A Jardin
email: axel.jardin@cea.fr

D Mazon

M O'mullane

J Mlynar

V Loffelmann

M Imrisek

M Chernyshova

T Czarski

G Kasprowicz

A Wojenski

C Bourdelle

P Malard

Tomographic Capabilities of the new GEM based SXR diagnostic of WEST

Keywords: Soft X-Rays, Tomography, Tokamak plasmas, WEST

The tokamak WEST (Tungsten Environment in Steady-State Tokamak) will start operating by the end of 2016 as a test bed for the ITER divertor components in long pulse operation. In this context, radiative cooling of heavy impurities like tungsten (W) in the Soft Xray (SXR) range [0.1 keV; 20 keV] is a critical issue for the plasma core performances. Thus reliable tools are required to monitor the local impurity density and avoid W accumulation. The WEST SXR diagnostic will be equipped with two new GEM (Gas Electron Multiplier) based poloidal cameras allowing to perform 2D tomographic reconstructions in tunable energy bands. In this paper tomographic capabilities of the Minimum Fisher Information (MFI) algorithm developed for Tore Supra and upgraded for WEST are investigated, in particular through a set of emissivity phantoms and the standard WEST scenario including reconstruction errors, influence of noise as well as computational time.

Introduction

The tokamak WEST, for Tungsten (W) Environment in Steady-State Tokamak, will start operating by the end of 2016 as a test bed for the ITER divertor components in long pulse operation. In this context, radiative cooling of heavy impurities like W is a critical issue for the plasma core performances [START_REF] Pütterich | Calculation and experimental test of the cooling factor of tungsten[END_REF]. Thus reliable tools are required to monitor the local impurity density and avoid W accumulation. To do so, plasma tomography in the Soft X-ray (SXR) range 0.1 keV -20 keV is a useful tool to observe radial and poloidal distribution of impurities. Unfortunately, SXR tomography is an ill-posed inverse problem [START_REF] Anton | X-ray tomography on the TCV tokamak[END_REF] in tokamak plasmas due to the limited number of Lines of Sight (LoS) and presence of noise in the measurements. Thus a priori information is generally used as additional constraint imposed on the plasma SXR emissivity to obtain a physically meaningful solution. The SXR diagnostic of WEST will be based on Tikhonov regularization with a Minimum Fisher Information (MFI) method adapted from the former Tore Supra tokamak [START_REF] Mazon | Soft x-ray tomography for real-time applications: present status at Tore Supra and possible future developments[END_REF][START_REF] Jardin | Comparison of two regularization methods for SXR tomography at Tore Supra[END_REF]. Unfortunately, most of the former SXR LoS of Tore Supra are lost due to the WEST upper divertor. Thus the design of the SXR diagnostic has been refurbished [START_REF] Mazon | Design of soft X-ray tomographic system in WEST using GEM detectors[END_REF][START_REF] Mazon | GEM detectors for WEST and potential application for heavy impurity transport studies[END_REF] in order to be adapted to the WEST configuration, see Fig. 1. The WEST SXR diagnostic will be equipped with two new Gas Electron Multiplier (GEM) based poloidal cameras with a total of ~ 200 pixels, including 50 µm thick Beryllium windows that cut off SXR spectrum below 1 -2 keV. They will work in "photon-counting mode" [START_REF] Chernyshova | Conceptual design and development of GEM based detecting system for tomographic tungsten focused transport monitoring[END_REF][START_REF] Czarski | Serial data acquisition for the X-ray plasma diagnostics with selected GEM detector structures[END_REF] in contrast with former silicon barrier diodes working in "current mode". Thus the system will perform 2D tomographic reconstructions with spectral resolution in tunable energy bands. In this work tomographic capabilities of the MFI method initially developed for Tore Supra and upgraded for WEST are investigated. The paper is structured as follows. First, the main features of the tomography algorithm are described. Secondly, a set of emissivity phantoms is used to assess quality of the reconstruction, including computational time and the influence of experimental noise. Then, SXR tomographic reconstructions in a WEST scenario [START_REF] Bourdelle | WEST Physics Basis[END_REF] are presented. Finally, conclusions and perspectives are given for WEST operations.

SXR plasma tomography principles 1.1 Tikhonov regularization

In this paper, The SXR plasma emissivity is discretized in the poloidal cross-section of the diagnostic on a matrix of N p × N p = N p 2 square pixels. The tomographic reconstruction of the emissivity elements 𝜀 𝑗 from the line-integrated measurements m i is an inverse problem defined by the set of equations:

𝑚 𝑖 = ∑ 𝑇 𝑖𝑗 𝜀 𝑗 𝑗 + 𝑚 𝑖 ̃ (2.1)
where 𝑚 𝑖 ̃ denotes the experimental noise on the i-th channel and T ij are the response matrix coefficients. T ij corresponds to the i-th chord length in the j-th pixel in the Line of Sight (LoS) approximation, as defined by the pinhole-detector geometry. A simple least-square minimization of the residual 𝜒 2 (𝜺) = ‖𝒎 -𝑇. 𝜺‖ 2 would not be applicable here due to the ill-conditioned nature of the problem with quite few lines of sight and presence of experimental noise. In order to obtain a physically meaningful solution, a priori information is added through a regularization term 𝑅 = 𝜺 𝒕 𝐻𝜺 in the functional  = 𝜒 2 + 𝜆𝑅, which can be minimized using vector differentiation [START_REF] Anton | X-ray tomography on the TCV tokamak[END_REF]:

𝜺 𝟎 = 𝑎𝑟𝑔 𝑚𝑖𝑛 𝜺 (𝜒 2 (𝜺) + 𝜆 𝜺 𝒕 𝐻𝜺 ) = ( 𝑇. 𝑇 + 𝜆𝐻 𝑡 ) -1 . 𝑇 𝑡 . 𝒎 (2.2)
where the superscript t denotes the matrix transpose operation, H is the regularization operator and λ is the regularization parameter. Thus the solution of the tomographic reconstruction is a compromise between minimization of the residual and regularization of the solution, determined by the value of the free parameter λ. The choice of optimal λ values is discussed in section 1.3.

The Minimum Fisher Information method

The Minimum Fisher Information (MFI) method used here was first developed for Tore Supra [START_REF] Jardin | Comparison of two regularization methods for SXR tomography at Tore Supra[END_REF] and is adapted for the WEST configuration. The regularization operator H is defined as:

𝐻 = (1 -𝜏) 𝛻 ⫽ 𝑡 . 𝑊. 𝛻 ⫽ + 𝜏 𝛻 ⊥ 𝑡 . 𝑊. 𝛻 ⊥ (2.3)
where 𝜵 denotes a discrete approximation of the gradient and W is a ponderation matrix:

{ 𝑊 𝑖𝑗 = 1 𝜀 𝑖 𝛿 𝑖𝑗 , 𝜀 𝑖 > 𝜀 𝑚𝑖𝑛 𝑊 𝑖𝑗 = 1 𝜀 𝑚𝑖𝑛 𝛿 𝑖𝑗 , 𝜀 𝑖 < 𝜀 𝑚𝑖𝑛 (2.4)
with 𝛿 𝑖𝑗 the Kronecker's delta and ε min > 0 the lower bound used for the zero emissivity regions. The main upgrade from [START_REF] Jardin | Comparison of two regularization methods for SXR tomography at Tore Supra[END_REF] consists in the decomposition of 𝜵 in two components 𝛻 ⫽ and 𝛻 ⊥ respectively parallel and perpendicular to the magnetic flux surfaces. The associated factor of anisotropic smoothing τ is introduced with 0 ≤ 𝜏 ≤ 0.5 in order to account for the privileged parallel transport direction. The effect of τ on the reconstruction is presented in Fig. (3).

Role of the regularization parameter λ

As introduced in Eq. (2.2), the regularization parameter λ quantifies the balance between overfitting of measurements and oversmoothing of the solution. Several methods exist to determine an optimal λ value like e.g. the L-curve corner selection [START_REF] Hansen | Analysis of discrete ill-posed problems by means of the L-curve[END_REF]. In this work, λ is calculated with a dynamic regula-falsi method introduced in [START_REF] Loffelmann | Minimum Fisher Regularization adapted to real-time tomography[END_REF] such that the residual equals the experimental noise level. To do so, a normalized residual is defined as:

𝜒 𝑁 2 (𝜆) = 1 𝑁 𝑚 ∑ [ 𝑚 𝑖 -𝑚 𝑖 𝑟𝑒𝑐 (𝜆) 𝜎 𝑖 ] 2 𝑖 ≈ 1 (2.5)
where N m denotes the number of measurements, 𝜎 𝑖 2 is the variance of the noise level on the i-th channel and 𝒎 𝒓𝒆𝒄 = 𝑇. 𝜺 𝒓𝒆𝒄 are the retrofit measurements. The benefit of this method is that only structures of emissivity above the noise level survive the reconstruction process.

Tomographic tests

Tomographic tests prior to experimental use are essential to assess the performances and limits of the method. In this paper, the quality of the tomographic reconstruction will be assessed thanks to phantom models of emissivity and using the figure of merits RMS em , which represents the root mean square of the reconstruction error:

𝑅𝑀𝑆 𝑒𝑚 = √ 1 𝑁 𝑝 2 ∑ (𝜀 𝑖 𝑚𝑜𝑑 -𝜀 𝑖 𝑟𝑒𝑐 ) 2 𝑖 (3.1)
where ε i mod denotes the emissivity in the i-th element of the model, ε i rec is the emissivity in the ith element of the reconstructed tomogram, and N p ² is the total number of pixels.

Phantom models of emissivity

A set of 4 phantom models (Gaussian, hollow, banana and peaked) is used to mimic various experimental emissivity profiles such as impurity poloidal asymmetries, central accumulation [START_REF] Casson | Theoretical description of heavy impurity transport and its application to the modelling of tungsten in JET and ASDEX upgrade[END_REF] or hollow shape after a sawtooth crash. Emissivity phantoms give the advantage of knowing the initial emissivity distribution in comparison with experimental reconstructions. One example of each phantom model is presented in Fig. 2.1(a-d). Synthetic SXR measurements are derived from these emissivity phantoms considering 2% of Gaussian noise level, see Fig. 2.2(a-d), and used as input for the tomography. Resulting tomograms are presented in Fig. 2.3(a-d), with an anisotropy factor 𝜏 = 10 -2 . Effect of τ value on the reconstruction is discussed in next section 3.2.

Effect of the anisotropy factor τ

As introduced in Eq. (2.3), the anisotropy factor 0 ≤ 𝜏 ≤ 0.5 gives a privileged parallel transport direction. Given the magnetic equilibrium is well determined, such anisotropic regularization is better adapted to the plasma configuration and leads to an increase of the quality of the reconstruction. This is illustrated in Fig. 3 with the banana phantom model, for τ values from τ = 0.01 (anisotropic smoothing) to τ = 0.5 (isotropic smoothing) and 2% of noise. The disadvantage of introducing the magnetic equilibrium in the reconstruction is the associated increase of the computational time in the regularization process as presented in Fig. 5.

The optimized regularization parameter λ

The optimized regularization parameter λ is determined as described in section 1.3, such that the normalized residual 𝜒 𝑁 2 (𝜆) ≈ 1 with a tolerance of 5% on convergence of 𝜒 𝑁 2 . This method allows finding a regularization parameter quite close to the best solution in terms of RMS em minimization, as illustrated in Fig. 4 with the Gaussian phantom model and for different noise levels from 1% to 10%.

where the W fractional abundances f W,q and line radiation (𝐾 𝑊,𝑞 𝜂,𝑏𝑏 ) are computed using respectively ionization-recombination coefficients and photon emissivity coefficients from the OPEN-ADAS database. Analytic formulae from [START_REF] Kunze | Introduction to plasma spectroscopy[END_REF] are used to compute Bremsstrahlung (𝐾 𝑊,𝑞 𝜂,𝑓𝑓 , 𝐾 𝐻 𝜂,𝑓𝑓 ) and radiative recombination (𝐾 𝑊,𝑞 𝜂,𝑓𝑏 ) contributions. Deuterium is fully ionized where the SXR emissivity is significant inside the separatrix. Dielectronic recombinations are not included here as well as transport effect on 𝐿 𝑊 𝜂 [START_REF] Vezinet | Impurity density derivation from bandpass soft x-ray tomography: applicability, perspectives and limitations[END_REF] for simplicity. W line radiation is the dominant contribution overall.

Associated SXR tomography

The tomographic algorithm is then tested on the W dominated emissivity, as well as on the Deuterium emissivity only to account for a pure plasma case. The resulting reconstruction error maps are presented on Fig. 7.1-2(c) and show that the tomography is valid for both emissivity reconstruction due to impurities and main ions in a realistic case with < 10% reconstruction error in the region of interest where 𝑇 𝑒 ≳ 1 -2 𝑘𝑒𝑉.

Figure 1 .

 1 Figure 1. WEST plasma coverage of the (a) former Tore Supra diodes (b) new GEM based SXR cameras. The red part of the former viewing angle is lost due to the WEST upper divertor.

Figure 2 .

 2 Figure 2. Phantoms of emissivity with 1(a-d) models, 2(a-d) associated measurements, 3(a-d) reconstructed profiles for (a) Gaussian, (b) Hollow, (c) Banana, and (d) Peaked shapes.

Figure 3 .

 3 Figure 3. Effect of anisotropy factor τ on the reconstruction of the Banana phantom with (a) τ = 0.01 (b) τ = 0.10, (c) τ = 0.25 and (d) τ = 0.50.

Figure 4 .

 4 Figure 4. λ optimization with the Gaussian phantom and different noise levels with (a) residual X N ², regularization term R and (b) reconstruction error RMS em versus λ. Vertical dashed lines correspond to the solution of Eq. (2.5).

Figure 6 .

 6 Figure 6. (a) Temperature and density profiles of the WEST standard scenario (b) filtered cooling factors computed in the SXR range and (c) extrapolated SXR emissivity in the poloidal cross-section of the diagnostic.

Figure 7 .

 7 Figure 7. Tomographic inversion of 1(a-c) Tungsten and 2(a-c) Deuterium emissivity calculated in section 4.1, with (a) SXR emissivity, (b) associated measurements assuming 2% Gaussian noise, and (c) 2D reconstruction error map.

  where 𝐿 𝑊 𝜂 and 𝐿 𝐻 𝜂 denote the filtered cooling factors of tungsten and deuterium. They are calculated according to Eq. (4.2):

	𝐿 𝑊 𝜂 = ∑ 𝑓 𝑊,𝑞 . (𝐾 𝑊,𝑞 𝜂,𝑏𝑏 + 𝐾 𝑊,𝑞 𝜂,𝑓𝑏 + 𝐾 𝑊,𝑞 𝜂,𝑓𝑓 ) 𝑞 { 𝐿 𝐻 𝜂 = 𝐾 𝐻 𝜂,𝑓𝑓

Choice of the plasma spatial resolution

In this section, we discuss the optimal choice of the plasma spatial resolution N p x N p . First, the Tikhonov regularization is by nature only adapted to under-determined problems. As a result we should use a number of pixels N p ² > N LoS ~ 200, thus N p ≥ 15. Then a set of 25 phantoms of the 4 models with different sizes as introduced in section 3.1 is used to assess the quality of the reconstruction against the grid size, as presented in Fig. 5(a) for 3 different Gaussian noise levels of 2, 5 and 10% in measurements. The benefit and computational cost of anisotropic regularization is highlighted in Fig 5(b-c). Fig. 5(a) shows a global increase of the quality of the reconstruction with N p . On the other hand, the computational time is an increasing exponential function of N p . N p > 50 is time consuming (> 1s) and does not lead to a significantly better reconstructions while N p < 25 leads to quite poor reconstructions without any benefit in computational time (~10-50ms). As a result, Np ≥ 25 could be used for real-time or automatic post-processing analysis with dedicated fast computing units like e.g. FPGA, while Np ≥ 50 could be used for finer analysis.

Application to the standard WEST scenario 4.1 A scenario from WEST physics basis

In this section, we apply the minimum Fisher method on SXR emissivity profiles extrapolated from the standard WEST physics basis [START_REF] Bourdelle | WEST Physics Basis[END_REF] H-mode scenario with P = 12 MW of Radio Frequency heating power and I p = 0.6 MA of plasma current. Electron density n e and temperature T e profiles are shown in Fig. 6(a). In this scenario, the tungsten density profile n W is assumed to be flat with n W ~ 10 16 m -3 and its concentration c W = 5.10 -4 at the separatrix. For simplicity, the spectral response of the GEM detector will be assumed here to be η = 1 in the SXR range ℎ𝜈 ∊ [0.1 -20 keV], including the 50µm thick Beryllium filter that cuts off energies below 1 -2 keV. The GEM spectral response characterization is the subject of ongoing parallel work [START_REF] Jardin | Synthetic SXR diagnostic using GEM detectors on WEST[END_REF]. The SXR emissivity is calculated as follows, considering only W impurities:

[𝐿 𝐻 𝜂 (𝑇 𝑒 ) + 𝑐 𝑊 . 𝐿 𝑊 𝜂 (𝑇 𝑒 )] (4.1)

Summary

In this paper, the SXR tomography originally used for Tore Supra has been adapted and upgraded for the WEST configuration. In particular, the regularization parameter λ is now routinely optimized using the 𝜒 𝑁 2 (𝜆) ≈ 1 method, and the magnetic equilibrium can be included with the anisotropy factor 0 ≤ 𝜏 ≤ 0.5 to improve the reconstruction, but with an increase of the associated computational cost. Tomographic capabilities for the new SXR diagnostic of WEST have been well-tested using a set of phantom models of emissivity and including influence of noise in the measurements. Finally, the tomographic reconstruction has been validated for the standard scenario from WEST physics basis including the presence of W impurities.


Julie Barbeau

Loïc Lemiègre

Allan Quelen

Virginie Malard

Haifei Gao

Cristine Gonçalves

Mathieu Berchel

Paul-Alain Jaffrès

Chantal Pichon

Patrick Midoux
email: patrick.midoux@cnrs-orleans.fr

Thierry Benvegnu
email: thierry.benvegnu@ensc-rennes.fr

Synthesis of a Trimannosylated-Equipped Archaeal Diether Lipid for the Development of Novel Glycoliposomes

Keywords: Archaeal trimannosyl lipid, liposomes, dendritic cell binding, mannose receptor

An archaeal diether lipid possessing a tri-antenna of α-D-mannopyranoside linked via an oligoethylene spacer to a (2S)-2-(phytanyloxy)-3-(hexadecyloxy)propanoic acid backbone (TriMan-Diether) was designed and synthesized. This new mannosylated lipid inserted in liposomes would show both DC-targeting and adjuvant properties thanks to the TriMan structure and the diether tail part, respectively.

Introduction

Ethers of lipids or archaeolipids naturally present in the membranes of Archaea are characterized by the presence of ether linkages between a glycerol and phytanyl aliphatic chains. 1 They include those made with synthetically derived lipids that have the unique structural characteristics of natural lipids, for example: (1) regularly branched phytanyl chains linked through ether bonds to sn-2,3 carbons of glycerol; (2) monopolar archaeol (diether) lipids and/or bipolar tetraether lipids linked to two glycerol entities in an antiparallel manner (caldarchaeol) or in a parallel manner (isochaldarcheol); (3) neutral, negatively charged or zwitterionic unusual polar heads.

Glycolipid analogues with archaeal tetraether macrocycles bearing monovalent or trivalent lactose or mannose head groups at one or the two terminal ends were synthesized. [2][3][4] Such glycolipids incorporated into liposomes allowed specific interactions with cell membrane receptors. Archaeal lipids exhibit adjuvant properties independent on Toll-like receptors activation which can be benefit to boost the immune response. [START_REF] Benvegnu | Plusquellec in Carbohydrate Chemistry, Chemical and Biological Approaches[END_REF]3,[START_REF] Sprott | [END_REF] Liposomes comprising these lipids, also called archaeosomes, possess an intrinsic adjuvant effect and mannosylated archaeal liposomes containing ovalbumin were able to induce anti-ovalbumin CD8+ T cell response in mice. [START_REF] Sprott | [END_REF] The preparation of semi-synthetic compounds comprising ether lipids extracted from Archaea requires the establishment of archaea cultures, methods of extraction and isolation difficult to achieve at large scale. It is therefore necessary to synthetize ethertype compounds linked to a sugar group having the advantageous properties of lipids from Archaea.

Liposomes are very effective carriers to transport molecules of interest (drugs and nucleic acids) within cells. For better efficacy, liposomes must be recognized by the target cells, especially those used for vaccine proposals must be recognized and internalized by antigenpresenting cells notably dendritic cells (DCs) to deliver antigen. [START_REF] Benvegnu | Plusquellec in Carbohydrate Chemistry, Chemical and Biological Approaches[END_REF] The targeting of DCs can be 7 Carbohydrate-based targeting of the above mentioned receptors is an obvious manner to enhance specificity and uptake of liposomes. 8 For this purpose, mono-or disaccharides are usually used. Indeed, the synthesis of high mannose structures and complex oligosaccharides in general that exhibit high affinity for membrane lectins is difficult, expensive and yields are weak. The binding affinity of monovalent carbohydrate ligands such as mannose typically is weak (K ≈ 10 -3 M -10 -4 M). It can be increased by 1 or 2 orders of magnitude by coupling several monosaccharide units per protein, polymer or liposome. The multivalent decoration with monosaccharide units of low affinity increases the apparent affinity of the monosaccharide for its receptor. 9 Natural oligosaccharides form antennary structures that allow higher affinity than monosaccharides. When compared, mono-, di-, and tetra-antennary mannosylated lipid derivatives revealed that liposomes containing multibranched mannosylated lipids displayed higher binding affinity for MR compared to monomannosylated analogues. 10 Di-antennary mannosylated lipids were as efficient as the tetra-antennary lipids suggesting that the di-antennary structure was sufficient. The present work relates for the first time to the synthesis of an archaeal-type diether lipid 1 (TriMan-Diether) derived from (2S)-2-(phytanyloxy)-3-(hexadecyloxy)propanoic acid linked via an oligo(ethylene glycol) spacer to a tri-antenna of alpha D-mannopyranoside. This lipid was designed to bring a sufficient flexibility to the head-group orientation with the objective of making the accessibility of mannose moieties optimal. The presence of both linear and branched chains represents an appropriate compromise between membrane fluidity and rigidity. A relatively short oligo(ethylene glycol) spacer-arm was selected to join the hydrophilic and the hydrophobic regions. This linker was considered to be sufficiently long to authorize a high degree of carbohydrate ligand exposure at the liposome surface.

The trimannosylated ligand is based on a pentaerythritol skeleton as an anchoring point for the construction of the tri-antenna structure. This tetraol was used for grafting three dipropylene glycol spacers followed by the fixation of the mannosyl units at their terminal ends.

The length of the di-propylene glycol spacer is a key parameter for ensuring an optimized ligand-cell receptor binding since it will impact the distance (d) between the mannose residues. The preferred conformation of the tri-antenna structure for carbohydrate-cell recognition was found to require generally a distance (d) higher than 2 Å, 11 this condition being met in the case of TriMan-Diether lipid 1.

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 5

Results and discussion

Synthesis of Trimannosyl diether lipid

The strategic plan for the synthesis of TriMan-Diether 1 shown in Figure 1 involved a suitable synthesis of trimannosyl cluster 2 and its subsequent grafting onto amino PEGylated diether (H 2 N-PEG 350 -Diether) 3 (Scheme 2). This convergent synthetic pathway is advantageous for the preparation of various triglycosylated lipids as it allows to module easily the length of the oligo(ethylene) spacer. Indeed the introduction of the tri-antenna moiety into the lipid backbone was performed in the final steps of the synthesis.

The construction of trimannosyl cluster 2 possessing three di-propylene glycol spacers was achieved in a seven-step procedure (Scheme 1) from commercially available pentaerythritol allyl ether 4 (technical grade, 70%) after a purification step by silica gel chromatography.

Quantitative benzylation of 4 was performed in DMF using 1.5 equiv of benzyl bromide in the presence of NaH (0.3 equiv). Hydroboration reactions with 9-BBN (9borabicyclo[3.3.1]nonane) followed by the in situ conversion to the corresponding triol under oxidative/basic conditions provided triol 6 (83%). As expected, hydroboration of the allyl groups in 5 proceeded strictly regioselectively with the sterically demanding 9-BBN reagent.

Triallylation of 6 with allyl bromide gave compound 7 in good yield (78%). The same hydroboration/oxidation reaction using the 9-BBN/H 2 O 2 , NaOH conditions converted totally the triallylated derivative 7 into the corresponding triol 8. Subsequent mannosylation of 8 was carried out using 2,3,4,6-tetra-O-benzoyl-α-mannosyl trichloroacetimidate 9 as the glycosyl donor in the presence of TMS triflate as the catalyst. As reported in the literature, 12 the use of benzoylated trichloroacetimidate instead of the corresponding acetylated mannosyl donor completely avoided the orthoester formation. The mannosylation reaction was performed using various amounts of glycosyl donor (5 to 20 equiv in mol). The best results were obtained when 10 equiv of mannosyl trichloroacetimidate 9 was used (82% yield) that (1/1) gave the totally unprotected TriMan-Diether 1 in 97% yield. This trimannosylated diether lipid was easily and fully characterized by NMR ( 1 H and 13 C) and high-resolution mass spectrometry.

Binding of TriMan-liposomes on dendritic cells

Then we tested whether TriMan-Diether 1 can trigger specific binding of liposomes on dendritic cells via the mannose receptor. DC2.4 cells are mouse dendritic cells that express the mannose receptor. 14 TriMan-Diether (5%) was incorporated in cationic liposomes made with three lipophosphoramidates with a polar head containing an imidazolium, histamine and fluorescein group in the percentage of 47.25%, 47.25% and 0.5%, respectively (see experimental part). Those TriMan-liposomes exhibited a size of 213 ± 16 nm and a positive charge of 58 ± 5 mV. When DC2.4 cells were incubated at 4°C with various concentrations of TriMan-liposomes, the fluorescence intensity of the cells (MFI) increased with the liposomes concentration showing the binding of those liposomes on DC2.4 cells (Figure 2). When the cells were preincubated with mannose before incubation with TriMan-liposomes in the presence of mannose, the MFI decreased. The inhibition was 70% in the presence of 500 mM mannose in

Conclusion

We have developed a synthetic route for the preparation of a novel archaeal diether lipid functionalized with a mannose triantennary cluster (TriMan-Diether) designed as a multivalent ligand for the interaction with dendritic cells through their mannose-specific receptors. The convergent synthetic pathways were based on a peptidic-type coupling reaction between a PEGylated diether amine and a trimannosyl cluster possessing a carboxylic acid function. When 5% of this TriMan-Diether is inserted in liposomes it promotes a strong

Experimental

General methods.

Commercially available chemicals were used without further purification (except alcohol 4 which was purified by flash chromatography) and solvents were carefully dried and distilled prior to use. Unless otherwise noted, non-aqueous reactions were carried out under a nitrogen atmosphere. Analytical TLC was performed on Merck 60 F254 silica gel non-activated plates.

A solution of 5% H 2 SO 4 in EtOH was used to develop the plates. Merck60H (5-40µm) silica gel was used for flash chromatography. 1 H and 13 C NMR spectra were recorded at 400 and 100 MHz, respectively, on a Bruker Avance III. MS spectra were recorded on a Waters Micromass Q-TOF equipped with a Z-spray ion source or on a Shimadzu LCMS 2020 and optical rotation were recorded on a Perkin-Elmer 341 polarimeter.

Synthesis of TriMan-Diether (1).

Triallyl-benzyl-pentaerythritol (5).

To a suspension of NaH (2.6 g, 90 mmol) in anhydrous DMF (100 mL), alcohol 4 (7.70 g, 300 mmol) was added at 0°C and the reaction mixture was stirred during 2h at the same temperature. Benzyl bromide (7.7 g, 450 mmol) was added dropwise at 0°C and the reaction mixture was stirred during 17h at room temperature. MeOH (6 mL) was added dropwise at 0°C, solvents were removed under reduced pressure and the crude material was dissolved in

Tri(3-hydroxypropyl)-benzyl-pentaerythritol (6).

To a solution of 5 (10 g, 28.8 mmol) in dry dioxane (50 mL) was added at 0 °C 9borabicylco[3.3.1]nonane (0.5 M in THF; 519 mL, 0.259 mol) and the reaction mixture was stirred at room temperature for 24 h. An aqueous solution of sodium hydroxide (577 mL, 3 M) and a solution of H 2 O 2 (115 mL, 30%) were added at 0 °C and the resulting mixture was stirred at room temperature for further 12 h. The reaction mixture was extracted with EtOAc and the combined organic phases were dried (MgSO 4 ) and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (EtOAc/Cyclohexane/MeOH: 10:5:1) to yield 6 (9.57 g, 83%) as a colourless oil.

Tri(3-allyloxypropyl)-benzyl-pentaerythritol (7).

Triol 6 (7.8 g, 19.4 mmol) was dissolved in anhydrous DMF (100 mL) and this solution was added at 0°C dropwise to a suspension of KH (3.9 g, 97 mmol) in DMF (100 mL). The reaction mixture was stirred during 10 min before the slow addition of allyl bromide (8.35 05, 45.48, 67.47, 68.22, 69.60, 69.68, 71.86, 73.26, 116.68, 127.22, 127.25, 128.19, 134.99, 139.02

Tri(3-hydroxypropyloxypropyl)-benzyl-pentaerythritol (8).

To a solution of 7 (7.9 g, 15.0 mmol) in dry dioxane (50 mL) was added at 0 °C 9borabicylco[3.3.1]nonane (0.5 M in THF; 272 mL, 0.136 mol) and the reaction mixture was stirred at room temperature for 24 h. An aqueous solution of sodium hydroxide (500 mL, 3 M) and a solution of H 2 O 2 (60 mL, 30%) were added at 0 °C and the resulting mixture was stirred at room temperature for further 12 h. The reaction mixture was extracted with EtOAc and the combined organic phases were dried (MgSO 4 ) and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (EtOAc/Cyclohexane/MeOH: 10:5:1) to yield 8 (8.51 g, 99%) as a colourless oil. Rf = 0.1 (EtOAc/Cyclohexane/MeOH: 10:5:1); 1 H NMR (CDCl 3 , 400 MHz) δ (ppm) 1.79 (12H, p, J = 7.24-7.35 (7H, m, Har). 13 C NMR (CDCl3, 400 MHz) δ (ppm) 29.96, 32.01, 45.47, 61.93, 68.09, 68.29, 69.52, 69.72, 70.10, 73.26, 127.24, 127.28, 128.20, 138.96.

TriMan-CH 2 OBn (10).

To a mixture of the mannosyl donnor 9 (10 g, 13.4 mmol) and triol 8 (1.03 g, 1.79 mmol) in dry dichloromethane (5 mL) was added a solution of trimethylsilyl trifluoromethane sulfonate (640 µL, 0.179 mmol; 5% in DCM) and the reaction mixture was stirred at room temperature for 12 h. NaHCO 3 (2 g) was added and the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (Cyclohexane/EtOAc: 6:4) to yield 10 (3.44 g, 82%) as a yellowish solid. Rf = 0. 73, 30.04, 45.46, 62.84, 65.58, 66.94, 67.30, 68.22, 68.31, 68.77, 69.71, 70.15, 70.53, 73.24, 97.62, 127.20, 127.23, 128.18, 128.28, 128.42, 128.55, 128.98, 129.09, 129.35, 129.72, 129.77, 129.83, 129.87, 133.02, 133.13, 133.40, 139.05, 165.39, 165.45, 165.48, 166.13.

TriMan-COOH (2).

Debenzylation 66.05, 66.94, 67.37, 68.11, 68.67, 68.78, 70.14, 70.53, 71.46, 97.61, 128.28, 128.42, 128.56, 128.99, 129.09, 129.35, 129.72, 129.78, 129.83, 129.87, 133.03, 133.14, 133.41, 165.39, 165.44, 165.49, 166.13 Oxidation. Previous alcohol (614 mg, 0.275 mmol) was dissolved in EtOAc (10 mL).

Aqueous solutions of KBr (56 µL, 0.5 M, 0.028 mmol) and TEMPO (15 mg, 0.096 mmol)

were added, followed by the addition of NaOCl (1.2 mL, 0.84 mmol) at 0°C. After 3h at room temperature, the reaction mixture was acidified with 5% HCl until pH 3 before the addition of NaO 2 Cl (560 µL, 1.68 mmol). The reaction mixture was stirred overnight at room temperature and then was extracted with EtOAc. The combined organic phases were dried (MgSO 4 ) and concentrated under reduced pressure to give the carboxylic acid 2 (614 mg, 99%). Rf = 0. 60, 29.77, 52.86, 60.34, 62.79, 65.46, 66.85, 67.31, 67.87, 68.59, 68.73, 69.07, 70.13, 70.49, 97.55, 128.24, 128.38, 128.52, 128.92, 129.00, 129.27, 129.68, 129.73, 129.78, 133.02, 133.12, 133.37, 133.39, 165.39, 165.42, 165.50, 166.15, 173.45 13 C NMR (CDCl 3 , 100 MHz) δ (ppm) 14.10, 19.58, 19.65, 19.72, 22.60, 22.69, 22.66, 24.36, 24.47, 24.78, 26.03, 27.94, 29.84, 32.76, 32.78, 29.34, 29.46, 29.52, 29.63, 29.68, 31.89, 37.26, 37.36, 37.39, 37.45, 37.50, 39.32, 38.67, 50.63, 69.72, 69.83, 70.3-70.7, 71.47, 71.68, 80.48, 170.57 (ppm) 14.10, 19.58, 19.65, 19.72, 22.61, 22.71, 22.66, 24.34, 24.45, 24.76, 26.00, 27.95, 29.77, 29.85, 32.79, 29.32, 29.45, 29.54, 20.61, 29.66, 31.88, 37.23, 37.34, 37.37, 37.43, 39.31, 38.70, 40.45, 66.89, 69.72-70.53, 71.45, 71.70, 80.47, 170.63 12, 19.62 ,19.68, 19.75, 22.63, 22.69, 22.72-29.56, 29.65, 29.70, 29.74, 29.84, 29.91, 31.10, 31.43, 31.63, 31.92, 32.80, 53.03, 60.40, 62.85, 65.53, 66.91, 67.36, 67.92, 68.61, 68.78, 68.95, 70.15, 70.55, 80.53, 97.64, 128.30, 128.44, 128.57, 129.00, 129.10, 129.35, 129.73, 129.79, 129.84, 129.88, 133.05, 133.16, 133.43, 165.4, 165.50, 166.14, 170.69.

TriMan-PEG 350 -diether (1).

Freshly prepared MeONa solution in MeOH (5.3M, 48.8 µL, 0.258 mmol) was added to a solution of 14 (435 mg, 0.139 mmol) in CH 2 Cl 2 /MeOH (100 mL, 1:1) and the reaction mixture was stirred overnight. Amberlite IR-120 H + was added and filtered off. The solvent was removed under reduced pressure to furnish 1 (255 mg, 97% 13.66, 19.26, 19.30, 19.33, 19.37, 19.40, 19.44, 22.22, 22.31, 22.50, 24.24, 24.31, 24.65, 25.92, 27.83, 29.21, 29.32, 29.39; 29.48, 29.50, 29.53, 29.64, 29.68, 29.72, 31.27, 31.78, 32.62, 32.64, 32.67, 36.59, 36.65, 36.72, 37.13, 37.23, 37.25, 37.30, 37.36, 37.39, 38.67, 38.95, 39.25, 52.25, 61.45, 64.22, 67.06, 67.52, 67.69, 68.41, 69.43, 69.48, 69.53, 69.61, 70.09, 70.11, 70.23, 70.33, 70.35, 70.37, 70.70, 71.11, 71.15, 71.33, 71.65, 72.56, 80.34, 80.29, 100.08, 171.49, 173.69 The amount of imidazole in liposomes was determined by the Pauly colorimetric assay. 18 The amount of TriMan-Diether incorporated in liposomes was determined using the colorimetric resorcinol/sulfuric assay and a standard curve with mannose was used to determine the amount of TriMan-Diether. 19 The size and the ζ potential of liposomes were measured by using SZ-100 Analyser (Horiba Scientific, les Ulis, France).

  of liposomes with ligands that are selectively recognized by surface receptors of DCs and permit their endocytosis. DCs express several sugar receptors called membrane lectins and most of them are involved in antigen capture and presentation. The Mannose receptor (MR) (CD206) recognizes mannose-and fucose-terminated glycans, Man9-, 3-sulfo-LewisA, tri-GlcNAc, and palmitoyl-phosphatidylinositol dimannoside (PIMs) and induces clathrin-depend endocytosis. Other receptors including the DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), dectin-1, dectin-2 and Langerin bind also high-mannose-(Man9GlcNAc2-, Man4-, Manα1-3(Manα1-6)Manα1-), fucose-(αfucose-1-4GlcNAc-, Lacto-N-fucopentaose III-) and N-acetylglucosamine (GlcNAc2Man3-)terminated glycans as well as Lewis-type blood antigens (Leb or Lex trisaccharide), βglucans.

Figure 1 .

 1 Figure 1. Structure of the trimannosylated diether lipid (TriMan-Diether) 1.

M

  Scheme 1. Synthesis of the trimannosyl cluster 2.

  64 µM TriMan-liposomes indicating that the binding was mediated by the mannose receptor. The inhibition level varied with the mannose concentration (Figure2insert). The high concentration of free mannose required for a significant inhibition was indicative of a good affinity of the TriMan structure for the mannose receptor.

Figure 2 .

 2 Figure 2. Binding of TriMan-liposomes onto DC2.4 cells and their inhibition by

  dendritic cells mediated via the mannose receptor. This new mannosylated lipid that would show both DC-targeting and adjuvant properties thanks to the TriMan structure and the Diether tail part respectively can be benefit to boost the immune response.

  washed twice with water and brine. The combined organic phases were dried (MgSO 4 ) and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (Cyclohexane/EtOAc: 9:1) to yield 5 (10.45 g, quant.) as a colourless oil. 1 H NMR (CDCl 3 , 400 MHz) δ (ppm) 3.50 (6H, s, C-CH 2 -O), 3.56 (2H, s, C-CH 2 -O), 3.95 (6H, dt, J = 5.3, 1.5 Hz, CH 2 -CH=CH 2 ), 4.65 (2H, s, Ph-CH 2 ), 5.12 (3H, dq, J = 10.4, 1.7 Hz, CH 2 =), 5.24 (3H, dq, J = 17.2, 1.7 Hz, CH 2 =),5.87 (3H, m, CH=), 7.24-7.35 (7H, m, Har). These analytical data are in total agreement with previously published data.3

  1 H NMR (CDCl 3 , 400 MHz) δ (ppm) 1.75 (6H, p, J = 5.5, 5.3 Hz, CH 2 -CH 2 -CH 2 ), 3.28 (3H, broad s, OH), 3.42 (6H, s, C-CH 2 -O), 3.44 (2H, s, C-CH 2 -O), 3.56 (6H, t, J = 5.5 Hz, O-CH 2 -CH 2 ), 3.70 (6H, t, J = 5.3 Hz, CH 2 -OH), 4.62 (2H, s, Ph-CH 2 ), 7.24-7.35 (7H, m, Har). These analytical data are in total agreement with previously published data.

3

 3

  mmol) at the same temperature. The reaction mixture was stirred during 24h at room temperature and water (100 mL) was added carefully. The product was extracted three times with Et 2 O and the combined organic phases were washed with brine, dried (MgSO 4 ) and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (Cyclohexana/EtOAc: 85:15) to yield 7 (8.63 g, 78%) as a colourless oil. Rf = 0.15 (Cyclohexane/EtOAC: 9:1); 1 H NMR (CDCl 3 , 400 MHz) δ (ppm) 1.81 (6H, q, J = 6.4 Hz,CH 2 -CH 2 -CH 2 ), 3.40 (6H, s, C-CH 2 ), 3.44-3.50 (14H, m, CH 2 -O), 3.95 (6H, ddd, J = 5.7, 1.5, CH 2 -CH=), 4.48 (2H, s, Ph-CH 2 ),5.15 (3H, ddd, J = 10.3, 3.3, 1.5 Hz, CH 2 =CH),5.26 (3H, ddd, J = 17.3, 3.5, 1.5 Hz, CH 2 =CH),5.90 (3H, ddt, J = 10.3, 6,8, 5.7 Hz, CH=CH 2 ), 7.24-7.35 (7H, m, Har);13 C NMR (CDCl 3 , 100 MHz) δ (ppm) 30.

5. 7

 7 Hz, CH 2 -CH 2 -CH 2 ), 2.62 (3H, broad s, OH), 3.42 (6H, s, C-CH 2 ), 3.49-3.43 (14H, m, CH 2 -O), 3.55 (6H, t, J = 5.7 Hz, CH 2 -O), 3.73 (6H, t, J = 5.4 Hz, CH 2 -OH), 4.64 (2H, s, Ph-

3 (

 3 Cyclohexane/EtOAc: 6:4); m.p. = 61°C; [α] 20 D = -32.4 (c 0.9 CHCl 3 ); 1 H NMR (CDCl 3 , 400 MHz) δ (ppm) 1.77-1.84 (6H, m, J = 6.4 Hz, CH 2 -CH 2 -CH 2 ), 1.91-2.00 (6H, m, CH 2 -CH 2 -CH 2 ), 3.41-3.57 (26H, m, CH 2 -O), 3.57-3.68 (3H, m, CH 2 -O), 3.90-3.96 (3H, m, CH 2 -O), 4.40-4.50 (3H, m, H5), 4.46-4.50 (5H, m, Ph-CH 2 , H6b), 5.08 (3H, d, J = 2.0 Hz, H1), 5.70 (3H, dd, J = 3.2, 1.8 Hz, H2), 5.91 (3H, dd, J = 10.1, 3.3 Hz, H3), 6.11 (3H, t, J = 10.1 Hz, H4), 7.24-8.11 (65H, m, Har); 13 C NMR (CDCl 3 , 100 MHz) δ (ppm) 29.

2 (

 2 ) δ (ppm) 1.83 (6H, p, J = 6.3 Hz, CH 2 -CH 2 -CH 2 ), 1.96 (6H, p, J = 6.2 Hz, CH 2 -CH 2 -CH 2 ), 3.51 (6H, t, J = 6.3 Hz, CH 2 -O), 3.53 (6H, t, J = 6.1 Hz, CH 2 -O), 3.56 (6H, m, CH 2 -O), 3.64 (6H, s, C-CH 2 ), 3.66 (3H, td, J = 9.7, 6.4 Hz, CH 2 -O), 3.94 (3H, td, J = 9.7, , CH 2 -O), 4.47-4.41 (3H, m, H5), 4.49 (3H. dd. J = 12.0. 4.2 Hz, H6b), 4.71 (3H, dd, J = 12.0, 2.4 Hz. H6a), 5.10 (3H, d, J = 1.7 Hz, H1), 5.70 (3H, dd, J = 3.3, 1.7 Hz, H2),5.92 (3H, dd, J = 10.1, 3.3 Hz, H3), 6.13 (3H, t, J = 10.0 Hz, H4), 7.24-7.59 (36H, m, Har), 7.82- 8.11 (24H, m, Har);13 C NMR (CDCl 3 , 100 MHz) δ (ppm) 29.

  were prepared by mixing at 5.4 mM in ethanol the O,O-dioleyl-N-[3N-(N- methylimidazolium iodide)propylene] Phosphoramidate,15 the O,O-dioleyl-N-histamine Phosphoramidate,16 the ({Dioleyloxyphosphoryl)amino} methyl)-triazolyl}-PEGfluorescein,17 and TriMan-Diether in the percentage of 47.25%, 47.25%, 0.5% and 5%, respectively. Ethanol solution was then evaporated until formation of a film. The film was hydrated for 12 h at 4°C in 1 mL of 10 mM HEPES buffer, pH 7.4, vortexed and then the suspension was sonicated for 15 min at 37 kHz using a Bioblock ultrasonic bath (Bioblock Scientific, Illkirch, France). Liposomes were dialysed (Dialysis Tubing Cellulose membrane; MWCO: 12.4 kDa; size: 33 × 21 mm, Sigma) at 4°C for 6 h and then overnight against 500 mL 10 mM HEPES buffer, pH 7.4. The lipid concentration was determined with Nile Red.

4. 4 .

 4 Cells and cell culture. The murine DC line (DC2.4 cells) was kindly given by Dr P. Jeannin and Dr Yves Delneste (INSERM UMR 892 Nantes-Angers, France). 20 DC2.4 and HeLa cells (CCL2, ATCC, Rockville MD, USA) were grown at 37°C in a humidified atmosphere containing 5% CO 2 in DMEM (DC2.4) or in MEM (HeLa) supplemented with 10% fetal calf serum (PAA Laboratories, Les Mureaux, France), 100 Units/mL of penicillin and 100 µg/ mL of streptomycin (Fischer Bioblock, Illkirch, France).

  were harvested with trypsin, washed first with complete medium and then with cold phosphate-buffered saline (PBS). Cells (2 x 10 5 in 0.5 ml) in suspension were incubated for 2h at 4°C with the fluorescent liposomes at the indicated final concentration. For binding inhibition, cells in suspension were pre-incubated for 30 min at 20°C with various mannose concentrations. Then, cells were put on ice and the fluorescent liposomes were added at the indicated final concentration and incubated for 2 h at 4°C still in the presence of mannose, Then, the cells were washed with cold PBS and the cell-associated fluorescence intensity was measured with a flow cytometer (BD LSR, Becton Dickinson; λex = 488 nm; λem = 530/30 nm). The fluorescence intensity is expressed as the mean value of the fluorescence intensity (MFI) of 10,000 cells.Electronic Supporting InformationNMR data of intermediates and final product relative to the synthesis of TriMan-Diether 1.

  ; MS (ESI) calculated for C 30 H 48 O 7 [M+Na] + 543.33 found 543.35.

  CHCl 3 ); 1 H NMR (CDCl 3 , 400 MHz) δ (ppm) 1.81 (6H, p, J = 6.4 Hz, CH 2 -CH 2 -CH 2 ), 1.93 (6H, p, J = 6.4 Hz, CH 2 -CH 2 -CH 2 ), 3.04 (1H, t, J = 6.1 Hz, OH), 3.43 (6H, s, C-CH 2 ),3.47

	ACCEPTED MANUSCRIPT
	white solid. Rf = 0.4 (Cyclohexane/EtOAc/MeOH: 6:3:1); m.p. = 69°C; [α] 20	D = -33.6 (c 2.2
	(6H, t, J = 6.3 Hz, CH 2 -O), 3.50 (6H, t, J = 6.5 Hz, CH 2 -O), 3.55 (6H, m, CH 2 -O), 3.68 (5H,
	M m, CH 2 -O), 3.91 (3H, dt, J = 9.7, 6.4 Hz, CH 2 -O), 4.39-4.43 (3H, m, H5), 4.47 (3H, dd, J = 12.0, 4.2 Hz, H6a), 4.68 (3H, dd, J = 12.0, 2.4 Hz, H6b), 5.07 (3H, d, J = 1.7 Hz, H1), 5.69 (3H, dd, J = 3.3, 1.7 Hz, H2), 5.91 (3H, dd, J = 10.1, 3.3 Hz, H3), 6.11 (3H, t, J = 10.1 Hz, H4), 7.23-7.41 (20H, m, Har), 7.41-7.58 (20H, m, Har), 7.82-8.08 (20H, m, Har); 13 C NMR A N U S C R I P T (CDCl3, 100 MHz) δ (ppm) 29.69 (C8), 29.95 (C5), 44.81 (C3a), 62.84 (C6'), 65.56 (C9),
	. A mixture of 10 (3.44 g, 1.49 mmol) and palladium on activated carbon (340 A C C E P T E D
	mg, 10% w/w) in CH 2 Cl 2 /MeOH (30 mL, 4:1) was stirred overnight at room temperature
	under hydrogen atmosphere. The reaction mixture was filtered on Celite® and the filtrate was
		14

concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (Cyclohexane/EtOAc: 6:4) to yield the corresponding alcohol (2.61 g, 79%) as a

  ; HRMS (ESI) calculated for C 125 H 126 O 37 [M+Na] + 2241.78702, found 2241.7899; Elemental analysis calculated for C 125 H 126 O 37 %C 67.42 %H 5.72, found %C 67.53, %H 5.62.

N 3 -PEG 350 -diether (13).

  ; Elemental analysis calculated for C 125 H 124 O 38 %C 67.20 %H 5.59, found %C 67.15, %H 5.70. DIEA (188 µL, 1.08 mmol) was added to a mixture of 12 (508 mg, 0.831 mmol) and TBTU (347 mg, 1.08 mmol) in dry CH 2 Cl 2 (15 mL) under nitrogen atmosphere. After 20 min at room temperature, a solution of N 3 -PEG 350 -NH 2 (291 mg, 0.831 mmol) in dry CH 2 Cl 2(5 mL)

	4.2.7. was added and the reaction mixture was stirred for 12h. An aqueous solution of HCl 1N was
	added and the organic phase was washed with water. The combined organic phases were dried
	(MgSO 4 ) and concentrated under reduced pressure. Flash chromatography on silica gel
	(DCM/MeOH: 98:2) yielded 13 (700 mg, 89 %) as a yellow oil; Rf = 0.36 (DCM/MeOH:
	95:5); 1 H NMR (CDCl 3 , 400 MHz) δ (ppm) 0.83-0.87 (m, 18H, 6 CH 3 ), 1.04-1.78 (m, 52H,
	24 CH 2 , 4 CH), 3.37-3.40 (m, 2H, CH 2 -N 3 ), 3.41-3.50 (m, 4H, CH 2 -N, CH 2 -O), 3.53-3.57
	(m, 4H, CH 2 -O), 3.58-3.69 (m, 23H, CH 2 -O), 3.75-3.78 (m, 1H, CH 2 -O), 3.88-3.90 (dd, J =
	2.51, 5.92 Hz, 1H, CH-O), 7.02-7.05 (m, 1H, NH);

  ; MS (ESI) calculated for C 53 H 108 N 2 O 9 [M+H] + 917.81, found 917.80.

	ACCEPTED MANUSCRIPT
	O), 4.40-4.44 (3H, m, H5), 4.48 (3H, dd, J = 4.1, 12.0 Hz, H6b), 4.69 (3H, dd, J = 2.1, 12.1
	Hz, H6a), 5.09 (3H, d, J = 1.8 Hz, H1), 5.69 (3H, dd, J = 1.6, 3.4 Hz, H2), 5.91 (3H, dd, J =
	3.2, 10.3 Hz, H3), 6.11 (3H, t, J = 10.3 Hz, H4), 7.24-8.11 (60H, m, Har); 13 C NMR (CDCl 3 ,
	100 MHz) δ (ppm) 14.
	4.2.9. Perbenzoyl-TriMan-PEG 350 -diether (14). DIEA (112 µL, 0.643 mmol) was added to a mixture of 2 (575 mg, 0.257 mmol) and TBTU (124 mg, 0.386 mmol) in dry CH 2 Cl 2 (70 mL) under nitrogen atmosphere. After 20 min at room temperature, a solution of diether-PEG 350 -NH 2 3 (335 mg, 0.365 mmol) in dry CH 2 Cl 2 (50 mL) was added and the reaction mixture was stirred for 12h. An aqueous solution of HCl 1N was added (until pH 1) and the organic phase was washed with water. The combined organic phases were dried (MgSO 4 ) and concentrated under reduced pressure. Flash chromatography on silica gel (Cyclohexane/EtOAc/MeOH: 49:49:2) yielded 14 (500 mg, 62 %) as a gum. Rf = 0.6 (DCM/MeOH: 9:1); 1 H NMR (CDCl 3 , 400 MHz) δ (ppm) 0.83-0.89 A N U S C R I P T (m, 21H, CH M A C C E P T E D
	17

3 ), 1.00-1.59 (m, 68H, CH 2 ), 1.79-1.86 (6H, q, CH 2 -CH 2 -O), 1.93-1.99 (6H, q, CH 2 -CH 2 -O), 3.40-3.68 (65H, m, CH 2 -O),

3.75-3.78 (m, 1H, CH-O), 3.88-3.95(4H, m, CH 2 -

Acknowledgements

We thank David Gosset (P@CYFIC platform at CBM) for his technical assistance in flow cytometry

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

Highlights :

• The synthesis of a trimannosyl archaeal diether-like lipid is described.

• The preparation of liposomes including the glycolipid is proposed.

• Binding of the glycoliposomes to dendritic cells is evaluated.