stable in size during the storage at 4 °C for at least seven days, apart from P15AzPdPC, which shows vesicle fusion. In contrast, r15AzPdHPC self-assembles in large sheet-like and non-extrudable aggregates of several microns, which is due to the existence of both the terminal azido moiety and the hexadecyl chain in sn-2 position of the glycerol. The aggregation behavior of all azidolipids as a function of temperature revealed that those azidolipids bearing the azido group in the middle part of the alkyl chain (P10AzSPC and r12AzSHPC) depict no transition between 5–75 °C. Both lipids are in the liquid-crystalline state (Lα phase) at ambient temperature and reveal a lamellar repeat distance of d = 66.8 Å and d = 58.2 Å for P10AzSPC and r12AzSHPC. In contrast, the other two azidolipids with a terminal


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azido moiety show a transition from a lamellar gel (Lβ) phase to the liquid-crystalline (Lα) phase at Tm = 36.3 °C and Tm = 41.9 °C for P15AzPdPC and r15AzPdHPC. In the case of r15AzPdHPC, SAXS measurements show a lamellar repeat distance of d = 47.6 Å (for T < Tm) and d = 66.1 Å (for T > Tm). The small d-value at temperatures below Tm could only be interpreted with a bilayer, where the lipid molecules are arranged in an interdigitated fashion (LβI phase). We further assume that the position of the νasN3 vibrational band can be used to decide whether an alkyl chain interdigitation exists or not. The position of this IR band is indicative of the hydration state of the azido function, i.e. the higher the frequency of νasN3 band the higher the hydration of the azido group. A value of νasN3 > 2100 cm–1, as found for P15AzPdPC and r15AzPdHPC at temperatures below Tm, could be linked to well-hydrated azido moieties, which is only possible if the terminal azido groups are arranged in the headgroup region of the lipid aggregates (interdigitated gel phase). A value of νasN3 < 2100 cm–1, as found for all azidolipids in the Lα phase, could be connected to less hydrated azido groups, which are now placed in the middle, more hydrophobic part of the lipid bilayer. Finally, we could show that the azide group can be activated by UV-irradiation leading to several decomposition products of the azidolipid. Nevertheless, at least on example of our novel azide-modified lipid is capable to study peptide/lipid interactions. Liposomes of a P10AzSPC:DMPC mixture including 2mol% of a transmembrane model peptide (KLAW23) were irradiated with 254 nm and a cross-link between peptide and azidolipid was identified by mass spectrometry. For future peptide/lipid or protein/lipid interaction studies using the cross-linking/MS approach, we would suggest the use of modified phospholipids that exhibit (i) two acyl chains (ester bonds) rather than an acyl and an alkyl chain (ether bond) and (ii) a photo-reactive group


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positioned in the middle of the hydrophobic part of the lipid molecule. The fact that a terminal modification of the alkyl chain and/or presence of an ether linkage in the sn-2 position of the glycerol trigger phase interdigitation, as found for r15AzPdHPC, should be kept in mind for further studies. This alkyl chain interdigitation could also cause problems when these modified membrane lipids are mixed with classical, bilayer-forming phosphatidylcholines and, consequently, further studies regarding the miscibility of both lipid components are mandatory. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website: Synthetic procedures, analytical data of prepared compounds, and further TEM, DSC, DLS, FTIR, SAXS, and MS measurements. AUTHOR INFORMATION Corresponding Authors *SD: E-mail: [email protected], phone: +49-345-5525196, fax: +49-3455527026. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was financially supported by a grant from the Deutsche Forschungsgemeinschaft (DFG) project DR 1024/1-1. S.D. thanks Dr. Bernd Rattay (Institute of Pharmacy, Martin Luther University Halle-Wittenberg) for providing azidostearic acids.


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Azide-Modified Membrane Lipids: Synthesis ... - ACS Publications

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