Nolwenn Fougeron

Aurélien Macron

Hélène Pillet

Wafa Skalli

Pierre-Yves Rohan

Aurélien

P-Y Rohan

Finite Element mesh generation of the pelvis from bi-Planar X-ray images

Keywords: FEM, Hexahedral mesh, Subject specific, Pelvis, Bi-planar X-rays

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

Finite Element Modelling is becoming an ever more important tool in the field of biomedical engineering for many applications including the investigation of the mechanisms responsible for the onset of pathologies, for surgical planing and for the evalution of the impact of medical devices on patient outcome.

Finite Element models are generally built from patient medical image data, such as computed tomography (CT) or magnetic resonance imaging (MRI) [START_REF] Linder-Ganz | Strains and stresses in sub-dermal tissues of the buttocks are greater in paraplegics than in healthy during sitting[END_REF]). However, image segmentation and finite element mesh design are long and tedious processes that represent major bottlenecks for the fast generation of patient-specific Finite Element Meshes. Many attempts have been made in the literature to circumvent this problem and today several fast and robust methods have been developed for automatically generating tetrahedral meshes of arbitrary geometries. However, for a wide range of applications, hexahedral-based meshes are preferred: First, to achieve the same solution, accuracy for a given analysis requires far more tetrahedral elements than hexahedral elements, and this leads to higher computational costs (both time and memory). Second, it is well known that for incompressible and/or nearly incompressible materials, 4-noded tetrahedra with linear shape functions tend to lock and become overly stiff, generally producing acceptable displacement results but relatively inaccurate results for stresses.

Recent developments in 3D reconstruction techniques from calibrated bi-planar X-ray imaging provide a promising alternative tool for patientspecific 3D modelling of the spine, lower limb, pelvis [START_REF] Mitton | 3D reconstruction of the pelvis from bi-planar raiography[END_REF]) and external envelope.

The aim of this study is to develop a methodology for the automatic hexahedral subject specific Finite Element mesh design of the pelvis based on 3D reconstruction from bi planar X-rays. The model obtained should preserve acceptable mesh quality to allow for mechanical simulations.

Methods

Generic Mesh design

A generic 3D-reconstruction model of the pelvis (anatomical atlas) was used to design the generic Finite Element mesh. Half of the surface mesh of the generic 3D reconstruction (sacrum and one ilium wing) was patched using NURBS surfaces in Geomagic Studio version 12 software (Geomagic U.S. Corp., Research Triangle Park, NC, USA). The NURBS surfaces were then imported into ANSYS® Mechanical APDL, Release 15.0 (ANSYS Inc., Canonsburg, Pennsylvania, USA) and used to define cube subvolumes of the pelvic geometry. Each subvolume was meshed with linear hexahedral elements (8-node SOLID45 elements) in ANSYS. Special attention was paid to the discretisation of the NURBS so as to have a conforming mesh. The complete volume was defined as the union of the sub-volumes meshed with hexahedral elements. The ilium wing was then duplicated by symmetry and the full pelvic mesh was defined as the union of all the meshes (figure 1(c)). Mesh regularization was performed using an optimization process based on four criteria: jacobian condition number, skewness, warping factor and parallel deviation. The objective function was calculated as a weighted sum of the element metrics. This optimisation procedure was performed using conjugate gradient method and the corresponding displacement field was applied to the internal nodes at each step. In addition, specific anatomical landmarks were identified on the surface mesh [START_REF] Besnault | Modélisation par éléments finis du bassin humain en configuration de chocs automobiles[END_REF].

Images acquisition and 3D reconstruction

Low-dose biplanar X-ray images of a 25 years old female (156 cm tall, and weighing 54.9 kg) was extracted from the database of the Institut de Biomécanique Humaine Georges Charpak. 3D reconstruction of the pelvis was performed (Figure 1(a) and Figure 1(b)) from the deformation of a generic model embedding the same anatomical landmarks as those defined on the Finite Element mesh [START_REF] Mitton | 3D reconstruction of the pelvis from bi-planar raiography[END_REF].

Personalisation of the mesh using kriging interpolation

The generic pelvic Finite Element mesh was then deformed using the kriging interpolation method [START_REF] Trochu | A Contouring Program Based on Dual Kriging Interpolation[END_REF] to conform to the morpho-realistic 3D-reconstruction from bi-planar X-ray radiographs of the volunteer. Kriging interpolation used the above mentioned control points to obtain subject specific mesh (Figure 1 (e)). The entire process was implemented in a custom-made routine on MATLAB® Release 2014b (The MathWorks, Inc., Natick, Massachusetts, United States).

Mesh quality evaluation

ANSYS® shape checking was performed over the following criteria: aspect ratio (AR), parallel deviation (PD), maximum angle (MA), jacobian ratio (JR) and warping factor (WR)

Results

The generic Finite Element mesh is composed of 3014 hexahedrons (Figure 1(d)). The results of the shape checking are provided in Table 1 for the generic model before regularization (GM1), for the generic model after regularization (GM2) and for the subject specific model (SSM). Our results show that the regularization process improved the quality of the generic mesh repairing all errors (GM2 vs GM1). In the end, only less than 1% of the elements remain within the warning thresholds of parallel deviation. . Our results also show that the personalisation process increased the proportion of warnings but no errors occurred (SSM vs GM2). The subject specific mesh was obtained in 0.43 s. Average point-to-surface error between the 3D reconstruction and the personalised FE mesh was 1.2 mm (max 11.6 mm).

Model

Discussion

In this contribution, a new methodology has been proposed for the generation of personalised hexahedral Finite Element mesh of the pelvis from bi planar Xrays. Bi-planar X-ray imaging and the associated 3D reconstruction and clinical parameters computation provide a promising alternative to CT-scan and MR images and, as far as the authors are aware of, this is the first attempt to generate a personalised Finite Element mesh of the pelvis from this imaging modality.

Our results show that a high quality mesh (zero error) can be obtained in a fast way (less than half a second). This is promising for clinical applications. Results also show that the kriging interpolation technique seems to be well-suited for the purpose of computing a mesh deformation driven by anatomical landmarks. This proposed application.

Future works will include the definition of two generic Finite Element meshes: one for males and one for females. The robustness of the method will also be evaluated by testing the proposed method on a larger database.

Figure 1

 1 Figure 1 (a) EOS images of the subject. (b) Reconstruction of the subject geometry from X-rays. (c) Generic model geometry. (d) Generic mesh after regularization. (e) Subject specific mesh.

Acknowledgements

The authors would like to thank the Fondation de l'Avenir for their financial support.


Nolwenn Fougeron
email: nolwenn.fougeron@ensam.eu

Pierre-Yves Rohan

Aurélien Macron

Christophe Travert

Hélène Pillet

Wafa Skalli

Specific Finite Element Mesh Generation of the Pelvis from Biplanar X-ray Images: Application to 120 clinical cases

Keywords: Biplanar X-rays, Finite Element Modelling, Hexahedral Mesh, Pelvis, Subject Specific

Several Finite Element (FE) models of the pelvis have been developed to comprehensively assess the onset of pathologies and for clinical and industrial applications. However, because of the difficulties associated with the creation of subject-specific FE mesh from CT scan and MR images, most of the existing models rely on the data of one given individual. Moreover, although several fast and robust methods have been developed for automatically generating tetrahedral meshes of arbitrary geometries, hexahedral meshes are still preferred today because of their distinct advantages but their generation remains an open challenge. Recently, approaches have been proposed for fast 3D reconstruction of bones based on X-ray imaging. In this study, we adapted such an approach for the fast and automatic generation of all-hexahedral subject-specific FE models of the pelvis based on the elastic registration of a generic mesh to the subject-specific target in conjunction with element regularity and quality correction. The technique was successfully tested on a database of 120 3D reconstructions of pelvises from biplanar X-ray images. For each patient, a full hexahedral subject-specific FE mesh was generated with an accurate surface representation.

Introduction

Finite Element Modelling of the pelvis is becoming an ever more important tool for investigating the mechanisms responsible for the onset and the development of pathologies [START_REF] Filardi | Finite element analysis of sagittal balance in different morphotype: forces and resulting strain in pelvis and spine[END_REF]. It also constitutes a valuable tool for the development and evaluation of medical devices [START_REF] Watson | The effect of boundary constraints on finite element modelling of the human pelvis[END_REF], haptic medical training devices [START_REF] Buttin | Biomechanical simulation of the fetal descent without imposed theoretical trajectory[END_REF] and have the potential of improving both prevention strategies [START_REF] Linder-Ganz | Strains and stresses in sub-dermal tissues of the buttocks are greater in paraplegics than in healthy during sitting[END_REF]) and surgical planning [START_REF] Liu | Design and biomechanical study of a novel adjustable hemipelvic prosthesis[END_REF]. FE models of the pelvis are generally built from patient-specific Computed Tomography (CT) data [START_REF] Anderson | Subject-specific finite element model of the pelvis: development, validation and sensitivity studies[END_REF] which, in addition to the lengthy acquisition times, requires intensive user intervention at multiple stages of the segmentation process and for the FE mesh design [START_REF] Salo | Computed tomography landmark-based semi-automated mesh morphing and mapping techniques: generation of patient specific models of the human pelvis without segmentation[END_REF]. For this reason, existing models generally rely on the data of only one individual and, except for a few attempts, the question of personalization in a framework compatible with clinical routine remains an open question.

Moreover, although several fast and robust methods have been developed for automatically generating tetrahedral meshes of arbitrary geometries [START_REF] Si | Tetgen, a delaunay-based quality tetrahedral mesh generator[END_REF], hexahedral meshes are still preferred today because of their distinct advantages over standard displacement-based tetrahedral elements namely (i) reduced order of convergence for strains and stresses [START_REF] Payen | Improved stresses for the 4-node tetrahedral element[END_REF] and (ii) stability issues associated to shear locking, volumetric locking [START_REF] Joldes | Suite of finite element algorithms for accurate computation of soft tissue deformation for surgical simulation[END_REF]). Yet the automatic generation of all-hexahedral FE meshes for complex geometries remains an open challenge and research is still ongoing [START_REF] Gregson | All-hex mesh generation via volumetric polycube deformation[END_REF].

Recent developments in 3D reconstruction techniques from calibrated biplanar X-ray imaging provide a promising alternative tool for patient-specific validated 3D modelling of the pelvis [START_REF] Ghostine | Influence of patient axial malpositioning on the trueness and precision of pelvic parameters obtained from 3D reconstructions based on biplanar radiographs[END_REF]. Unlike CT scanner or MRI systems where the patient is in a supine position, this technique provide biplanar images of the subject in a standing position.

The aim of this contribution is to develop a methodology for the automatic hexahedral subject specific FE mesh design of the pelvis based on 3D reconstruction from biplanar X-rays suitable for FE simulations.

Materials and methods

Biplanar X-ray acquisition and 3D reconstruction of the pelvis

Images and associated 3D semi-automatic reconstructions were derived from EOS biplanar radiographs [START_REF] Dubousset | EOS: a new imaging system with low dose radiation in standing position for spine and bone & joint disorders[END_REF]) (EOS Imaging, Paris, France) of 120 subjects (57 females, 63 males) collected in previous studies [START_REF] Surets | Procédé pour la fabrication d'un dispositif médical externe[END_REF][START_REF] Chaibi | Fast 3D reconstruction of the lower limb using a parametric model and statistical inferences and clinical measurements calculation from biplanar X-rays[END_REF][START_REF] Quijano | Three-dimensional reconstruction of the lower limb from biplanar calibrated radiographs[END_REF][START_REF] Pillet | A reference method for the evaluation of femoral head joint center location technique based on external markers[END_REF][START_REF] Sangeux | Which method of hip joint centre localisation should be used in gait analysis?[END_REF][START_REF] Ohl | 3d scapular orientation on healthy and pathologic subjects using stereoradiographs during arm elevation[END_REF][START_REF] Amabile | Invariance of head-pelvis alignment and compensatory mechanisms for asymptomatic adults older than 49 years[END_REF][START_REF] Bendaya | Good vs poor results after total hip arthroplasty: an analysis method using implant and anatomic parameters with the EOS imaging system[END_REF][START_REF] Zhang | Investigation of 3D glenohumeral displacements from 3D reconstruction using biplane X-ray images: Accuracy and reproducibility of the technique and preliminary analysis in rotator cuff tear patients[END_REF][START_REF] Ghostine | Influence of patient axial malpositioning on the trueness and precision of pelvic parameters obtained from 3D reconstructions based on biplanar radiographs[END_REF]. Ages, weights and heights were comprised between 20 and 76 years old, 42 and 120 kg and 150 and 190 cm. For each patient, the 3D geometry of the pelvis was reconstructed using previously described validated techniques [START_REF] Ghostine | Influence of patient axial malpositioning on the trueness and precision of pelvic parameters obtained from 3D reconstructions based on biplanar radiographs[END_REF]) by experienced users. Each reconstruction takes around 20 minutes.

Generic mesh design

Female and male pelvises were distinguished due to their anatomical differences. The generic anatomical atlases of both pelvises used for the 3D reconstructions were used to design the respective generic FE meshes. Non-uniform rational B-spline (NURBS) surfaces were generated over the triangular surface meshes of the anatomical atlases using Geomagic Studio (Geomagic U.S. Corp., Research Triangle Park, NC, USA). Owing to the complex shape of the sacrum the surface mesh was closed at the entrance of the sacral canal. The surface patches were then used to define a virtual partitioning of the pelvic volume into cubic interior volumes in ANSYS Mechanical APDL (ANSYS Inc., Canonsburg, Pennsylvania, USA) and meshed using linear hexahedral elements.

Kriging interpolation

For each subject, the personalised hexahedral FE mesh was obtained by computing the geometrical deformation of the source mesh (surface nodes of the generic FE mesh) to the target mesh (nodes of the 3D surface mesh reconstruction). This deformation was performed using a dual kriging algorithm [START_REF] Trochu | A contouring program based on dual Kriging interpolation[END_REF]. A particular attention was paid to the following functional areas: the acetabulum, the sacral promontory and the ischial tuberosities where higher density of control points was attributed. The kriging transformation was then applied to whole hexahedral FE mesh to define the patient specific mesh of the pelvis (Figure 1).

Mesh regularisation

Mesh regularization of generic meshes was carried out to correct eventual excessive mesh spatial distortions. The algorithm uses an optimisation-based method to minimize the errors measured using four mesh quality metrics namely jacobian condition number, skewness, warping factor and parallel deviation. Briefly, an objective function defined as the weighted sum of the four metrics is minimised using the conjugate gradient method. The resulting mesh quality was subsequently analysed using standard ANSYS Mechanical APDL mesh quality metrics: Aspect Ratio, Parallel Deviation, Maximum Angle, Jacobian Ratio and Warping Factor.

Point-to-surface comparison

For verification purposes, the personalised surface FE mesh was compared to the initial 3D reconstruction surface mesh. Point (nodes of source FE mesh) to-surface (target 3D reconstruction mesh) distances were computed in 3 zones: (i) contact zones (ii) zones of high curvature and (iii) the remaining zones.

Results

Generic FE Mesh

The generic female FE mesh and male FE mesh are composed of 3014 and 2746 linear hexahedral elements respectively. Table 1 summarizes the mesh quality metrics before and after mesh regularization.

Personalised FE Mesh

Table 2 summarises the average and maximal values of the quality metric for the 120 personalised FE meshes for both the female subject specific meshes and male subject specific meshes. Overall, the kriging interpolation does not introduce irregular or low quality elements that need to be repaired before proceeding to FE analysis. Each of the subject specific FE hexahedral meshes was automatically computed in less than 0.5 s (Figure 2).

FE surface mesh accuracy

The average point-to-surface distances between the personalised surface FE mesh and the initial 3D reconstruction surface mesh was less than 2 mm. The maximum (local) error was 9.6 mm for the females subject specific FE meshes and 11 mm for males subject specific FE meshes and were localized in non-functional areas (Table 3). The maximum error computed in the contact zones was inferior to 6 mm for the acetabulum and at the base of the sacrum and lower than 3 mm in the zones of high curvature such as the ischion.

Table 3. Average point-to-surface distances over the 120 personalised FE meshes. A surface was generated from the mesh of all the patient pelvis. This surface was compared to the 3D reconstruction of those pelvis in terms of point-tosurface distances.

Discussion

Generation of subject-specific FE models of the pelvis is still an open question today because they are traditionally generated from clinical CT scans -thereby exposing subjects and patients to high levels of irradiation -and generally require intensive user intervention at multiple stages of the segmentation process [START_REF] Salo | Computed tomography landmark-based semi-automated mesh morphing and mapping techniques: generation of patient specific models of the human pelvis without segmentation[END_REF]. In this contribution, a new methodology for the generation of subject-specific all-hexahedral FE mesh of the pelvis from biplanar X-rays imaging has been proposed. Biplanar X-ray imaging exposes individuals to much lower doses irradiation than CT-scans and can be performed in the standing position providing a promising alternative to CT-scan. Moreover, the semi-automatic 3D reconstruction process only lasts about 20 min. Combined with the fully automatic process of FE mesh generation from 3D reconstruction proposed in this contribution (which takes less than half a second) the total time to generate the personalised all-hexahedral FE mesh of the pelvis from biplanar X-rays imaging takes around 20 minutes which represents a significant decrease in both time and operator effort.

The results obtained on a database composed of 120 subjects with widely different morphologies suggest algorithm robustness. Surface errors of the subject-specific FE meshes were inferior to 1.5 mm on average and lower than 6 mm in functional areas (acetabulum, base of the sacrum and ischium). These functional areas were the focus of more specific attention due to their functional role in the biomechanical response owing to the crucial role they play in the transmission of movements and forces in both joint regions and with the surrounding soft tissues. The initial 3D reconstruction surface mesh is also known to contain inaccuracies inherent to reconstruction process. In cases where high precision is needed, digitally reconstructed radiographs could be used [START_REF] Ghostine | Influence of patient axial malpositioning on the trueness and precision of pelvic parameters obtained from 3D reconstructions based on biplanar radiographs[END_REF].

Other research teams have reported satisfactory results with atlas-based mesh morphing techniques for the fast generation of subject-specific FE mesh [START_REF] Bucki | A fast and robust patient specific finite element mesh registration technique: application to 60 clinical cases[END_REF]. As compared to similar approaches, the proposed methodology allows to circumvent the segmentation of either CT scan or clinical MRI which, except for a few attempts [START_REF] Bijar | Atlas-based automatic generation of subject-specific finite element tongue meshes[END_REF], constitutes a major bottleneck for the fast generation of FE mesh.

The method proposed in this paper is promising for clinical application because it is fast, fully automatic and allows to build subject-specific all-hexahedral FE meshes for the pelvis in a postural position. The model opens new perspectives for bringing new insights on the biomechanics of the pelvis and on the osteo-articular system in general. It has also been shown recently that locating the joint axes based on biplanar radiographs allows to have a more accurate kinematics and kinetics prediction [START_REF] Sangeux | Which method of hip joint centre localisation should be used in gait analysis?[END_REF][START_REF] Sauret | On the use of knee functional calibration to determine the medio-lateral axis of the femur in gait analysis: comparison with EOS biplanar radiographs as reference[END_REF]. Defining the personalised FE mesh from biplanar X-ray images would also allow to use more accurate boundary conditions for the biomechanical simulations by taking advantage of the EOS-gait analysis coupling.

Financial disclosure

The auhors would like to thank the Fondation de L'Avenir for their financial support (grant number AP-RM-2016-030).

Figure 1 .

 1 Figure 1. Design of the patient specific mesh. (a) EOS images and the 3D semi-automatic reconstruction associated to the patient (lasts 20 min). (b) Virtual Generic 3D reconstruction of the pelvic bone. (c) Generic FE mesh. (d) Result of the kriging of the FE mesh on the subject-specific 3D reconstruction.

  values are shown because no errors occurred after the kriging. Av means average number of elements above the warning thresholds for the 120 subjects and Max means maximum number of elements above the warning thresholds among the 120 subjects.

Figure 2 .

 2 Figure 2. (a) Illustration of 8 generic female pelvic FE meshes (b) Illustration of 8 subject specific female pelvic FE meshes.

Table 1 .

 1 Quality of the generic meshes.TableLayout

	Models		Male model 1 † Male model 2 † Female model 1 † Female model 2 †
	Aspect ratio	Warning	0	0	0	0
		Error	0	0	0	0
	Parallel deviation	Warning	72	28	91	19
		Error	0	0	0	0
	Maximum angle	Warning	240	0	265	0
		Error	0	0	0	0
	Jacobian ratio	Warning	24	0	20	0
		Error	3	0	2	0
	Warping factor	Warning	138	0	249	0
		Error	1	0	0	0
	†Index 1 are models before regularization and index 2 are models after regularization. Warn means number of elements
	above the warning thresholds and Error the number of elements above the error thresholds.

Table 2 .

 2 Resulting quality of the subject specific meshes.Table Layout values are shown because no errors occurred after the kriging. Av means average number of elements above the warning thresholds for the 120 subjects and Max means maximum number of elements above the warning thresholds among the 120 subjects.

		Male subject-specific model	Female subject-specific model
	Tolerance †	Average	Maximum	Average	Maximum
	Aspect ratio	0	0	0	0
	Parallel deviation	34	45	41	50
	Maximum Angle	6	45	3	41
	†Only warning

Table Layout

 Layout

	Model	Male subject-specific mesh Female subject-specific mesh
	Average (mm)	1	1.3
	Standard deviation (mm)	1.6	1.8
	Maximum (mm)	11	9.6

Computer Methods in Biomechanics and Biomedical Engineering, 0 (2017), © 2018 Informa UK Limited, trading as Taylor & Francis Group 10.1080/10255842.2018.1469624

Conflict of interest

The authors declare no potential conflict of interests.

Author's ORCID

Hélène Pillet

AUTHOR QUERIES

Query: AQ1: Please review the table of contributors below and confirm that the first and last names are structured correctly and that the authors are listed in the correct order of contribution. This check is to ensure that your names will appear correctly online and when the article is indexed.


Changping Zheng

Teodora Bavaro

Sara Tengattini

Andrea Gualla Mascherpa

Matthieu Sollogoub

Yongmin Zhang
email: yongmin.zhang@upmc.fr

Marco Terreni
email: marco.terreni@unipv.it[

Chemoenzymatic Synthesis of Glycoconjugates Mediated by Regioselective Enzymatic Hydrolysis of Acetylated 2-Amino Pyranose Derivatives

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Glycoproteins and glycolipids are involved in many physiological and pathological processes. [1] Consequently, synthetic non-natural glycoconjugates are largely investigated for developing new therapeutic strategies, [2] including the use of glycolipids with anticancer activity [3] or carbohydratebased vaccines. [4][5][6][7] In particular, sialylated oligosaccharides are involved in cell-cell adhesion processes [8] and many of these oligosaccharides are considered as tumor associated carbohydrate antigens (TACAs) because overexpressed in cancer cells. Specific glycosphingolipid types, which are detectable in normal cells, are more highly expressed in tumors. The high level of expression on the surface of tumour cells causes an antibody response towards these glycosphingolipids, which are therefore considered as targets for cancer therapy [9] . For example the lactosyl-ganglioside GM3 (NeuAcα3Galβ4Glcβ1Cer), is recognized as an important melanoma-associated antigen, and may have a role in metastasis. [10,11] Similarly, Sialyl-Le X , sialyl-α-1,6-galactosamine (Sialyl-Tn antigen), and other sialylated TACAs with a core of N-acetyl glucosamine or galactosamine, are over-expressed in a wide variety of human cancers. [12] Glycoconjugated lipid analogues of GM3, obtained by sialylation of glucose at C-6, recently have been reported as a new class of potential anticancer agents. [3] Semi-synthetic glycoproteins (neoglycoproteins) can instead provide immunogens from which therapeutic agents can be derived. [13,14] In particular, neo-glycoproteins have been largely investigated as therapeutic anticancer vaccines [15,16] based on TACAs. Sialyl-Tn has been proposed as therapeutic vaccine against prostate cancer, and its assembly with other TACAs has been proposed for developing multivalent glycoconjugate vaccines. [17] However, the study of glycoconjugates is limited by the complex procedures required for the preparation of protected building blocks bearing free hydroxyl groups in the desired positions. In presence of glucosamine or galactosamine, the synthesis of oligosaccharides is currently performed protecting the amino as acetyl-amide, [START_REF] Van Kasteren | Proc, Natl. Acad. Sci[END_REF][START_REF] Gamblin | [END_REF] as naturally existing in the TACAs structure. However, protection as phthalimide is often preferred. [20] At the anomeric position of the acceptor, different groups can be used, including thio-alkyl/aryl leaving groups, because after glycosylation of the free hydroxyl group, these products can be used as sugar donors for a further glycosylation reaction. [21] In addition, for the synthesis of neo-glycoproteins should be done the preparation of building blocks with an appropriate reactive linker at anomeric position, required for conjugation with the protein carrier.

One of the most used strategies implies the use of 2-iminomethoxyethyl thioglycosides (IME) that selectively reacts with lysines on the protein surface.

The use of enzymatic regioselective hydrolysis of peracetylated sugar has been proposed as an important tool for developing simple and efficient synthesis of sugar building blocks. [22] This strategy was employed for the preparation of mannopyranose glycans with the anomeric thiocyanomethyl group, that were used for protein glycosylation after activation as anomeric IME-linker. [23,24] Peracetylated monodeprotected galactopyranoses have also been used for the preparation of sialylated glycoconjugate lipids. [25] The most used biocatalyst for regioselective deacetylation is the lipase from Candida rugosa (CRL).

However, other enzymes were also used. In particular, Acetyl xylan esterase from Bacillus pumilus (AXE), has been successfully employed in the preparation of advanced building blocks by regioselective deacetylation of acetylated disaccharides. [26] The use of this chemoenzymatic approach has never been investigated for the preparation of glyco-derivatives (neo-glycoproteins and glycolipids) using TACAs with a core of glucosamine or galactosamine, such as the STn antigen. Consequently, in this work, we have studied the regioselectively enzymatic hydrolysis of 2-amino-pyranoside derivatives (Scheme 1), exploring the effect on the enzyme activity and selectivity induced by the presence of different reactive groups in anomeric position and by the protection of the 2-amino function.

Scheme 1

The results obtained demonstrated that this approach is versatile and can be conveniently used for the preparation of different building blocks bearing only a free hydroxyl group at C-6 with presenting different functional groups at anomeric position, including the thio-cyanomethyl group currently employed for protein glycosylation. These intermediates can be conveniently glycosylated with different sugar donors, allowing the preparation of glycans used for the synthesis of glycoconjugated derivatives (neo-glycoproteins and glycosphingolipids) including sialylated products such as the STn antigen.

Results and Discussion

Enzymatic hydrolysis of different substrates

In this work, two immobilized enzymes have been compared (CRL and AXE) for the preparation of various glucosamine and galactosamine building blocks. A screening has been performed at low substrate concentration (10 mM) and in presence of 20% of acetonitrile in order to ensure the complete solubility and hydrolysis of the substrates (Table 1).

The esterase AXE was used immobilized by covalent attachment on epoxy carrier since covalent immobilization generally ensures the stabilization of enzymes such as esterases or proteases. In the case of CRL, this enzyme was used immobilized by adsorption on hydrophobic carrier because this method is appropriate to obtain good stability and activity of lipases. [22] Except for 2-deoxy-2-acetamido-1,3,4,6-tetra-O-acetyl-α-D-glucopyranoside 1 (since AXE was reported to catalyze hydrolysis the anomeric position of pyranoses with acetoxy group), [27] considering the other N-acetylated substrates 2-5, the esterase AXE allowed best performances. In fact, the pure αanomer of 1, with the anomeric acetyl leaving group, can be used as substrate for immobilized CRL according to the procedure previously reported. [28,29] In this work, we used CRL immobilized on Sepabeads-OD (a C18 hydrophobic carrier) and the obtained results (Table 1) were similar (94% of 2deoxy-2-acetamido-1,3,4-tri-O-acetyl-α-D-glucopyranose 12) to those previously reported using CRL immobilized on octyl-agarose (a C8 hydrophobic carrier). However, CRL-Sepabeads-OD permitted much higher reaction rate providing almost quantitative hydrolysis of 1 in 24 hours. When the same reaction condition was used, the CRL immobilized on C8 agarose provided only 75% of the substrate hydrolyzed in 48 hours. The enzymatic hydrolysis of 1 was performed up to 20 mM concentration (about 8 g/L) and 92% yield of 12 was obtained after 24 hours. In addition, the hydrolysis of 1 with CRL was also tested using the α and β mixture (5/1) avoiding the separation of the two anomers after the chemical synthesis of this substrate (Table 1). The enzyme can be completely selective to provide the pure α anomer of 12 in 80% yield, corresponding to almost complete hydrolysis of the α-form of 1.

In this case, the β anomer remains completely unreacted and can be easily separated from the α-12.

Using AXE, methyl 2-deoxy-2-acetamido-3,4-di-O-acetyl-β-D-glucopyranoside 13 was prepared in 94% yield (Table 1), much better compared with the result obtained by means of CRL (70% yield).

Similarly, yields ranging from 65% to 90% of various sugar building blocks have been obtained with AXE, starting from N-acetyl glucosamine derivatives with an anomeric S-alkyl group, including the thio-cyanomethyl reactive linker used for the glycosylation of proteins. In fact, cyanomethyl 2-deoxy-2acetamido-3,4,-di-O-acetyl-1-thio-β-D-glucopyranoside 14 was prepared in 90% yield. This reaction was scaled up to 50 mM concentration (about 20 g/L) of substrate 3 (see Table S1 1).

Synthesis of disaccharides and glycoconjugated derivatives

The different building blocks prepared by enzymatic hydrolysis have been then used as intermediates for the preparation of disaccharides. Starting from 14, products 23 and 24 (with the anomeric thiocyanomethyl reactive linker) have been synthesized in almost pure anomeric form with good yields (Scheme 2).

Scheme 2

These compounds, after activation to obtain the corresponding deprotected iminomethoxyethyl thioglycosides, (see Supporting Information), have been used for the preparation of neo-glycoproteins 23a and 24a in 80% and 100% yields respectively, as resulting by MS analysis (Figure 1) considering the % of glycosylated protein (RNase-A selected as model of carrier protein). These results demonstrate that the proposed approach, mediated by regioselective hydrolysis catalyzed by AXE, is suitable for the preparation of neo-glycoproteins with glycans having a glucosamine core.

Figure 1

However, when the building block 14 was used for the preparation of sialylated glyco-derivatives (potentially advantageous for the preparation of sialylated neo-glycoprotein), the glycosylation with sialyl xanthate afforded product 25 (55% yield) in 3/2 α/β mixture (Scheme 2). Similarly, sialylation of N-acetyl glucosamine and galactosamine building blocks 12 and 21, allowed preparation of the disaccharides 26 and 27 as a mixture of α/β anomers.

The difficulties intrinsic in the stereo-controlled synthesis of α-sialosides have been largely reported and complex procedures are required in order to obtain pure or almost pure anomers. [30] In fact, α/β mixtures are obtained in different ratio using monodeprotected sugar acceptors, regardless the protection used for the C-2 amino group of the pyranose acceptor or using precursors with the 2-azido group. [31,32] In our work, this effect was observed regardless the kind of sialyl-donor used in the glycosylation of 2-deoxy-2-acetamido glucose or galactose building blocks. In addition, the sialylation of 21 was investigated in different conditions (including the use of sialyl chloride donor), but in all cases similar yields and an anomeric mixtures of 27 was obtained (see Supporting Information).

Surprisingly, high stereoselectivity was obtained when N-phthalimido glucosamine or galactosamine intermediates were considered for the sialylation after enzymatic deprotection at C-6. Accordingly, it was possible to perform the preparation of sialyl-disaccharides with different reactive groups at C-1, in pure or almost pure α-form (Scheme 3). Product 28 (with 1-α-acetoxy) was obtained in 70% yield as almost pure α anomer, allowing results much better compared with those previously reported. [31] Similarly, pure α anomer of 29 (with 1-β-methoxy) was obtained in 64% yield.

Scheme 3

Much poorer performances were observed when sialyl xanthate was used as donor for sialylation of Nphthalimido-glucosamine 19 and galactosamine 22 (with 1-β-thioethyl), probably as consequence of the activation of the anomeric S-alkyl group in the reaction condition used that causes the formation of by-products. In fact, starting from N-phthalyl-glucosamine 19, pure 30 was obtained only in 32% yield, and the sialyl-Tn derivative 31 was produced in 30% yield from N-phthalyl-galactosamine 22 (see Supporting Information). For improving the yield in the sialylation of N-phthalimido building blocks, the sialyl chloride was investigated as donor in the glycosylation of intermediates 19 and 22 (Scheme 3). In this condition, 65% and 70% yields were obtained respectively for product 30 and sialyl-Tn precursor 31 (in both cases as 95/5 mixture of α/β isomers).

These results suggest that the presence of an acetamido group at position C-2, unlike the 2-phthalimido group, could be responsible for the formation of the β-anomer during sialylation. In particular, the 2acetamido group could be involved in the formation of a hydrogen bond with the carboxy-ester of the sialic acid donor. Accordingly, this effect was not observed in the glycosylation of N-acetamidoglucosamine intermediate 14 with the Gal-TCA or Man-TCA. In fact, disaccharides 23 and 24 were obtained in pure β and α-form, respectively (Scheme 2). Consequently, the use of 2-phthalimido pyranoses should be preferred for the synthesis of sialyl derivatives.

Finally, starting from the sialylated disaccharide 28 (Scheme 3), after removing the anomeric acetyl group (yielding 75% of the crude product 32) and activation as trichloroacetimidate (yield in 60% of 33 after purification), the glycosylation of the 3-O-benzoyl-azidosphingosine building block provided the sialylated glycosphingolipid 34 in 61% yield.

Conclusions

In summary, a new chemoenzymatic synthetic strategy has been investigated and optimized for the preparation of neo-glycoproteins and glycolipids with a core of glucosamine or galactosamine, which are interesting as potential anticancer agents. A regioselective enzymatic hydrolysis can be conveniently used for the synthesis of 2-amino-pyranose building blocks bearing free hydroxyl group in primary position, starting from different acetylated 2-deoxy-2-acetamido or 2-deoxy-2-phthalimido gluco-and galactopyranoses. However, depending on the product desired, the appropriate starting material (substrate) and enzyme should be selected.

Thus, AXE can be used for the synthesis of neo-glycoproteins with an N-acetyl glucosamine core. In fact, the hydrolysis catalyzed by this enzyme provided different intermediates with a β-1-thio-(S-alkyl) group in very good yields (from 65% to 95%). For example, cyanomethyl 2-deoxy-2-acetamido-3,4,-di-O-acetyl-1-thio-β-D-glucopyranoside 14 was prepared in 90% yield and, after glycosylation, two disaccharides with the thiocyanomethyl reactive linker at anomeric position were obtained and used for the preparation of neo-glycoproteins.

CRL provides C-6 monodeprotected 2-N-phthalyl pyranose building blocks in good yields (from 65% to 85%) with different groups at anomeric position that can be used for the preparation of new sialylated glycoderivatives. Accordingly, the synthesis of protected Sialyl-Tn antigen 31 has been performed in two steps (56% yield) while the glycosphingolipid 34 has been prepared in 3 steps in 40% yield.

The monodeprotected compounds obtained in this work can be used as intermediates for the preparation of different products as potential new anticancer agents. Different glycosphingolipids will be prepared using this synthetic route and investigated as anticancer GM3 analogues.

Experimental Section

Materials and general experimental methods All the new compounds were fully characterized by 1 H and 13 C NMR, as well as HRMS. All the known products were characterized only by 1 H NMR by comparison with literatures.

Synthesis of substrates 1-11

Synthesis of substrates 1-11 has been performed using conventional chemical procedures. Experimental detail and analytical characterization are reported in the Supporting Information.

Immobilization of Candida rugosa lipase (CRL)

Immobilization of CRL was performed slightly modifying the procedure previously reported. [33] Briefly, the crude extract of CRL (2 g) was diluted in 25 mM phosphate buffer pH 7 (32 mL) and kept stirring for 30 min. 5 g of octadecyl-Sepabeads TM , previously washed with the same buffer, was added and the suspension was stirred at r.t. for 3 h. The enzyme derivative (CRL-OD) was then filtered and washed with distilled water.

The activity of the enzyme was measured at pH 7.0 and r.t. in the hydrolysis of tripropionin according to the literature. [34]

Enzymatic hydrolysis

The enzymatic hydrolysis of substrates 1-11 (10 mM)

Analytical data of enzymatic products 12-22 ( 1 H, 13 C NMR and MS)

2-acetamido-1,3,4-tri-O-acetyl-2-deoxy-α-D-glucopyranoside (12). R f = 0.36 (CH 2 Cl 2 -MeOH, 95:5).

The 1 H NMR is accordance with the literature previously reported. [28] Methyl-2-acetamido-3,4-di-O-acetyl-2-deoxy-β-D-glucopyranoside (13). The 1 H NMR is in accordance with the literature previously reported. [35] R f = 0.30 (CH 2 Cl 2 -MeOH, 95:5). Cyanomethyl 2-acetamido-3,4-di-O-acetyl-2-deoxy-1-thio-β-D-glucopyranoside (14). R f = 0.28 (CH 2 Cl 2 -MeOH, 95:5). The 1 H NMR is accordance with the literature previously reported. [36] 1,3,4-tri-O-acetyl-2-phthalimido-2-deoxy-α-D-glucopyranoside (17). R f = 0.36 (Toluene-EtOAc, 3:2).

The 1 H NMR is accordance with the literature previously reported. [37] Methyl -3,4-di-O-acetyl-2-phthalimido-2-deoxy-β-D-glucopyranoside (18). The product is as colourless oil. R f = 0.40 (Hexane-EtOAc, 1:1). 1 H NMR (400 Hz, CDCl 3 ): δ 7.88-7.84 (m, 2H, phthalimido protons H), 7.75-7.73 (m, 2H, phthalimido protons), 5.83 (dd, J=9.8, 1.6 Hz, 1H, H-3), 5.34 (d, J=9.6 Hz, 1H, H-1), 5.12 (t, J=9. 2 Hz, 1H, H-4), 4.28 (dd, J=8.8, 2.6 Hz, 1H, H-2), 3.84-3.81 (m, 1H, H-5), 3.71-3.64 (m, 2H, H-6), 3.45 (s, 3H, OCH 3 ), 2.07 (s, 3H, OAc), 1.87 (s, 3H, OAc). 13 C NMR (100 Hz,

CDCl 3 ): δ 20.67 (OAc), 20.92 (OAc) Ethyl -3,4-di-O-acetyl-2-phthalimido-2-deoxy-1-thio-β-D-glucopyranoside (19). R f = 0.41 (Hexane-EtOAc, 3:2). The 1 H NMR is accordance with the literature previously reported. [38] Phenyl -3,4-di-O-acetyl-2-phthalimido-2-deoxy-1-thio-β-D-glucopyranoside (20). R f = 0.38 (Hexane-EtOAc, 3:2). The 1 H NMR is accordance with the literature previously reported. [38] 1 H NMR (400 MHz, CDCl 3 ): δ 7.94-7.75 (m, 4H, phthalimido protons), 7.48-7.26 (m, 5H, Ph), 5.85 (dd, J=9.5 Hz, 1H, H-3), 5.79 (d, J=10.6 Hz, 1H, H-1), 5.14 (t, J=9.6 Hz, 1H, H-4), 4.39 (t, J=10.6 Hz, 1H, H-2), 3.84-3.61 (m, 3H, H-5, H-6), 2.05 (s, 3H, OAc), 1.87 (s, 3H, OAc). (21). R f = 0.30 (EtOAc-Hexane, 1:1).

2-acetamido-1,3,4-tri-O-acetyl-2-deoxy-α-D-galactopyranoside

The 1 H NMR is accordance with the literature previously reported. [28] 1 H NMR (400 MHz, CDCl3): δ 6. 25 (d, J=3.6 Hz, 1H, H-1), 5.58 (bd, 1H, NH), 5.45 (bdd, 1H, H-4), 5.30 (dd, J=8.7, 3.1 Hz, 1H, H-3), 4.80 (ddd, J=11.0, 9.8, 3.6 Hz, 1H, H-2), 4.08 (t, J=12.8 Hz, 1H, H-5), 3.50-3.64 (m, 2H, H-6), 2.23 (s, 3H, OAc), 2.18 (s, 3H, OAc), 2.08 (s, 3H, OAc), 1.96 (s, 3H, NAc).

Ethyl -3,4-di-O-acetyl-2-phthalimido-2-deoxy-1-thio-β-D-galactopyranoside (22). The product is as a white solid, m.p.: 78-80 ℃. R f = 0.35 (Hexane-EtOAc, 3:2). 1 H NMR (400 Hz, CDCl 3 ): δ 7.87-7.85 (m, 2H, phthalimido protons), 7.76-7.74 (m, 2H, phthalimido protons), 5.85 (dd, J=10.6, 3.2 Hz, 1H, H-3), 5.50 (d, J=3.0 Hz, 1H, H-4), 5.48 (d, J=10.4 Hz, 1H, H-1), 4.63 (t, J=10.5 Hz, 1H, H-2), 3.98-3.95 (m, 1H, H-5), 3.78, 3.55 (2m, 2H, H-6) ), 123.77, 123.86, 134.42, 134.47, 134.54 (aromatic C), 167.60, 168.11, 169.80, 171.29 (C=O).

ESI-HRMS (m/z) calcd for C 20 H 23 NNaO 8 S [M+Na] + : 460.1042, found: 460.1025.

Synthesis of disaccharides 23-31

Cyanomethyl- (2',3',4',6'- Cyanomethyl- (2',3',4',6'-tetra-O-acetyl-α-D-mannopyranosyl) CHCN, Hb-6'), 3.30 (d, J=17.6 Hz, 1H, CHCN), 2.14, 2.13, 2.11, 2.09, 2.04, 1.98, 1.96 (7s, 21H, OAc). Cyanomethyl O-(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5- (25). 3.81,3.79,COOCH 3 ;3.51,3,25,SCH 2 CN;2.64,2.48,OAc,NAc. 13 C NMR ( 100MHz, CDCl 3 ): δ 171. 93-167.49, C=O; 115.18, CN; 98.42, 98.32, C-2; 83.53, 83.50, C-1'; 52.94, 52.77, COOCH 3 ; 51.12, C-2'; 49.33, C-5; 37.59, C-3; 23.22-20.61, NAc, OAc; 14.20 O- (methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero- 3,4-tri-O-acetyl-2-deoxy-α-D-glucopyranose (26). The method in the literature [39] was used with some modifications: A mixture of sialyl xanthates (137 mg, 0. (22 µL, 0.19 mmol) in the dry CH 2 Cl 2 (0.12 mL) was added by running the solution down the cold wall of the reaction flask. Finally, the mixture was stirred for 2.5 h at -68 o C. After that, the mixture was diluted with a suspension of silica gel (0.5 g) in EtOAc (3 mL), filtered through celite, washed with saturated aqueous NaHCO 3 and water, dried with MgSO 4 , and concentrated under reduced pressure. The residue was chromatographed (MeOH-EtOAc 1:20 to 1:5) to give disaccharide 26 (58 mg, 60%) as α and β isomer in the ratio of 2:1 from 1 H NMR. R f = 0.29 (MeOH-EtOAc 1:10). 1 H NMR (400 MHz, CDCl 3 ): δ 6. 22, 6.16, H-1'; 3.81, 3.79 COOCH 3 ; 2.63, 2.47, H-3eq; 2.20-2.05, OAc; 1.94-1.89 NAc. 13 C NMR (100 MHz, CDCl 3 ): δ 171. 93-167.49, C=O; 98.42, 98.32, C-2; 90.82, 90.68, C-1'; 52.94, 52.77, COOCH 3 ; 51.21, 51.12, C-2'; 49.33, C-5; 37.59, C-3; 23.22-23.08, NAc; 21.18-20.61 O- (Methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α/β-D-galacto-2nonulopyranosylonate)-( 2→6) -2-acetamido-1,3,4-tri-O-acetyl-2-deoxy-α-D-galactopyranose (27) O-(methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2nonulopyranosylonate)-( 2→6) -1,3,4-tri-O-acetyl-2-phthalimido-2-deoxy-α-D-glucopyranoside (28) 13 C NMR (100 MHz, CDCl 3 ): δ 171. 17, 170.73, 170.40, 170.32, 170.22, 170.18, 169.83, 169.56, 169.52, 167.79 21.29, 21.15, 20.99, 20.96, 20.92, 20.85, 20.81 (CH 3 , OAc). ESI-HRMS (m/z) calcd for C 34 H 47 NO 22 Na [M+Na] + : 931.2596, found: 931.2590. O-(methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5- -3,4-di-O-acetyl-2-phthalimido-2-deoxy-β-D-glucopyranoside (29) 13 C NMR (100 MHz, CDCl 3 ): δ 171. 17, 170.72, 170.39, 170.33, 170.22, 170.18, 169.83, 169.56, 169.51, 167.80 (C=O), 134.51, 123.81 (aromatic C), 98.46 (C-2), 91.90 (C-1'), 72.47, 70.74, 69.26, 69.14, 68.01, 67.50, 67.33 21.29, 21.15, 20.99, 20.96, 20.92, 20.85 (CH 3 , OAc). ESI-HRMS (m/z) calcd for C 34 H 47 NO 22 Na [M+Na] + : 903.2647, found: 903.2658.

tetra-O-acetyl-β-D-galactopyranosyl)-(1→6)-2-acetamido-3,4-di-O-acetyl-2- deoxy-1-thio-β-D-glucopyranoside-(23).
dideoxy-D-glycero-α/β-D-galacto-2- nonulopyranosylonate)-(2→6)-2-acetamido-3,4-di-O-acetyl-2-deoxy-β-D-glucopyranose
α/β-D-galacto-2- nonulopyranosylonate)-(2→6)-2-acetamido-1,

Methyl

dideoxy-D-glycero-α-D-galacto-2- nonulopyranosylonate)-(2→6)
Ethyl-O- (methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2nonulopyranosylonate)-( 2→6) -3,4-di-O-acetyl-2-phthalimido-2-deoxy-1-thio-β-D-glucopyranoside (30).

The method in the literature was used [40] with some modifications: The acceptor 19 (20 mg

hydrolyzed in 4

 4 and 24 hours, respectively. Lower yield was obtained in the hydrolysis of phenyl 2deoxy-2-phthalimido-3,4,6-tri-O-acetyl-1-thio-β-D-glucopyranoside 9, because this reaction proceeded slowly (38% yield of product 20 in 24 hours).Finally, the enzymatic hydrolysis of galactosamine derivatives was also investigated. The hydrolysis of 2-deoxy-2-acetamido-1,3,4,6-tetra-O-acetyl-α-D-galactopyranose 10 (Table1), obtained by chemical synthesis in α and β mixture (5/1), has been performed using CRL. This reaction provided 2-deoxy-2acetamido-1,3,4-tri-O-acetyl-α-D-galactopyranose 21 (with the anomeric O-acetyl group) with 70% yield in pure α form (the β anomer is not hydrolyzed by CRL) and was scaled up by improving the substrate concentration up to 10 g/L (70% yield after purification). The hydrolysis of the ethyl 2-deoxy-2-phthalimido-3,4,6-tri-O-acetyl-1-thio-β-D-galactopyranoside 11 proceeded much faster compared with the fully acetylated galactosamine 10, and ethyl 2-deoxy-2-phthalimido-3,4-di-O-acetyl-1-thio-β-D-galactopyranoside 22 was obtained in very good yield (80% after purification) after only 5 hours of reaction (Table

  2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-α-D-glucopyranoside (1) and 2-acetamido-2-deoxy-Dgalactopyranoside, Candida rugosa lipase were purchased from Sigma-Aldrich (Italy, Milan). The octadecyl-Sepabeads TM was kindly supplied by Resindion (Italy). Immobilized AXE was kindly donated by ACS Dobfar (Italy). All chemicals were purchased as reagent grade and used without further purification. All chemical reactions were carried out under N 2 atmosphere and anhydrous conditions with freshly distilled solvents, unless otherwise noted. Reactions were monitored by thin-layer chromatography (TLC) on a pre-coated plate of silica gel 60 F254 (Merck) and detection by staining with sulfuric acid. Solvents were evaporated under reduced pressure and below 40 °C (water bath).Column chromatography was performed on silica gel 60 (230-400 mesh, Merck). 1 H NMR and 13 C NMR spectra were recorded at 400 MHz with Bruker AVANCE DRX 400 spectrometer. The chemical shifts were referenced to the solvent peak, 7.26 ppm ( 1 H) and 77.16 ppm ( 13 C) for CDCl 3 at 25 °C, and coupling constants were given in Hz. High-resolution mass spectra (HRMS) were recorded with a Bruker micro-TOF spectrometer in electrospray ionization (ESI) mode, using Tuning-Mix as reference.

  was carried out in 50 mM phosphate buffer (15 mL) containing 20% acetonitrile (for 6 and 7, 25% of acetonitrile for complete solubility of the substrates) under mechanical stirring. The reaction started after the addition of 1 g of immobilized enzyme CRL-OD (1200 UI/g) or AXE (90 UI/g). During the reaction, the pH of the solution was maintained by automatic titration. The course of the hydrolysis reaction was monitored by HPLC (HPLC analysis: 30-50% acetonitrile in phosphate buffer (10 mM) at pH 4, flow rate 1.0 mL/min, λ = 210 nm) and TLC. After complete or almost-complete consumption of the substrate, the reaction was stopped by biocatalyst filtration, and the obtained products were isolated by extraction with ethyl acetate followed by purification by flash chromatography (product 12, 13 and 14: CH 2 Cl 2 -MeOH, 95:5, product 15: CH 2 Cl 2 -Et 2 O, 7:3, product 16: EtOAc-Hexane, 4:1, product 17: Toluene-EtOAc, 3:2, product 18 and 21: Hexane-EtOAc, 1:1, product 19, 20 and 22: Hexane-EtOAc, 3:2) and characterized by NMR spectra and mass spectrometry.

2 , 3 , 4 , 6 - 37 (

 234637 Tetra-O-acetyl-α-D-galactopyranose trichloroacetimidate (0.137 g, 0.278 mmol) and compound 14 (0.05 g, 0.139 mmol) were co-evaporated with toluene (2×10 mL) and dried under reduced pressure. The mixture was then dissolved in dry CH 2 Cl 2(25 mL) in presence of 4Å molecular sieves and cooled at 0 °C. The BF 3 •Et 2 O (0.034 mL, 0.278 mmol) was added and the reaction allowed to warm to r.t. After 2.5 h, the reaction mixture was quenched with Et 3 N, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (EtOAc-Et 2 O, 3:2) to afford disaccharide 23 as a white solid (0.065 g, 68 %). R f = 0.EtOAc-Et 2 O, 3:2).White solid: m.p.: 106-108 ℃. 1 H NMR (400 MHz, CDCl 3 ): δ 5.71 (d, J=10.0 Hz, 1H, NH), 5.21-5.10 (m, 2H, H-4', H-3), 5.01 (dd, J=10.5, 3.0 Hz, 1H, H-2'), 4.94 (t, J=9.1 Hz, 1H, H- 3'), 4.64 (d, J=10.1 Hz, 1H, H-1), 4.56 (d, J=7.8 Hz, 1H, H-1'), 4.21-4.09 (m, 4H, H-2, H-4, H-6a, H- 6a'), 3.93-3.87 (m, 2H, H-6b, H-6b'), 3.79-3.73 (m, 2H, H-5, CHCN), 3.65-3.60 (m, 1H, H-5'), 3.27 (d, J=17.3 Hz, 1H, CHCN), 1.96-2.14 (7s, 21H, OAc).13 C NMR (100 MHz, CDCl 3 ): δ 14.55 (SCH 2 CN),20.39-29.57 (7CH 3 ), 52.70 (C-2), 61.38 (C-6), 67.14 (C-6'), 68.50 (C-5'), 68.81 (C-4), 68.85 (C-2'), 70.88, 70.89 (C-3', C-4'), 73.39 (C-3), 77.77 (C-5), 83.48 (C-1), 101.44 (C-1'), 116.86 (SCH 2 CN), 169.56-171.40 (7C=O). ESI-HRMS (m/z) calcd for C 28 H 38 N 2 NaO 16 S [M+Na] + : 713.1840, found: 713.1859.

  -(1→6)-2-acetamido-3,4-di-O-acetyl-2- deoxy-1-thio-β-D-glucopyranoside(24). 2,3,4,6-tetra-O-acetyl-α-D-mannopyranose trichloroacetimidate (0.20 g, 0.417 mmol) and compound 14 (0.075 g, 0.208 mmol) were co-evaporated with toluene (2×10 mL) and dried under reduced pressure. The mixture was then dissolved in dry CH 2 Cl 2 (25 mL) in presence of 4Å molecular sieves and cooled at 0 °C. The BF 3 •Et 2 O (0.058 mL, 0.417 mmol) was added and the reaction allowed to warm to r.t. After 2.5 h, the reaction mixture was quenched with Et 3 N, filtered and concentrated in vacuum. The residue was purified by flash column chromatography (EtOAc-Et 2 O, 3:2) to afford disaccharide 24 as a white solid (0.1 g, 70 %). R f = 0.39 (EtOAc-Et 2 O, 3:2). White solid: m.p.: 99-102 ℃. 1 H NMR (400 MHz, CDCl 3 ): δ 5.92 (d, J=9.6 Hz, 1H, NH), 5.31- 5.01 (m, 5H, H-4', H-3', H-1', H-4, H-4', H-5'), 4.66 (d, J=10.6 Hz, 1H, H-1), 4.58 (dd, J=13.6, 2.1 Hz, 1H, H-3), 4.28-4.03 (m, 5H, H-2, H-2', H-6, Ha-6'), 3.86 (t, J=9.6 Hz, 1H, H-5), 3.71-3.65 (m, 2H,

13 C

 13 NMR (100 MHz, CDCl 3 ): δ 14.72 (SCH 2 CN), 20.97-20.77 (6CH 3 , OAc), 23.21 (CH 3 , NAc), 52.96 (C-2), 62.33 (C-6, C'-6) , 62.62 (C'-2), 65.73, 68.67, 69.70, 70.13 (C-3, C'-3, C'-4, C'-5, C-4), 75.30 (C-5), 83.09 (C-1), 100.06 (C'-1), 116.17 (SCH 2 CN), 169.71-171.80 (7C=O). ESI-HRMS (m/z) calcd for C 28 H 38 N 2 NaO 16 S [M+Na] + : 713.1840, found: 713.1828.

  (C-6, C-5', C-4', C-4, C-8, C-3', C-7), 62.60 (C-9), 62.30 (C-6'), 60.53 (OCH 3 ), 53.67 (COOCH 3 ), 53.05 (C-2'), 49.50 (C-5), 37.89 (C-3), 23.33 (CH 3 , NAc),

Table 1 .

 1 Enzymatic Hydrolysis of Different Derivatives of Glucosamine and Galactosamine

	Substrates	Products	CRL: yield (h)	AXE: yield (h)
	1: R,R 4 =OAc; R 1 ,R 3 =H; R 2 =NHAc	12	94% (24)	10% (3) a
	1 b : R,R 4 =OAc; R 1 ,R 3 =H; R 2 =NHAc	12	80% (24)	-
	2: R,R 3 =H; R 1 =OMe; R 2 =NHAc R 4 =OAc	13	70% (24)	94% (24)
	3: R,R 3 =H; R 1 =SCH 2 CN; R 2 =NHAc; R 4 =OAc	14	68% (48)	90% (24)
	4: R,R 3 =H; R 1 =SEt; R 2 =NHAc; R 4 =OAc	15	<10% (48) a	85% (12)
	5: R,R 3 =H; R 1 =SPh; R 2 =NHAc; R 4 =OAc	16	<10% (48) a	65% (6)
	6: R,R 4 =OAc; R 1 ,R 3 =H; R 2 =NPhth	17	76% (6)	<10% (24) a
	7: R,R 3 =H; R 1 =OMe; R 2 =NPhth; R 4 =OAc	18	85% (4)	12% (8) a
	8: R,R 3 =H; R 1 =SEt; R 2 =NPhth; R 4 =OAc	19	64% (24)	30% (96) a
	9: R,R 3 =H; R 1 =SPh; R 2 =NPhth; R 4 =OAc	20	38% (24)	<10% (194) a
	10 b : R,R 3 =OAc; R 1 ,R 4 =H; R 2 =NHAc	21	70% (30)	-
	11: R,R 4 =H; R 1 =SEt; R 2 =NPhth; R 3 =OAc	22	80% (5)	-

a Yield evaluated by HPLC analysis without isolation of the product. b α/β=5/1. Experimental conditions: substrate 10 mM, volume 15 mL, phosphate buffer 50 mM, acetonitrile 20% (for substrates 6 and 7, 25% of acetonitrile was used), pH 5, r.t., enzyme 1 g of CRL-OD (1200 UI/g) or AXE (90 UI/g).

  The sialyl xanthates (248 mg, 0.42 mmol) and compound 14 (100 mg, 0.28 mmol) were dissolved in the CH 3 CN (4.5 mL) stirred for 1 h with 4Å powdered molecular sieves. Then it cooled to -40 °C, and NIS (187 mg, 0.83 mmol) and TfOH(36 µL, 0.46 mmol) were added. After 2 h, the reaction was quenched with Et 3 N.The mixture was diluted with CH 2 Cl 2 , filtered through celite, washed with 20% aqueous Na 2 S 2 O 3

	solution, dried over MgSO 4 , and concentrated under reduced pressure. The residue was purified by
	column (Cy-EtOAc, 1:4) to obtain the disaccharide 25 (127 mg, 55%) as α and β isomer in the ratio of
	3:2 from 1 H NMR. R f = 0.35 (Cy-EtOAc 1:4, twice). 1 H NMR (400 MHz, CDCl 3 ): δ 4.63, 4.62, H-1';

  , SCH 2 CN. ESI-HRMS (m/z) calcd for C 34 H 47 NO 22 Na [M+Na] + : 870.2579, found: 870.2571.

  , OAc. ESI-HRMS (m/z) calcd for C 34 H 47 NO 22 Na [M+Na] + : 857.2804, found: 857.2800.

  . A mixture of sialyl xanthates (343 mg, 0.58 mmol) and compound 21 (40 mg, 0.29 mmol) with 4Å powdered molecular sieves were dissolved in the dry CH 3 CN (6.0 mL) and CH 2 Cl 2 (3.0 mL) stirring at r.t. for 1 h. Further, AgOTf (123 mg, 0.47 mmol) and DTBP (115 µL, 0.53 mmol) were added, and the mixture was cooled to -68 o C and kept protected from light. Then PhSCl (54 µL, 0.47 mmol) in the dry CH 2 Cl 2 (0.3 mL) was added by running the solution down the cold wall of the reaction flask. Finally, the mixture was stirred for 3 h at -68 o C. After that, the mixture was diluted with a suspension of silica gel (1.25 g) in EtOAc (7.5 mL), filtered through celite, washed with saturated aqueous NaHCO 3 and water, dried with MgSO 4 , and concentrated under reduced pressure. The residue was chromatographed

	(MeOH-EtOAc 0:1 to 1:9) to give disaccharide 27 (150 mg, 64%) as α and β isomer in the ratio of 1:1
	from 1 H NMR. R f = 0.26 (EtOAc, twice). 1 H NMR (400 MHz, CDCl 3 ): δ 6.18, H-1'; 3.78, 3.76
	COOCH 3 ; 2.50, 2.43, H-3eq; 2.18-1.85, OAc, NAc. 13 C NMR (100 MHz, CDCl 3 ): δ 172.06-167.15,
	C=O; 98.84, 98.58, C-2; 90.32, C-1'; 53.12, 52.03, COOCH 3 ; 49.33, C-2'; 48.00, C-5; 37.88, 36.57, C-
	3; 23.26-20.81, NAc, OAc. ESI-HRMS (m/z) calcd for C 34 H 47 NO 22 Na [M+Na] + : 857.2804, found:
	857.2815.

  cooled to -68 o C and kept protected from light. Then PhSCl (337 µL, 1.38 mmol) in the dry CH 2 Cl 2 (0.86 mL) was added by running the solution down the cold wall of the reaction flask. Finally,

	the mixture was stirred for 3 h at -68 o C. After that, the mixture was diluted with a suspension of silica
	gel (3.6 g) in EtOAc (22.5 mL), filtered through celite, washed with saturated aqueous NaHCO 3 and
	water, dried with MgSO 4 , and concentrated under reduced pressure. The residue was chromatographed
	(Cy-EtOAc 1:3) to give disaccharide 28 (540 mg, 70%). R f = 0.31 (Cy-EtOAc 1:3, twice). 1 H NMR
	(400 MHz, CDCl 3 ): δ 7.84-7.81 (m, 2H, phthalimido protons), 7.73-7.71 (m, 2H, phthalimido protons),
	6.52 (dd, J=9.2, 2.2 Hz, 1H, H-3'), 6.24 (d, J=3.9 Hz, 1H, H-1'), 5.35-5.15 (m, 3H, H-4', H-7, H-8),
	4.89-4.82 (m, 1H, H-4), 4.68 (dd, J=8.2, 3.6 Hz, 1H, H-2'), 4.28-4.23 (m, 2H, H-5', H-9a), 4.03-3.99
	(m, 4H, Ha-6', H-5, H-6, H-9b), 3.80 (s, 3H, COOCH 3 ), 3.40 (dd, J=11.4, 1.6 Hz, 1H, Hb-6'), 2.65 (dd,
	. A
	mixture of sialyl xanthates (912 mg, 1.54 mmol) and acceptor 17 (370 mg, 0.85 mmol) with 4Å

powdered molecular sieves were dissolved in the dry CH 3

CN (17.2 mL)

and CH 2 Cl 2 (8.6 mL) stirring at r.t. for 1 h. Further, AgOTf (360 mg, 1.38 mmol) and DTBP (337 µL, 1.55 mmol) were added, and the mixture was J=12.7, 4.4 Hz, 1H, H-3eq), 2.14 (s, 3H, OAc), 2.13 (s, 3H, OAc), 2.09 (s, 3H, OAc), 2.05 (s, 3H, OAc), 2.03 (s, 3H, OAc), 2.02 (s, 3H, OAc), 2.01 (s, 3H, OAc), 1.96 (t, J=3.6 Hz, 1H, H-3ax), 1.85 (s, 3H, NAc).

  Cl 2 (0.13 mL) was added by running the solution down the cold wall of the reaction flask. Finally, the mixture was stirred for 3 h at -68 o C. After that, the mixture was diluted with a suspension of silica gel (0.42 g) in EtOAc (2.5 mL), filtered through celite, washed with saturated aqueous NaHCO 3 and water, dried with MgSO 4 , and concentrated under reduced pressure. The residue was chromatographed (Cy-EtOAc 1:3) to give disaccharide 29 (55 mg, 64%). R f = 0.47 (Cy-EtOAc 1:3, twice).1 H NMR (400MHz, CDCl 3 ): δ 7.88-7.86 (m, 2H, phthalimido protons), 7.76-7.74 (m, 2H, phthalimido protons), 5.78 (dd, J=9.1, 1.2 Hz, 1H, H-3'), 5.44 (d, J=10.9 Hz, 1H, H-1'), 5.32-5.17(m, 3H, H-4', H-7, H-8), 4.94- 4.85 (m, 1H, H-4), 4.68 (dd, J=9.2, 3.6 Hz, 1H, H-2'), 4.28-4.23 (m, 2H, H-5', H-9a), 4.06-4.01 (m, 4H, Ha-6', H-5, H-6, H-9b), 3.80 (s, 3H, COOCH 3 ), 3.40 (dd, J=11.4, 1.6 Hz, 1H, Hb-6'), 3.31 (s, 3H, OCH 3 ), 2.65 (dd, J=12.7, 4.4 Hz, 1H, H-3eq), 2.13 (s, 3H, OAc), 2.09 (s, 3H, OAc), 2.05 (s, 3H, OAc), 2.03 (s, 3H, OAc), 2.02 (s, 3H, OAc), 2.01 (s, 3H, OAc),1.96 (t, J=3.6 Hz, 1H, H-3ax), 1.86 (s, 3H, NAc).

	. A
	mixture of sialyl xanthates (117 mg, 0.20 mmol) and acceptor 18 (40 mg, 0.10 mmol) with 4Å
	powdered molecular sieves were dissolved in the dry CH 3 CN (1.66 mL) and CH 2 Cl 2 (0.83 mL) stirring

at r.t. for 1 h. Further, AgOTf (50 mg, 0.20 mmol) and DTBP (45 µL, 0.20 mmol) were added, and the mixture was cooled to -68 o C and kept protected from light. Then PhSCl

(24 µL, 0.21 mmol)

in the dry CH 2

  , 0.046 mmol) and acetochloroneuraminate methyl ester (70 mg, 0.138 mmol) in the dry CH 2 Cl 2 (4.6 ml) were stirred with 4Å powdered molecular sieves for 1 h at r.t.. Then it was cooled to -45 o C and kept protected from light, and AgOTf (83 mg, 0.322 mmol) was added. The mixture was gradually warmed to r.t. during 12 h, and further stirred at r.t. for 3 h. At last, filtered by celite and concentrated under reduced pressure. Regioselective hydrolysis of acetylated glucosamine and galactosamine derivatives 1-11 catalyzed by immobilized CRL and AXE (Table 1). BF 3 •Et 2 O , CH 2 Cl 2 , N 2 , 0 °C, (23, 68% yield; β>95%); b) 14, Man-TCA, BF 3 •Et 2 O, CH 2 Cl 2 , N 2 , 0°C, (24, 63% yield; α>95%); (ii) a) MeONa/MeOH, r.t., 24 h, 50% yield IME-glycans; b) sodium tetraborate buffer, pH 9.5, 25 °C, molar ratio IME-glycan/ribonuclease A 100:1, 6 h; 23a yield 81%; 24a yield>98%; (iii) a) 14, sialyl xanthate, NIS, TfOH, CH 3 CN; -40 °C; 25 yield 55% (α/β 3/2); b) 12, sialyl xanthate, PhSCl, AgOTf, CH 3 CN and CH 2 Cl 2 (2/1), N 2 ; -68 °C; 26 yield 60% (α/β 2/1); c) 21, sialyl xanthate, PhSCl, AgOTf, CH 3 CN and CH 2 Cl 2 ( 2/1), N sialyl xanthate, PhSCl, AgOTf, CH 3 CN and CH 2 Cl 2 (2/1), N 2 , -68°C; 28 yield 70% (>98%); 29 yield 64% (>98%); 30 yield 32% (>98%); 31 yield 30% (>98%); b) sialyl chloride, AgOTf, CH 2 Cl 2 , N 2 , -40 °C; 30 yield 65% (95%); 31 yield 70% (95%). (ii) 28, NH 2 CH 2 CH 2 NH 2 , AcOH, THF, r.t., 3 h, 32 (75% crude). (iii) 32, CCl 3 CN, DBU, CH 2 Cl 2 , -5 °C, 2 h, 33 (60%, α/β 4/1). (iv) 33, 3-O-

	Captions
	Scheme 1. Scheme 2. (i) a) 14, Gal-TCA, benzoyl-azidosphingosine, BF 3 •Et 2 O,	CH 2 Cl 2 ,	-30	°C,	1.5	h,	34	(61%).

2 ; -68 °C; 27 yield 64% (α/β 1/1).

Figure 1. MS spectra obtained in the glycosylation of RNase A with the glycans IME-23 and IME-24 to obtain neo-glycoproteins 23a (A) (average number of incorporated saccharides: 2.3) and 24a (B) (average number of incorporated saccharides: 4.1). Scheme 3. (i) a)

Acknowledgments

Authors would like specially to acknowledge Dr. Roberto Pavesi (ACS DOBFAR; Italy) that kindly provided immobilized AXE. Authors also thank the China Scholarship Council (CSC) for a Ph.D. fellowship to Changping Zheng. Financial supports from the Centre National de la Recherche Scientifique (CNRS) and the Sorbonne Université in France are gratefully acknowledged. Keywords: enzymatic hydrolysis • sialyl-Tn antigen • glycosphingolipids • neo-glycoproteins

Chemoenzymatic approach, mediated by Acetyl xylan esterase (AXE) from Bacillus pumilus and Candida rugosa lipase (CRL), permits the concise synthesis of neo-glycoproteins and glycosphingolipids, respectively.

Immobilized CRL