Bruno Payre
email: bruno.payre@univ-tlse3.fr

Etienne Gontier

Ahmed Jarray

Yves Martinez

Jean Pierre Laugier

Alexandre Delalleau

Brigitte Gaillard-Martinie

Isabelle Anselme

Dominique Goudouneche

Isabelle Fourquaux

B P A Y R E

E G O N T I E R

A . J A R R A Y ‡, Y . M A R T I N E Z J P L A U G I E R #

A D E L A L L E A U

B . M . G A I L L A R D † †, I . A N S E L M E ‡ ‡ D G O U D O U N È C H E

I F O U R Q U A U X

C G U I L B E A U -F R U G I E R

M B Delisle

C Guilbeau

A new HPF specimen carrier adapter for the use of high-pressure freezing with cryoscanning electron microscope: two applications: stearic acid organization in a hydroxypropyl methylcellulose matrix and mice myocardium

Keywords: Cryogenic scanning electron microscopy, Cryogenic fracture, Cryogenic freezing, High-pressure freezing, HPF specimen carrier adapter

Introduction

Cryoscanning electron microscope (cryo-SEM) examination of fully hydrated cryofixed chemical and biological specimens for morphological studies has been extensively used [START_REF] Wergin | Use of low temperature scanning electron microscopy to observe frozen hydrated specimens of nematodes[END_REF][START_REF] Nakatomi | Cryo-SEM and subsequent TEM examinations of identical neural tissue specimen[END_REF][START_REF] Nguyen | Thermoresponsive Properties of PNIPAM-Based Hydrogels: Effect of Molecular Architecture and Embedded Gold Nanoparticles[END_REF][START_REF] Engström | Chemical structures of plant hydrolyzable tannins reveal their in vitro activity against egg hatching and motility of haemonchus contortus nematodes[END_REF].

For the past decade, cryo-SEM observation has experienced a wonderful development thanks to the improvement of cryo-observation modules. However, cryopreparation of samples is key to a cryo-observation of quality and can be very challenging.

Tissue which has been cryofixated remains hydrated and specimen components are rapidly immobilized, which is not the case using critical point drying by carbon dioxide [START_REF] Polliack | Identification of human B and T lymphocytes by scanning electron microscopy[END_REF] or hexamethyldisilazane (HMDS) [START_REF] Hazrin-Chong | An alternative SEM drying method using hexamethyldisilazane (HMDS) for microbial cell attachment studies on sub-bituminous coal[END_REF]. Cryofixation involves immersing fresh

Summary

Cryogenic transmission electron microscopy of high-pressure freezing (HPF) samples is a well-established technique for the analysis of liquid containing specimens. This technique enables observation without removing water or other volatile components. The HPF technique is less used in scanning electron microscopy (SEM) due to the lack of a suitable HPF specimen carrier adapter. The traditional SEM cryotransfer system (PP3000T Quorum Laughton, East Sussex, UK; Alto Gatan, Pleasanton, CA, USA) usually uses nitrogen slush. Unfortunately, and unlike HPF, nitrogen slush produces water crystal artefacts. So, we propose a new HPF specimen carrier adapter for sample transfer from HPF system to cryogenic-scanning electronic microscope (Cryo-SEM). The new transfer system is validated using technical two applications, a stearic acid in hydroxypropyl methylcellulose solution and mice myocardium. Preservation of samples is suitable in both cases. Cryo-SEM examination of HPF samples enables a good correlation between acid stearic and hydrated samples into a liquid cryogen. This technique is commonly followed by freeze fracture and freeze drying [START_REF] Pieniazek | Scanning electron microscopy combined with image processing technique: analysis of microstructure, texture and tenderness in semitendinous and gluteus medius bovine[END_REF].

The technique of nitrogen slush cryofixation is also based on rapid freezing of the sample. However, the nitrogen liquid calefaction phenomenon resulting from this technique induces slow cooling by freezing and results in water crystals formation due to nucleation and ice crystal growth [START_REF] Studer | High pressure freezing comes of age[END_REF]. Usually, nitrogen slush fixation is used in SEM systems fitted with cryopreparation chamber.

Many cryofreezing techniques are proposed as nitrogen slush alternative. Studies have performed for freon [START_REF] Lyer | An improved specimen loader for cryo-scanning electron microscopy[END_REF], ethane [START_REF] Issman | Cryo-SEM specimen preparation under controlled temperature and concentration conditions[END_REF], propane [START_REF] Inoué | High resolution low-temperature scanning electron microscopy for observing intracellular structures of quick frozen biological specimens[END_REF], propane jet [START_REF] Walther | Recent progress in freeze-fracturing of high-pressure frozen samples[END_REF], and highpressure freezing (HPF) [START_REF] Walther | Recent progress in freeze-fracturing of high-pressure frozen samples[END_REF][START_REF] Fujita | method for efficient observation of intracellular membranes of monolayer culture cells by quick-freeze and freeze-fracture electron microscopy[END_REF] cryofixation.

Unlike the techniques above, HPF enables the freezing of specimen up to a thickness of about 200 µm without the use of cryoprotectant [START_REF] Walther | Recent progress in freeze-fracturing of high-pressure frozen samples[END_REF] (although preserved frozen sample thickness is dependent of sample kind [START_REF] Studer | Vitrification of articular cartilage by high-pressure freezing[END_REF]). The absence of cryoprotectant is essential for sublimation after cryofracture, because cryoprotectant slows down sublimation.

HPF uses high pressure and very low temperatures to achieve high cooling rates [START_REF] Bald | A device for the rapid freezing of biological specimens under precisely controlled and reproducible conditions[END_REF]. HPF is a technique to produce vitreous ice which preserves hydrated samples [START_REF] Dubochet | Electron microscopy of frozen-hydrated bacteria[END_REF][START_REF] Moor | Theory and practice of high pressure freezing[END_REF][START_REF] Studer | High-pressure freezing of soybean nodules leads to an improved preservation of ultrastructure[END_REF]. This technique prevents cryofixation artefacts and has been recommended for better preservation of hydrated samples [START_REF] Dubochet | Cryo-electron microscopy of vitrified specimens[END_REF][START_REF] Vanhecke | Close-to-native ultrastructural preservation by high pressure freezing[END_REF].

After HPF, sample cryofracture and freeze etching can be done before SEM examination. This freeze etching corresponds to a sublimation of frozen water (ice) at low temperature and high vacuum. Sublimation of surface ice under vacuum reveals details of the fractured face that were originally hidden [START_REF] Echlin | Handbook of Sample Preparation for Scanning Electron Microscopy and X-Ray Microanalysis[END_REF]. After deep etching, structures protruding from the remaining ice are generally not collapsed. This is probably a result of the retention of unfreezable water (also known as 'hydration' or 'bound' water) [START_REF] Given | Molecular behavior of water in a flour-water baked model system[END_REF].

Each step of cryopreparation can be challenging and is key to a suitable examination of samples. That is why many parts of cryopreparation system have been designed specifically to answer specific needs [START_REF] Inoué | High resolution low-temperature scanning electron microscopy for observing intracellular structures of quick frozen biological specimens[END_REF][START_REF] Lyer | An improved specimen loader for cryo-scanning electron microscopy[END_REF][START_REF] Issman | Cryo-SEM specimen preparation under controlled temperature and concentration conditions[END_REF] One of the limits for the use of HPF for SEM observation is linked to the lack of well adapted modules for transfer of HPF specimen carrier from HPF systems (EM-ICE, Leica, Nanterre, France, or, HPM100, Leica, Nanterre, France) to the SEM cryotransfer systems (Alto, Gatan, Pleasanton, CA, United States or PP300 and PP3010T Quorum, Laughton, East Sussex, UK). In order to allow this HPF specimen carrier transfer it is necessary to implement an adapter. However, this adapter has to be developed within specific constraints. Firstly, it is essential that the HPF specimen carrier sandwich is firmly fixated to the adapter, as it will be necessary to apply pressure on the sandwich to open it and to create a fracture using the scalpel. Secondly, the HPF carrier sample fixation has to be done in liquid nitrogen. Finally, the HPF specimen carrier adapter has to be locked to the specimen shuttle (this specimen shuttle is designed by Quorum and Gatan cryopreparation chamber).

Taking all these constraints into account, we designed an HPF specimen carrier adapter. To validate its use we tested two specimens: a chemical one [hydroxypropyl methylcellulose (HPMC) with stearic acid (SA)] and a biological one (mice myocardium).

Cellulose derivatives, such as HPMC, are often used in granulation processes as a protective colloid to coat hydrophobic particles such as SA [START_REF] Mahato | Pharmaceutical Dosage Forms and Drug Delivery[END_REF]. When dispersed in water, hydrophobic SA particles tend to minimize their interaction with the water and thus form large aggregates, whereas HPMC can be dissolved in water. HPMC polymer is able to form a gel network when mixed with water. Upon dispersing SA in the HPMC-water blend, the HPMC gel will entrap SA particles and prevent their aggregation, thus producing stable SA particles (Jarray et al., 2015). Particle dispersion is known to be stable when the particles remain dispersed over a long time (i.e. the total number of particles remains constant over time). As, HPMC-SA is hydrated, it is a good model to test cryofixation quality. It can be verified if, by examination, the HPMC-SA correlated to reality. To this purpose, we have calculated SA-surface in total surface and compared it to real SA concentration.

For biological sample, we chose mice myocardium, because cardiomyocyte morphology and inner cell structure organization are well established. It is therefore possible to evaluate technical quality of the technique thanks to the examination of the SEM-myocardial ultrastructure.

Materials and methods

HPMC-SA sample preparation

Aqueous polymeric solutions of HPMC-SA are prepared by dispersing SA in a solution of HPMC and water at 85°C, under agitation for 30 to 60 mins with a rotor-stator homogenizer (Ultraturrax T25, Janke and Kunkel, Staufen, Germany). The solution is then crystallized by cooling the suspension to room temperature. Solutions are thereafter degassed at 50 mbar for 2 h and stored at 5°C for at least 24 h.

Mice myocardial preparation

Studies were performed on 2-month-old male C57BL/ 6JOlaHsd mice (purchased from Envigo). Experimental animal protocols were carried out in accordance with the European and French regulation guidelines for animal experimentation and Figs. 3 and4) and settle down on the specimen shuttle (AL200077B, Quorum) which is slipped into the cryostage of cryochamber preparation or of SEM cryochamber (Fig. 2). The technical diagram, the picture and the 3D graphic of the new HPF specimen carrier adapter are shown in Fig. 3. The specimen shuttle, HPF specimen carrier adapter, and HPF specimen carrier constitute a combined unit (Fig. 4A). The HPF specimen carrier adapter is organized into two areas, a deposit area and a fixative area (Fig. 4B). Fixation of the HPF sandwich specimen carrier was achieved by slipping it onto slide channels 0.26 mm thick (Fig. 4B,C). This easy fixative method can be used in nitrogen liquid. Moreover, HPF sample fixative area is designed with same principle to accept two specimens reducing the whole procedure duration (Fig. S2).

Transfer of the HPF prepared sample

HPF specimen carrier adapter (Fig. 3), is first fixated by a screw to the specimen shuttle (Quorum; Figs. 2 and4) and dried carefully. The transfer occurs in a liquid nitrogen dewar. A large slusher pot full of liquid nitrogen is used to place the HPF specimen carrier (Leica) on the new HPF specimen carrier adapter. The following steps to introduce the sample in the cryopreparation chamber are well established and follow user manual steps (PP3000T, Quorum). and were approved by the local ethical committee. Mice were euthanized through intraperitoneal injection of pentobarbital sodium (160 mg/kg).

Immediately after removal, the heart is plunged in cold PBS during a few seconds thus stopping immediately heart beating. Then, the heart was cautiously slicedwith razor blade (fewmm thick) on ice bed without any torsions or pressures.

HPF-Cryofixation of HPMC or HPMC-SA or mice myocardium

The HPMC or HPMC-SA is inserted between two HPF specimen carriers dedicated for cryofracture (Leica) to create a 'sandwich' (Fig. 1), and it is loaded immediately into a HPM100 HPF machine (Leica). The sample is fixated and frozen within 5 ms at 2050 bar and samples are then transferred to cryovials under liquid nitrogen.

For mice myocardium, each myocardial piece is briefly immerged in hexadecane solution, and then inserted between two HPF specimen carriers, loaded into a HPM100 HPF machine (Leica) and fixated following the same procedure as HPMC.

New HPF specimen carrier adapter

The HPF specimen carrier adapter, made in aluminum, is designed to receive the HPF specimen carrier sandwich (Fig.1

Sample cryofracture

The fracture of the sandwich specimen carrier HPF is produced in a PP3000T cryochamber by contact with scalpel blades (Fig. 5A); the whole surface of the sample is fractured (Fig. 5B). If the separation of the sandwich specimen carrier occurs without fracture, it is possible to perform the fracture directly by contact of the sample with a scalpel blade, which will induce a small fracture area (Fig. 5C).

Freeze drying of the surface of the sample

Ice sublimation is necessary for surface sample observation. Ice is directly transformed into water vapor at -95°C in 30 mins. Sublimation is performed in the cryostage of cryopreparation chamber (Quorum).

Examination with scanning electron microscope

Samples are metalized in cryostage of Quorum cryochamber preparation with Platinium for 60 s at 10 mA. The platinium layer is thinner than 1 nm but allows examination at high voltage with better resolution. Sample was introduced into SEM chamber Quanta 250 FEG FEI SEM (Thermo Fisher, Hillsboro, OR, USA) and was observed at 1-5 kV with spot size 2 and 0.9-1,1 cm working distance. We used secondary electrons detector located in SEM chamber, so the optimal working distance recommended is around 1 cm. The temperature was kept below -140°C during SEM imaging.

SA counting by image analysis

The density of the SA in the HMPC mixture is measured with the ImageJ image processing software [START_REF] Rasband | ImageJ[END_REF]. We chose this software because previous studies validated it in a similar context [START_REF] Hermana | Adsorption and stabilizing effects of highly-charged latex nanoparticles in dispersions of weakly-charged silica colloids[END_REF] to determine the size distribution of adsorbed latex nanoparticles in colloid dispersion; Ellison and collaborators (2014) for the calculation of the mean particle diameter of coated silver nanoparticles; and [START_REF] Baghbanzadeh | Effects of hydrophilic CuO nanoparticles on properties and performance of PVDF VMD membranes[END_REF] to measure the surface roughness of membranes.

In ImageJ, in order to remove smooth continuous backgrounds from SEM images of HPMC-SA dispersions, we applied a 'rolling ball' of 30 pixels. To improve the counting and selection of high intensity objects, median (radius 2 pixels) and high pass (radius 0.7 pixel) filters are applied. These filters remove the background noise in the images and increase the intensities of the particle signals in the dispersions. A

Results

Sample transfer with our HPF specimen carrier adapter

Sample transfer is easy but some precautions need to be taken. A screw must be used to fixate the HPF specimen carrier threshold is applied to the images to select objects to be counted to those with intensity between 90 and 255 in grey levels (8 bit images), with a save of the count mask of the outlines of the counted objects. A second count mask is applied to select the counting area on the flat regions. adapter on the specimen shuttle. It is very important to dry the specimen carrier adapter before nitrogen liquid immersion to prevent water crystals in the slide channel. Ten minutes are necessary for the system to reach liquid nitrogen temperature; this is characterized by boiling around the end of the piece. The HPF Specimen carrier transfer from the HPF (or cryotube stockage) to the HPF specimen carrier adapter, occurs in an adapted box containing liquid nitrogen. Cryoelectron microscopy tweezers at liquid nitrogen temperature are used to put HPF specimen carrier on the deposit area of the HPF specimen carrier adapter and to push it on the slide channels to the fixative area. This is technically very simple, however the sliding of the carrier could be blocked by water crystals if this system isn't well dried or if the sample overflows from the HPF specimen carrier during HPF. Under these conditions, the specimen carrier can slide if two tweezers are used for a strong push. Increasing the size of the slide channel would be a technical solution to avoid crystal blocking but this solution increases the risk to drop the HPF specimen carrier during transfer.

The use of two HPF specimen carriers during HPF provides a specimen carrier sandwich. Specimen carrier sandwich separation on the SEM cryotransfer chamber is very easy with the scalpel blade. Therefore, the whole surface of the sample is cryofractured without contamination by water crystals. Should the fracture not occur, it has to be done manually in SEM cryotransfer chamber. However, extraction of the plastic support for HPF can sometime result in a premature cryofracture, which can be responsible for pollution of the specimen by the emergence of water crystals. This pollution can be removed in the SEM cryotransfer chamber by directly fracturing the specimen again with the scalpel blade or by sublimation.

HPMC and HPMC-SA examination

HPMC does not contain any perforations and has a smooth surface, meaning there is no water crystal artefacts. Some pleating can be observed (Fig. 6A). This data confirms the suitability of cryofreezing by HPF, compared to nitrogen slush which induces holes 'honeycomb-like structure' (Fig. S1).

When HPMC is mixed with SA, white crystals appear. Those crystals have been previously described as SA crystals (Jarray, 2015[START_REF] Jarray | Structure of aqueous colloidal formulations used in coating and agglomeration processes: mesoscale model and experiments[END_REF] and never occur in 'pure HPMC-water' (Fig. 6).

The general appearance of HPMC-SA mixtures in water at SA 2% (Fig. 6B) or at SA 10% (Fig. 6C) is quite same, even if the number of SA crystals increase significantly for the SA 10% mixture. White SA crystals are distributed in the HPMC-water with a size below 1 µm. Some of the SA crystals are outside (or partially outside) the HPMC-amorphous phase, whereas others are covered with the HPMC-water phase but are still artefacts and enough etching to distinguish cell inner structures details. As expected, HPF-myocardium is well preserved and cryofracture enables a near 3D-examination of cardiomyocytes organization in myocardium (Fig. 8A). Cardiomyocyte cytoplasm is more homogeneous with HPF than with nitrogen slush freezing, with fewer water crystals artefacts (Fig. S1). Contractile apparatus of the inner cardiomyocyte is discerned. Specific organization of interfibrillar mitochondria along contractile apparatus can be observed (Fig. 8B). At high magnification, Z lines appear and can be measured (Fig. 8C), inner mitochondrial structures are well seen and cristae can be detected (Fig. 8D).

Discussion

Cryo-SEM examination practices increased tremendously over the last decade. The crucial step for cryo-SEM observation of hydrated samples is cryopreparation. There are various techniques of cryofixation available. HPF is a cryofixation validated and adequate technique for hydrated sample particularly, samples size are preserved on around 200µm of thickness. Furthermore, this technique does not entail the use distinguishable from the swollen HPMC-water surface. There are no big agglomerates.

At SA 20% (Fig. 6D) the structure of the HPMC-SA mixture changes; the SA crystal distribution becomes polydispersed and it becomes difficult to distinguish SA crystals in the aggregates. There is also formation of big agglomerates of SA with different morphologies (Fig. 6D).

Image analysis

Quantification of total HPMC-water surface covered by SA crystals (compared to total surface and expressed in percentage), with the scientific image analysis program ImageJ [START_REF] Rasband | ImageJ[END_REF] demonstrates an increase of this SA surface correlated with SA concentration. SA crystals surface calculated is well correlated with SA concentration until SA 10% (Fig. 7). At SA 20%, the surface calculated is underestimated because of emergence of aggregates. of any cryoprotectant. Cryoprotectants are known to prevent a correct examination of the sample after cryofracture and after a long sublimation. Indeed, cryofracture allows observation of the cell surface and inside the inner cell, a near 3D-estimation of tissue level organization (depending of the random fracture) and is acclaimed for morphological examination. However, the transfer of cryosample in cryo-SEM can be challenging because of the lack of suitable HPF-cryotransfer system. In this study, we designed a new HPF specimen carrier adapter for HPF transfer sample.

Mice myocardium examination

SEM frozen tissue examination can be sensitive because this technique needs to optimize freezing without water crystals

Various technical constraints have been considered in the design of the carrier, linked to the steps of the cryopreparation and linked to the design of the existing specimen carrier or specimen shuttle currently produced by manufacturers. First, the HPF specimen carrier adapter should be locked to the specimen shuttle and must not show significant expansion under the action of low temperature. That is why we have chosen aluminum material because its shape doesn't change at low temperature. Its diameter is 10 mm to be adapted to 11 mm of specimen shuttle hole. Then the sample is frozen between two HPF specimen carriers. This 'sandwich' should SA with different morphologies, meaning that HPMC becomes unable to stabilize SA. Our data are in accordance with literature [START_REF] Jarray | Structure of aqueous colloidal formulations used in coating and agglomeration processes: mesoscale model and experiments[END_REF]. Overall, in our study, there is no water crystal perforation and the structure of the SA crystals in the HPMC polymer gel is well preserved. To confirm our observations, we counted SA crystals with ImageJ. Our data indicates that SA crystals are well dispersed in the HPMCwater mixture. They also show that the HPF technique is able to preserve the structure of dispersed particles. At SA 20% (w/w), we obtain 15.65% ± 1.85 of SA surface on the sample total surface. This value, lower than the real SA concentration in HPMC-SA (i.e. 20% (w/w)), can be explained by a polydispersity in the mixture; at high polydispersity the number of chosen images treated by ImageJ become less representative of the distribution of the particles, but also by SA-aggregates. That's why a high calculation error may be obtained.

Heart is a biological sample SEM-examined for more than 30 years and cardiomyocyte inner structures are well known. However, studies of 80's with various freezing techniques and more recent [START_REF] Ashraf | Preparation of normal and ischemic myocardial tissue for scanning electron microscopy[END_REF][START_REF] Dalen | An ultrastructural study of cryofractured myocardial cells with special attention to the relationship between mitochondria and sarcoplasmic reticulum[END_REF][START_REF] Richter | High-pressure freezing and freeze substitution of rat myocardium for immunogold labeling of connexin 43[END_REF], demonstrated that it can be difficult to observe myocardium without water crystals artefacts. Therefore testing the new HPF specimen carrier adapter with mice myocardium seemed challenging enough. Despite the challenge the result is that the HPF-myocardium is adequately preserved, without any water crystscals artefacts, allowing a good examination of sarcolemma, contractile apparatus, interfibrillar mitochondria with cristae details and also allows if necessary to measure sarcomeres length.

Conclusion

The HPF specimen carrier adapter we designed,is available for transfer of HPF specimen carrier. It can be used with cryotransfer system (Gatan et Quorum) and therefore can be used with cryo-FIB provided with these system.

The tests performed with this adapter, with HPF cryofreezed HPMC without and with SA, confirm that aqueous samples can be frozen and transferred adequately to allow examination but also quantitative analysis. Same quality is obtained with myocardium extending the adapter practice to biological samples. It has to be however noted that every tissues being different the protocol might not be relevant for other organs. We, however, hope that the HPF specimen carrier adapter will allow future biological tests. be fixated firmly to the HPF specimen carrier adapter and this fixation must be performed in liquid nitrogen. The two parts of sandwich should be separated creating a sample-fracture. This operation requires some manual pressure on the system and therefore implies a stress on the fixation system. That is why we have chosen a fixation system in slides which allows fixation in immersion in the liquid nitrogen and which also firmly fixates the HPF support carrier on the HPF support carrier adapter.

Use of our HPF specimen carrier adapter is easy without any damage or any disadvantage requiring redesigning the adapter. A few small difficulties, such as the separation of the two parts of the sandwich without generating fracture, are easily resolved by fracturing manually with the scalpel blade.

To validate our HPF specimen carrier adapter the author tested it in 'real' condition using two samples: a chemical and aqueous sample (HPMC) and a biological sample (mice myocardium). HPMC is a nonionic cellulose ether, which is soluble in water and has thermal gelation properties. Because of its mechanical properties, HPMC is largely used in pharmaceutical industries. It forms a film that can be used as a matrix containing various substances (e.g. nanoparticles or SA) [START_REF] De Moura | Properties of novel hydroxypropyl methylcellulose films containing chitosan nanoparticles[END_REF]. Its aqueous nature makes it a good candidate for cryotests. For biological sample, we chose myocardium because its morphology is well established and inner cardiomyocytes structures are well described. After cryofracture, ice etching is performed at -95°C during 30 min. At this temperature, amorphous ice does not present major modification due to recrystallization because artefactual modifications are not apparent at the magnification the samples are examined. According to DJ Prior data [START_REF] Prior | Making EBSD on water ice routine[END_REF], we can suppose that if ice grains are present, their size is smaller than 1 nm, and they are therefore not detectable. Anyway, these data concern water and dirty water freezing. For biological samples, the removal of water by etching leads artefactual morphological modifications due to the shrinking and collapsing structures [START_REF] Walther | Recent progress in freeze-fracturing of high-pressure frozen samples[END_REF] which hide recrystallization artefacts.

The superior quality freezing with HPF compared to nitrogen slush freezing needn't be demonstrated [START_REF] Yong | Extracellular matrix reorganization during cryo preparation for scanning electron microscope imaging of staphylococcus aureus biofilms[END_REF], and as expected we observed a suitable quality of cryofixation with HPF. After HPF, HPMC is homogenous and white crystals of SA are easily distinguishable. At high pressure, water is more viscous than at atmospheric pressure, which drastically reduces the nucleation and thus the growth rate of water crystals. Therefore our HPF test permits observation of HPMC structural details similar to its native state. This first step validates our adapter confirming no disturbance in transfer of the HPF cryosample.

Thereafter we have tested the stability of HPMC freezing by mixing HPMC with SA at different concentrations. As expected, at low SA concentrations in the HPMC-SA mixture, SA crystals are stable with good preservation of the morphology of the blend. But at SA 20%, we observed big agglomerates of

Fig. 1 .

 1 Fig. 1. HPF specimen carrier. In HPF (HPM100 or ICE, Leica, Nanterre, France) samples are frozen in HPF specimen carrier. (A) manufacturer diagram of this carrier with size of each component (in mms). (B) shows the cavity where the sample is inserted and the (C) shows the sandwich which allows to freeze the sample between two HPF specimen carrier. This technique allows a fracture when separating the two carriers and a sample protection from any water contamination.

Fig. 2 .

 2 Fig. 2. Specimen shuttle. This shuttle is used for cryopreparation chamber (ref AL200077B, Quorum PP3000T, Laughton, East Sussex, UK). It has a hole of 11 mm (A) in its centre and a screw that allows stubs binding suitable for various samples. It uses to perform samples freezing in nitrogen slush. Specimen shuttle is adapted for SEM cold stage, picture B shows the top view without the specimen shuttle and C with it. (D) overview of SEM chamber with the implement of the specimen shuttle.

Fig. 3 .

 3 Fig. 3. HPF specimen carrier adapter. This adapter is designed to be locked in the specimen shuttle and to fasten HPF the specimen carrier. (A) HPF specimen carrier adapter manufacturer technical diagram. (B) SEM picture of this adapter (C) 3D overview diagram and cutting diagram.

Fig. 4 .

 4 Fig. 4. Assembly of technical parts (specimen shuttle, HPF specimen carrier adapter, HPF specimen carrier). (A) overview of every parts assembled. HPF specimen carrier is fastened in HPF specimen carrier adapter, itself placed in the specimen shuttle. (B) SEM picture of HPF specimen carrier adapter areas. Deposit area is used to easily place the HPF specimen carrier. Slide channels enable to slip HPF specimen carrier to the fixative area. (C) 3D-overview and cutting diagrams depict HPF specimen carrier adaptor with HPF specimen carrier in fixative area.

Fig. 5 .

 5 Fig. 5. Specimen cryofracture by removing HPF specimen carrier sandwich top in cryochamber preparation with scalpel blades. (A) (top picture) sandwich HPF specimen carrier in HPF specimen carrier adaptor in the cryochamber PP3000T. (middle picture) scalpel blade pushing sandwich HPF specimen. (bottom picture): HPF specimen carrier bottom remaining after sample fracture and after removing the top of the HPF specimen carrier. (B) sample cryofractured due to HPF specimen carrier dissociation. (C) without sample cryofracture after HPF specimen carrier dissociation the cryofracture is produced manually with scalpel blade.

Fig. 6 .

 6 Fig. 6. SEM examination fractured surface of various SA concentration in HPMC. (A) HPMC in water [10% (w/w)] exhibits a smooth, sometime pleating surface. HPMC-SA in water under different SA concentrations; (B) HPMC-SA 2%, SA is spread in HPMC, inset: small SA crystal (*). (C) HPMC-SA 10%, SA crystals are numerous but still scattered, inset: small SA crystals (*). (D) HPMC-SA 20%, HPMC cannot keep isolated SA crystals and SA agglomerates appear. Inset: high magnification of an SA agglomerate (*). HPMC: hydroxypropyl-methylcellulose; SA: stearic acid. (Scale bar 5 µm).

Fig. 7 .

 7 Fig. 7. Measure of area covered by SA in HPMC at various SA concentrations. SA surface is measured from SEM pictures by imageJ and expressed as percentage of SA area compared to total area. There is a good correlation at SA 2% and SA 10% between SA concentration in HPMC mixture and the SA area measured. At SA 20%, SA area measure underestimated compared to the real SA concentration.

Fig. 8 .

 8 Fig. 8. SEM examination fractured of ultrastructure mice myocardium. (A) At low magnification, cardiomyocytes can be examined in the myocardium with a 3D view (sarcolemma is apparent: arrow). (B) intracellular structures of cardiomyocyte are easily recognized: contractile apparatus (Z) and interfibrillar mitochondria (m) are well distinguished. Intercellular space (is) is noticeable. (C) at high magnification, mitochondria (m) are well visualized and also their organization in the cardiomyocyte, between contractile apparatus. Z-line can be measured (Z; double arrow). (D) mitochondria are suitable cryofixated and cristae organization can be observed.

Acknowledgements

The authors thank the 'Mission pour l'interdisciplinarité' of Centre National de la Recherche Scientifique via the Réseau National des Centres Communs de Microscopie (RCCM) for supporting this work. The work was partly supported by funding from the Ministère de l'Enseignement Supérieur et de la Recherche (ANR-10-INBS-04 FranceBioImaging), Contrat Plan Etat Région Midi Pyrénées (CPER) et Fonds Européen de Développement Régional (FEDER) Midi Pyrénées (Microscopie Electronique pour la biologie, la santé et les agro biosciences en midi Pyrénées). We thank company Eclisse for HPF carrier adapter machining.

We want to greatly thank Miss Bartlett Juliette for her English reviewing.

Supporting Information

Additional supporting information may be found online in the Supporting Information section at the end of the article. Supplementary method: Cryofixation of HPMC, HPMC-SA or mice myocardium by pasty nitrogen. The sample is immerged into pasty nitrogen slusher pot of workstation. Under vaccum conditions, (primary pump) liquid nitrogen (-196°C) is transformed into pasty nitrogen (-210°C approximately). Sample transfer to column mounted preparation chamber PP3000T Quorum Technologies is performed by a cryotransfer-rod. Cryofracture is produced by scalpel blades in the cryochamber. Sublimation is performed until -95°C.

Fig. S1. SEM micrographs of HPMC-SA in water (10%-10%

(w/w) and myocardium using nitrogen slush. A: cryofixation using nitrogen slush is a slow freezing process that generates ice crystals, consequently, HPMC exhibits large pores after sublimation (arrow). SA crystal can indeed be observed (star). Bar scale: 2 µm. B: examination of cardiomyocyte nitrogen slush freezing is possible and enables to observe endoplamic reticulum and mitochondria tight contacts. Contractile apparatus is noticeable. Anyway, some crystallization artefacts and some distortion of structures can be distinguished (arrow). Bar scale: 2 µm.


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Hung-Huy Nguyen

] Bohari

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email: pierre.le-pogam-alluard@u-psud.fr

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email: kimphiphung@yahoo.fr

Sanctis A-C: Three Racemic Procyanidin Analogues From The Lichen Parmotrema sancti-angelii

Keywords: Procyanidin analogues, Lichen, Parmotrema sancti-angelii, cytotoxicity, biosynthesis

The phytochemical investigation of the lichen Parmotrema sancti-angelii afforded three racemic compounds featuring an original dibenzo-2,8-dioxabicyclo[3.3.1]nonane scaffold, sanctis A-C. These compounds were structurally characterized by extensive NMR analyses, comparison between experimental and theoretical NMR data and X-ray crystallography. These procyanidin A-like metabolites display a methyl group instead of a pendant aromatic ring at C-9, a so far unprecedented structural feature. A biosynthetic route to sanctis A-C is proposed herein.

Introduction

Lichens are symbiotic associations comprised of a fungus and a photobiont partner (green alga and/or cyanobacteria). This original consortium produces unique secondary metabolites endowed with various biological activities, paving the way for the phytochemical investigation of these fascinating organisms. [1] Figure 1. Structures of sanctis A-C (1-3). Only one enantiomer is represented per racemic pair.

In the course of our systematic research on lichen substances from Viet Nam, we have examined Parmotrema sancti-angelii (Lynge) Hale, a widely distributed species in the southern part of central Viet Nam. Previous studies on the chemical constituents of P. sancti-angelii reported on the presence of two depsides, atranorin and lecanoric acid, and one depsidone, α-collatolic acid. [2] The current phytochemical investigation of P. sanctiangelii led to the isolation of three racemic pairs of new metabolites bearing an unprecedented carbon skeleton comprising a dibenzo-2,8-dioxabicyclo [3.3.1]nonane core, namely sanctis A-C (1-3) (Figure 1), for which a putative biosynthetic scheme is proposed herein alongside eight known metabolites, the structures of which are collated in Figure S1 (Supporting Information).

Results and Discussion

Compound 1 was obtained as a colorless amorphous solid. The molecular formula was deduced to be C20H20O6 by HRESIMS at m/z 379.1153 [M+Na] + (calcd. 379.1152 for C20H20O6Na) (11 units of unsaturation). 1 H NMR and HSQC spectra of 1 showed a hydrogen-bonded hydroxyl group (δH 13.03), one phenolic hydroxyl group (δH 7.58), three aromatic protons (δH 6.37, 6.34 and 6.31), one methoxy group (δH 3.93), one deshielded methine proton (δH 4.47, 1H, t, J = 3.0 Hz), one diastereotopic methylene group (δH 2.11, 1H, dd, J = 13.0, 3.0 Hz and δH 2.16, 1H, dd, J = 13.0, 3.0 Hz) and three methyl groups (δH 2.43, 2.19 and 1.85, each 3H, s) (Table 1). The 13 C NMR spectrum revealed twenty carbons including one methyl ester group [δC 172.8 (-CO), 52.3 (-OCH3)], three aromatic methine carbons (δC 112.8, 110.9 and 109.4), four oxygenated aromatic quaternary carbons (δC 159.1, 156.9, 153.3 and 152.1), six quaternary carbons (δC 141.3, 138.5, 112.1, 110.9, 105.4 and 98.2), one upfield shifted methine carbon (δC 21.4), one methylene carbon (δC 30.9), and three methyl groups (δC 27.3, 24.0 and 20.4). The HMBC spectrum showed the correlations of the chelated hydroxyl group (δH 13.03, 4-OH) to signals at δC 105.4 (C-3), 112.1 (C-5), and 159.1 (C-4). A first methyl group (δH 2.43, H3-20) correlated to signals at δC 105.4 (C-3), 112.8 (C-1), 141.3 (C-2) and also showed a 4 J coupling to a signal at δC 172.8 (C-18). A first aromatic proton (δH 6.34, H-1) correlated to this methyl carbon (δC 24.0), as well as to signals at δC 105.4 (C-3) and 156.9 (C-6). HMBC cross peaks revealed the correlations of two aromatic methine protons at δH 6.37 (H-13) and 6.34 (H-15) to a second methyl group at δC 20. 4 (C-16). This methyl group also correlated to signals at δC 138.5 (C-14), 110.9 (C-13) and 109.4 (C-15). The H-13 proton correlated to a signal at δC 153.3 (C-12) and the H-15 proton revealed cross peaks to signals at δC 152.1 (C-10) and 110.9 (C-13). HMBC connectivities comprised H-7 methine that resonated at δH 4.47 and δC 21.4. Although H-7 displays a low chemical shift, the high field shift of the corresponding carbon indicates that it might be directly connected to carbon atoms in both aromatic rings. Likewise, the low field shift of H-7 is consistent with its being constrained within the deshielding shift of two aromatic systems. [3,4] Its COSY correlation with a methylene group (H2-8, δH 2.11 and 2.16) indicated that these groups were adjacent to one another, which was further supported by the multiplicity of the methine signal. The AB system of this methylene group implied that the mentioned methine was an asymmetric carbon. Furthermore, the HMBC spectrum showed that these methine and methylene protons correlated with carbons C-5, and C-11 and also with an oxygenated quaternary carbon C-9 at δC 98.2. Finally, a third methyl group at δC 27.3 (C-17) revealed long range heteronuclear cross peaks to the oxygenated quaternary carbon C-9, to the methylene carbon C-8 and finally to carbons C-6 at δC 156.9 and C-10 at δC 152.1 (Figure 2). Combined, these data account for 9 degrees of unsaturation, requiring two additional rings to be present. Overall, NMR data indicated the presence of eight carbon-oxygen bonds. Owing to both the molecular formula and constraints on connectivities among the different substructures, C-7-C-9 were deduced to be involved in a bicyclic 2,8dioxabicyclo [3.3.1]nonane framework that bridges the two aromatic rings by incorporating two ether functions. Besides fulfilling hydrogen deficiency index, this proposed structure was well supported by HMBC cross-peaks between H3-17 and C-6 and C-10 as well as by NOESY correlations between proton H-7 with protons H2-8, 4-OH and 12-OH, between H2-8 and H3-17 and also between 4-OH and 12-OH. The null optical activity of 1 indicated its racemic nature, necessarily corresponding to a mixture of (7R, 9S)-and (7S, 9R)-enantiomers owing to its bridged structure. A single crystal X-ray experiment with Mo Kα (λ = 0.71073 Å) (Figure 3) both corroborated the planar structure deduced from spectroscopic data and validated this relative stereochemistry. 13 C NMR spectral data of 2 (Table 1) were similar to those of 1. Long-range heteronuclear correlations between H-1 at δH 6.32 to C-5 (δC 115.7), C-6 (δC 155.3), C-3 (δC 113.0) and C-20 (δC 19.4) led to define a substitution pattern for a first ring identical to that evidenced through the so-called first spin system of 1 (Figure 2). 2). The methyl group anchored at C-12 would indeed be in close spatial proximity to both the methoxycarbonyl and the C-4 phenolic hydroxyl groups. The steric hindrance caused by the bulky methyl group could compromise the coplanarity of the hydrogenoxygen bond of the C-4 moiety, precluding the chelation of this phenolic group by the vicinal ester carboxyl carbon as in 1 (4-OH, δH 13.03). Therefore, in 2, this signal appeared as a phenolic hydroxyl group resonating at δH 8.94. This might also account for the much higher polarity of 2 compared to that of 1. On the basis of the aforementioned data, the planar structure of 2 was determined as depicted in Figure 1. Compound 2 was isolated as an optically inactive compound, having no optical rotation nor Cotton effects in the ECD spectrum, determining it to consist of a racemic mixture of the two (7S, 9R)-and (7R, 9S)enantiomers, as previously reported for compound 1.

Compound 3 was obtained as a white amorphous solid. Its molecular formula, C33H32O10, was established from the [M+Na] + peak at m/z 611.1926 (calcd. 611.1888 for C33H32O10Na). The comparison of 13 C NMR spectra of 3 and 1 revealed that the chemical structure of the former was highly reminiscent to that of the latter with an addition of thirteen carbons (Table 1). Among these additional carbons, the 13 C chemical shift values of nine of them were similar to those of an aromatic ring of 1 (C-10-C-16),

Accepted Manuscript

suggesting the presence of a second methyl β-orsellinate moiety in 3. The four remaining carbons corresponded to one methine (δH 3.93; δC 30.9, C-7'), one olefinic methine (δH 4.48; δC 100.7, C-8'), one oxygenated quaternary olefinic carbon (δC 149.1, C-9') and one methyl group (δH 1.85; δC 19.0, C-10'). Signal patterns related to the common dibenzo-2,8-dioxabicyclo[3.3.1]nonane substructure only differed for two positions (Figure 4 and Table 1). At first, the triplet methine proton at δH 4.47 (1H, J = 3.0 Hz, H-7) turned into a doublet methine proton at δH 4.35 (J = 2.0 Hz). A second difference was the disappearance of the diastereotopic methylene signals of H-8 [pair of doublet of doublets methylene protons at δH 2.11 (1H, J = 13.0, 3.0 Hz, H-8a), and 2.16 (1H, J = 13.0, 3.0 Hz, H-8b) in 1] to give rise to a doublet of doublets methine proton at δH 2.44 [(1H, J = 2.0, 1.5 Hz) in 3]. These differences indicated that the additional core was anchored at C-8 position. COSY cross-peaks between H-8 and H-7' further supported this vicinity. The H-7/H-8/H-7'/H-8' connectivity also appeared throughout both COSY correlations and HMBC cross peaks (Figure 4). Altogether, these spectroscopic data led to establish the planar structure of 3 as displayed in Figure 1. NOESY spectrum could grant some information as to its stereochemistry such as the close spatial proximity between H-7' and H-7. Unfortunately, no spectral evidence could lead to define the relative configuration of C-8, as NOE cross-correlations between H-8 and both H-7' and H-7 might be observed irrespective of the configuration of the former carbon. In an attempt to discriminate between possible diastereoisomers, 13 C NMR modeling of the eight possible structures was performed using the wellestablished calculation of the DP4 probability. [5] The DP4 application demonstrated the structural equivalence of diastereomer 7R*, 8R*, 9S*, 7'R* with 3 with 99.80% probability. The lack of optical activity indicated the racemic nature of 3. Although the minute amount of 3 precluded any attempt of purification, this deduction was confirmed by the observation of two distinct peaks with identical areas by chiral HPLC analysis (Supporting Information; Figure S23).

Accepted Manuscript

FULL PAPER Scheme 1. Proposed biosynthetic pathway for the formation of 1-3. Note that the functionalization of the aromatic core of the chromene intermediate may occur prior to the cyclization giving rise to the pyrone ring as well.

The similarities between compounds 1-3 suggest that they share common biosynthetic precursors. Sanctis A-C bear striking similarities to co-isolated compounds methyl-βorsellinate 9 and orcinol 11. The biosynthesis of structurally related A-type proanthocyanidols originates from flavanols as early precursors. [6,7] Due to the lack of a phenyl substituent on C-9, a similar biosynthetic scenario would rely on a chromene precursor (Scheme 1), which is quite interestingly encountered in the structure of 3. A dozen of chromones are known from lichen sources, being assumed to arise from the polyketide pathway upon condensation of an acetyl CoA with four malonyl CoA units. [8] While the occurrences of both 2-methyl and 5phenol groups are straightforward to delineate from a biosynthetic perspective, the origins of the 6-methylester and 7methyl substituents are conversely difficult to decipher. Nevertheless, some formerly described lichen chromones also reveal some such additional methyl moieties whose origin is not established neither, at different oxidation states. [START_REF] Huneck | Identification of Lichen Substances[END_REF] As to our specific example, the substitution pattern of the benzene ring of the putative chromone is identical to that of the co-isolated methyl-β-orsellinate 9. Tailoring enzymes might be responsible for the introduction of these substituents as recently evidenced for the methylation of the closely related orsellinic acid. [START_REF] Chooi | [END_REF] Later on, the biosynthetic route to sancti derivatives might rely on the oxidation of the chromene into the corresponding ortho-quinone methide. [6] The subsequent 1,4-Michael addition of orcinol 11 to this ortho-quinone methide intermediate would then afford a structure displaying a nucleophilic vinyl ether moiety that is likely to react with an array of electrophiles. According to this putative scheme, reaching aromatic ring substitution pattern of 1/3 or of 2 would depend on the side of the orcinol moiety triggering the nucleophilic attack on C-7 position of the chromene precursor. A nucleophilic attack initiated by the carbon located between the two phenolic groups might give rise to 1/3 regioisomery, as

Accepted Manuscript

displayed in Scheme 1. Conversely, if the reactive site would be either of the carbons comprised between one phenolic moiety and the methyl group (due to orcinol being a meso compound), the methyl and phenol groups would be inverted to give rise to 2. The addition of a proton to the nucleophilic vinyl ether moiety (C-9) might yield an oxocarbenium ion giving access to either 1 or 2 upon acetal closure. This process is not catalyzed by an enzyme and could give rise to the two enantiomers, depending on the face of attack in the first step. [6,11] Alternatively, the enol ether moiety could react with a second chromene-derived orthoquinone methide as an electrophilic molecule to afford a second oxocarbenium ion that could lead to 3 upon acetal closure. This second nucleophilic attack scenario is deemed to occur with a high degree of diastereoselectivity trans to the existing orcinyl substituent, consistently with the determined stereochemistry of 3. Recent biomimetic syntheses of trimeric flavonoids dragonbloodins A1/A2 described a highly similar mechanism. [11] While the 2,8-dioxabicyclo[3.3.1]nonane was seldom encountered in the structure of polyketide-derived fungal metabolites such as averufin, [12] the molecular context of this core comprising benzene rings on both sides of it makes it an unprecedented report not only among lichen metabolites but also in the wider realm of fungi. Structural homologies with flavanol-derived A-type proanthocyanidols account for both the plant-specific distribution of this structural series so far and for the consensual occurrence of an aromatic ring at the equivalent of position C-9. One exception to this being the example of myristicyclins, isolated from the Myristicaceae plant Horsfieldia spicata, containing a hydrogen atom at this specific position. [13] Instead, the presence of a methyl group in our metabolites makes our carbon skeleton unique, also being consistent with the fact that filamentous fungus produce no flavonoid-related compounds. [14] A further structural originality reported herein is the occurrence of a cyclic substituent on C-8 position of 3. It must however be pointed out that such analogues were reported to step in the biomimetic synthesis of trimeric flavonoids from Daemonorops drcao although later rearrangements occur so that the final products do not display this scaffold any longer. [11] The first isolation of these metabolites in the frame of this phytochemical investigation might be explained by the large amount of lichen presently investigated.

Compounds 1, 2 and 4 were evaluated for their cytotoxic potential against cervical HeLa, breast MCF-7 and lung NCI-H460 cell lines using the sulforhodamine B method [15] with camptothecin as the positive control (Supporting Information; Table S1). While 2 revealed a mild cytotoxic activity, 1 and 4 were inactive.

Conclusions

Chemical investigation of the methanolic extract of the lichen Parmotrema sancti-angelii led to the isolation and structure elucidation of three racemic pairs of procyanidin A analogues for which a biosynthesis pathway is proposed from readily available precursors. Their structure and configurations were assigned on the basis of an extensive set of spectroscopic techniques (1D and 2D NMR, HRMS) along with Goodman probabilities and Xray crystallography. The carbon skeleton of our newly reported compounds is unique given the pendent methyl group occurring at C-9, indicating their arising from chromene precursors. Instead, flavonoid-derived A-type procyanidins display an aromatic ring at this specific position. Likewise, the occurrence of an aryl substituent on C-8 represents a further structural novelty.

Experimental Section

General Experimental Procedures: NMR spectra were recorded on a Bruker Avance III 500 MHz spectrometer using residual solvent signal as internal reference: chloroform-d H 7.26, C 77.2 and acetone-d6 H 2.1, C 206.3, 30.6. The HR-ESI-MS data were recorded on a MicroTOF-Q mass spectrometer. Optical rotations were measured on a Kruss digital polarimeter. Electronic circular dichroism spectra were recorded at room temperature using a Jasco-J815 ECD spectrometer. Thin Layer Chromatography (TLC) analyses were performed on silica gel 60F254 plates with a thickness of 0.25 mm (Merck Millipore, Billerica, Massachusetts, USA). Spots were visualized either by spraying with 10% aqueous H2SO4 or with 5% ferric chloride solutions followed by heating. Open column chromatographies were carried out on silica gel 60 (40-63 µm) (HiMedia, Mumbai, India). Crystallographic analyses were carried out with convenient single crystals on an automatic diffractometer (Bruker Model D8 Quest) with Mo Kα radiation and graphite monochromatization (λ = 0.71073 Å) at 100 K. The HPLC system (Prominence Shimadzu, Marne-La-Vallée, France) was equipped with a Chiralpak IA column (250 x 4.6 mm, 5 µm) and consisted of a quaternary pump (LC20ADSP) and a diode array detector (SPD-M20A). The elution was performed under isocratic conditions using a 98/2 (v/v) heptane/isopropanol mixture as the mobile phase at a flow rate of 0.5 mL/min.

Extraction and isolation:

The thallus material was washed overnight under a flow of tap water, prior to being rinsed with distilled water to thoroughly cleanse out of all extraneous material. The lichen was then air-dried at ambient temperature to obviate thermally induced decomposition prior to being ground into a fine powder. The powdered lichen thallus (700 g) was macerated with methanol at room temperature to afford a crude methanol extract (120 g). This crude extract was applied to silica gel solid phase extraction, being successively eluted with the following solvents: petroleum ether (6090 o C) (PE), chloroform (C), ethyl acetate (EA) and methanol (M) to afford corresponding extracts: extract PE (24 g), extract C (20 g), extract EA (14 g) and extract M (30 g). Extract PE (24 g) was applied to silica gel column chromatography to be eluted with PEEA (stepwise 10:00:10), giving 5 fractions (E1E5). Fraction E4 was chromatographed, eluted with PEEA (stepwise 9:17:3) to afford 8 (40 mg), and 9 (300 mg). Extract C (20 g) was applied to silica gel column chromatography, eluted with PEEA (95:50:10) to give 4 fractions (C1C4). Fraction C1 (100 mg) was rechromatographed, eluted with PEC (8:2) to yield three compounds 1 (11 mg), 3 (7 mg) and 10 (8 mg). Fraction C2 (10.1 g) was also selected for further chromatography, eluted with CEA (9: 1) to provide 5 (300 mg), 6 (900 mg), and 7 (30 mg). Fraction C3 (1.1 g) was rechromatographed, eluted with CEA (9: 1) to afford 2 (4 mg) and 11 (15.5 mg). Extract EA (14 g) was applied to silica gel column chromatography, eluted with PEEA (5:50:10) to give 2 fractions (EA1EA2). Fraction EA2 (190 mg) was rechromatographed, eluted with chloroformacetone (9: 1) to give compound 4 (20 mg).

Accepted Manuscript

(±)-Sanctis A (1): Colorless crystals (chloroformethyl acetate); [α]D 20 0 (c 0.075, chloroform). HR-ESI-MS m/z 379.1153 [M+Na] + (calcd. for C20H20O6Na, 379.1152). 1 H and 13 C NMR data (CDCl3) see Table 1. COSY, HMBC, NOESY correlations see Figure 2. Standard TLC [START_REF] Elix | A catalogue of standardized chromatographic data and biosynthetic relationships for lichen substances[END_REF] RF values: RF (A): 0.84; RF (C): 0.72; RF (F): 0.89. Acid spray: yellow-orange. Vanillin/acid spray: purple.

(±)-Sanctis B (2): White amorphous powder; [α]D 20 0 (c 0.075, chloroform). HR-ESI-MS m/z 379.1152 [M+Na] + (calcd. for C20H20O6Na, 379.1152). 1 H and 13 C NMR data (Acetone-d6) see Table 1. HMBC correlations see Figure 2. Standard TLC [START_REF] Elix | A catalogue of standardized chromatographic data and biosynthetic relationships for lichen substances[END_REF] 1 H and 13 C NMR data (CDCl3) see Table 1. COSY, HMBC, NOESY correlations see Figure 4. Standard TLC [START_REF] Elix | A catalogue of standardized chromatographic data and biosynthetic relationships for lichen substances[END_REF] RF values: RF (A): 0.86; RF (C): 0.70; RF (F): 0.87. Acid spray: orange. Vanillin/acid spray: orange-brown. The structure was solved by intrinsic phase methods using SHELXT and refined on F 2 by using SHELXTL [START_REF] Sheldrick | SHELXTL-97 Programs for the Solution and Refinement of Crystal Structures[END_REF] with anisotropic parameters for all non-H-atoms. All the hydrogen atoms were refined isotropically in riding model. A twinning refinement with the proposed twin law [-1.000 0.000 0.000 0.010 1.000 0.000 -0.005 -0.177 -1.000] was done. This resulted in a BASF value of 0.097(2) and about 6.0% improvement in R1 value. The asymmetric unit consisted of two independent molecules which were two enantiomers (Figure 3). The two six-membered heterocyclic rings in both molecules adopted distorted envelope conformations. The bond distances C18A-O5A [1.226(8) Å] and C18B-O5B [1.222(9) Å] are in the typical range of C-O double bonds. The labile hydrogen atoms on O3A/O3B and O4A/O4B were refined as rotating groups in riding model. The obtained results are supported by suitable Q peaks found in the difference electron density map and also by the finding of intramolecular hydrogen bonds O3A-H3A…O4A, O4A-H4A…O5A, O3B-H3B…O4B and O4B-H4B…O5B. The other bond lengths and angles are in normal ranges and comparable to those in its analogue1,5,13-trimethyl-9-phenyl-8,10-dioxatetracyclo [7.7.1.0 2,7 .0 11,[START_REF] Elix | A catalogue of standardized chromatographic data and biosynthetic relationships for lichen substances[END_REF] ] heptadeca-2,4,6,11, 13,15hexaene. [START_REF] Vijayalakshmi | [END_REF] Computational Details: All DFT calculations have been performed using Gaussian 09W. After a conformational analysis of the 8 diastereoisomers realized with MarvinScketch 17.8.0 ChemAxon, a geometry optimization (B3LYP/6-31g(d)) was undertaken on the most stable conformer of each diastereoisomer. The absence of imaginary frequencies was confirmed by a frequency calculation at the same level. Calculation of the chemical shifts was performed using the same method at the 6-311+g(d,p) level in the gaz phase. Comparison of the experimental and predicted chemical shifts was realized using a in-house implementation if the DP4 Java code available from Goodman lab (http://wwwjmg.ch.cam.ac.uk/tools/nmr/DP4/). Biological assays: Determination of cytotoxic activities against the HepG2 (liver hepatocellular carcinoma), MCF-7 (human breast cancer) and NCI-H460 (human lung cancer) cell lines of tested samples was performed at the concentration of 100 µg/mL using the Sulforhodamine B (SRB) assay with camptothecin being used as the positive control. All cells were cultured in EMEM medium (Eagle's Minimal Essential Medium) supplemented with 10% foetal bovine serum (FBS), 1% of 2 mM L-glutamine, 50 IU/mL penicillin, 50 μg/mL streptomycin and cultures were fixed by adding 50 μL of cold 50% (w/v) trichloroacetic acid per well and incubated at 4°C for 1 h. The plates were washed five times with distilled water and air dried. The cells were then stained with 50 µL of SRB (0.4% w/v in 1% acetic acid) at room temperature for 30 min. The SRB solution was removed out of plates by rinsing 4 times with a 1% glacial acetic acid solution (200 μL/well). The plates were air-dried for 12-24 h. The bound SRB was dissolved to each well by adding 100 μL of 10 mM Tris Base (pH 10.5). The plates were shaken gently for 20 min and the optical density of each well was read using a scanning multiwall spectrophotometer at a test wavelength of 492 nm and a reference wavelength of 620 nm. The optical density (OD) of SRB in each well is directly proportional to the cell number. Cell survival was measured as the percentage absorbance compared to the control (non-treated cells) maintained at 37 °C in a 5% CO2 atmosphere with 95% humidity. Viable cells were counted and inoculated in 96-well plate with a density of 104 cells/100 μL/well. After 24 h, the cells were treated with pure compound while the control wells were only added with 100 μL medium. All experiments were performed in triplicate. The plates were incubated in an atmosphere of 5% CO2, 95% humidity at 37 °C for 48 h. Adherent cell cultures were fixed by adding 50 μL of cold 50% (w/v) trichloroacetic acid per well and incubated at 4°C for 1 h. The plates were washed five times with distilled water and air dried. The cells were then stained with 50 µL of SRB (0.4% w/v in 1% acetic acid) at room temperature for 30 min. The SRB solution was removed out of plates by rinsing 4 times with a 1% glacial acetic acid solution (200 μL/well). The plates were air-dried for 12-24 h. The bound SRB was dissolved to each well by adding 100 μL of 10 mM Tris Base (pH 10.5). The plates were shaken gently for 20 min and the optical density of each well was read using a scanning multiwall spectrophotometer at a test wavelength of 492 nm and a reference wavelength of 620 nm. The optical density (OD) of SRB in each well is directly proportional to the cell number. Cell survival was measured as the percentage absorbance compared to the control (non-treated cells).

X-ray crystallographic analysis of (±)-Sanctis

Supporting information: CCDC 1581250 contains the supplementary crystallographic data for compound 1. The data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc. cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

Figure 2 .

 2 Figure 2. Key HMBC and NOESY correlations for 1 and 2.

Figure 3 . ORTEP diagram of 1 Compound 2

 312 Figure 3. ORTEP diagram of 1

  The 2,8-dioxabicyclo[3.3.1]nonane system was deduced from HMBC correlations of proton H-7 at δH 4.52 to C-5 (δC 115.7), to C-10 (δC 152.2), C-11 (δC 117.1), C-12 (δC 138.1), C-8 (δC 32.8) and C-9 (δC 98.3). The only difference was the permutation of positions of the hydroxyl and the methyl groups in the second aromatic ring. In 2, the phenolic hydroxyl group (δH 8.03) and the methyl moiety (δH 2.50) were respectively assigned to C-14 and C-12, based on long range heteronuclear connectivities of the phenolic hydroxyl proton with C-14 (δC 157.2), C-13 (δC 111.3) and C-15 (δC 101.7), and of the methyl protons with C-12 (δC 138.1), C-11 (δC 117.1) and C-13 (δC 111.3) (Figure

Figure 4 .

 4 Figure 4. Key HMBC correlations for 3.

Lichen material :

 material Thalli of Parmotrema sancti-angelii (Lynge) Hale were separated from the bark of tea trees Camelia sinensis (L.) O. Ktze at Bao Loc city, Lam Dong province, Vietnam in July 2010 (11 o 29'-11 o 31' north latitude and 107 o 44'-107 o 46' east longitude). The lichen species was identified by Dr. Harrie J.M. Sipman (Berlin Museum). A voucher specimen (No USB021) was deposited in the herbarium of the Department of Organic Chemistry, University of Science.

  RF values: RF (A): 0.39; RF (C): 0.15; RF (F): 0.67. Acid spray: brown. Vanillin/acid spray: purple. (±)-Sanctis C (3): White amorphous powder; [α]D 20 0 (c 0.075, chloroform). HR-ESI-MS m/z 611.1926 [M+Na] + (calcd. for C33H32O10Na, 611.1888).

  A (1): Formula: C20H20O6, Mr = 356.36. Colorless crystal 0.05 x 0.30 x 0.40 mm, a = 7.6701(9), b = 11.2158(14), c = 19.323(3) Å, α = 92.767(3), β = 91.849(3), γ = 90.152(3)º. V = 1659.4(4) Å 3 , ρcalcd = 1.426 Mg/m -3 , µ = 0.105 mm -1 , Z = 4, triclinic, Pī ; λ= 0.71073 Å, T= 100.0(2)K, ω and φ scans, 21113 reflections collected (± h, ± k, ± l) [I ≥ 2σ (I)], 6161 independent reflections (Rint = 0.0626), 482 refined parameters, R = 0.1168, ωR 2 = 0.3380, max residual electron density 0.53 (-0.44) e.Å -3 . The data set was collected with a Bruker D8 Quest CMOS diffractometer, absorption correction: multi-scan (SADABS) at VNU-Hanoi University of Science.

Table 1 .

 1 1 H (500 MHz) and13 C NMR (125 MHz) Data for 1-3.

		Sanctis-A (1) (CDCl3)		Sanctis-B (2) (acetone-d6)		Sanctis-C (3) (CDCl3)
	position	δH, mult (J, Hz)	δc	δH, mult (J, Hz)	δc	δH, mult (J, Hz)	δc
	1	6.34, s	112.8	6.32, s	109.9	6.21, s	111.2
	2		141.3		135.9		140.9 [a]
	3		105.4		113.0		105.2
	4		159.1		154.4		159.2
	5		112.1		115.7		111.0
	6		156.9		155.3		157.3
	7	4.47, t (3.0)	21.4	4.52, d (3.0)	24.2	4.35, d (2.0)	24.9
	8	2.11, dd (13.0, 3.0)	30.9	2.08, m	32.8	2.44, dd (2.0, 1.5)	45.1
		2.16 dd (13.0, 3.0)
	9		98.2		98.3		101.0
	10		152.1		152.2		151.6
	11		110.9		117.1		113.5
	12		153.3		138.1		152.7
	13	6.37, br d	110.9	6.23, br d	111.3	6.31, br d	111.4
	14		138.5		157.2		138.3
	15	6.31, br d	109.4	6.15, br d	101.7	6.23, br d	109.1
	16	2.19, s	20.4	2.50, s	19.9	2.15, s	21.3
	17	1.85, s	27.3	1.72, s	27.3	1.84, s	24.9
	18		172.8		168.7		172.5
	19	3.93, s	52.3	3.79, s	51.7	3.92, s	52.2
	20	2.43, s	24.0	2.10, s	19.4	2.36, s	23.9
	1'					6.23, s	112.1
	2'						140.7 [a]
	3'						106.8
	4'						162.1
	5'						108.5
	6'						156.6
	7'					3.93, br d	30.9
	8'					4.48, br d	100.7
	9'						149.1
	10'					1.85, s	19.0
	11'						172.1
	12'					3.89, s	52.2
	13'					2.34, s	23.9
	4-OH	13.03, s		8.94, s		12.82, s
	12-OH	7.58, s				7.52, s
	14-OH			8.03, s
	4'-OH					11.94, s
	[a] Interchangeable

Acknowledgements

This research was supported by Vietnam's National Foundation for Science and Technology Development (NAFOSTED) grant #104.01-2013.17. This work was financially supported by Rachadapisek Sompote found Fund for Postdoctoral Fellowship, Chulalongkorn University. This work was also supported partly by University of Rennes 1 through a scholarship for Duong T.H. from January to April 2016. We are grateful to Dr. Harrie J.M. Sipman (BerlinMuseum) for identifying the Parmotrema specimens. The authors thank UKM Dip 2014-16 vote for X-ray analyses, Dr. Do T.M.L. (Saigon U., VN) for acquiring NMR data. Dr. Laurent Evanno is gratefully acknowledged for having improved the graphical representation of the newly reported metabolites.

Accepted Manuscript

Accepted Manuscript