David Bliman

Martine Demeunynck

Pierre Leblond

Samuel Meignan

Isabelle Bausanne

Sebastien Fort

Enzymatically Activated Glyco-Prodrugs of Doxorubicin Synthesized by a Catalysis-Free Diels-Alder Reaction

The severe side effects associated with the use of anthracycline anticancer agents continues to limit their use. Herein we describe the synthesis and preliminary biological evaluation of three enzymatically activatable doxorubicin-oligosaccharide prodrugs. The synthetic protocol allows late stage variation of the carbohydrate and is compatible with the use of disaccharides such as lactose as well as more complex oligosaccharides such as xyloglucan oligomers. The enzymatic release of doxorubicin from the prodrugs by both protease (plasmin) and human carboxylesterases (hCE1 and 2) was demonstrated in vitro and the cytotoxic effect of the prodrugs were assayed on MCF-7 breast cancer cells.

Introduction

Despite continuing efforts in oncogenic drug development, surgery remains an important form of treatment for solid tumours. This approach generally includes a postoperative drug therapy to prevent reoccurrence of the tumour. Currently used drugs for such adjuvant treatment include the anthracycline antibiotic Doxorubicin (Dox). [START_REF] Kim | Lethal Effect of Adriamycin on the Division Cycle of HeLa Cells[END_REF] While the details of the mechanism of action of anthracyclines are still under investigation, [START_REF] Pérez-Arnaiz | New Insights into the Mechanism of the DNA/Doxorubicin Interaction[END_REF] the cytotoxic activity is suggested to be a result of intercalation with DNA [START_REF] Yang | Doxorubicin, DNA torsion, and chromatin dynamics[END_REF] and inhibition of topoisomerase II. [START_REF] Pommier | DNA Topoisomerases and Their Poisoning by Anticancer and Antibacterial Drugs[END_REF] Due to the unselective nature of the anthracyclines, treatments are often associated with severe side effects such as cardiac toxicity. 5 This has led to extensive efforts in reducing the systemic toxicity of Dox and other anticancer drugs by different types of prodrug approaches. [START_REF] Srinivasarao | Principles in the design of ligand-targeted cancer therapeutics and imaging agents[END_REF] In these strategies, the drug is modified in a way that minimizes its off target activity while allowing activation at the intended site. Prodrug approaches for Dox include both non-covalent and covalent modifications. A notable example of noncovalent prodrugs of Dox is the inclusion of Dox in liposomes. [START_REF] Barenholz | Doxil® -The first FDA-approved nano-drug: Lessons learned[END_REF] Lipid encapsulation of the drug contributes to decreasing toxic side effects, since liposomes can penetrate endothelial lesions found in the neovasculature of tumors but not in normal blood vessels. FDA approved pegylated and non-pegylated liposomal Dox formulations, namely Doxil ® and Myocet ® respectively for treating Aids related Kaposi's sarcoma, ovarian cancer and breast cancer. [START_REF] Waterhouse | A Comparison of Liposomal Formulations of Doxorubicin with Drug Administered in Free Form[END_REF] The covalent approach typically involves modification either on the primary amine to form carbamates, [START_REF] Mejia Oneto | In Vivo Bioorthogonal Chemistry Enables Local Hydrogel and Systemic Pro-Drug To Treat Soft Tissue Sarcoma[END_REF][START_REF] Amir | Prodrug Activation Gated by a Molecular "OR" Logic Trigger[END_REF] or on the ketone functionality by forming hydrazones. The pHsensitive hydrazone can be cleaved in the acidic microenvironment of the tumour [START_REF] Estrella | Acidity Generated by the Tumor Microenvironment Drives Local Invasion[END_REF] and the carbamate can be attached to an enzymatically hydrolyzable oligopeptide. Both approaches have been used to design cell-targeting prodrugs through the conjugation to antibodies. [START_REF] Tolcher | Randomized Phase II Study of BR96-Doxorubicin Conjugate in Patients With Metastatic Breast Cancer[END_REF][START_REF] Dubowchik | Cathepsin B-Labile Dipeptide Linkers for Lysosomal Release of Doxorubicin from Internalizing Immunoconjugates: Model Studies of Enzymatic Drug Release and Antigen-Specific In Vitro Anticancer Activity[END_REF] The diverse functionalities of carbohydrates make them potential candidates for use in prodrugs. Amazingly, there are very few examples of glycosylated prodrugs reported in the literature to date. Carbohydrates have mainly been used as a triggering group for the drug release since they can be hydrolyzed by the glycosidases overexpressed by cancer cells. The most prominent example is that of glucuronic acid [START_REF] Houba | A novel doxorubicin-glucuronide prodrug DOX-GA3 for tumour-selective chemotherapy: distribution and efficacy in experimental human ovarian cancer[END_REF][START_REF] Legigan | Synthesis and Antitumor Efficacy of a β-Glucuronidase-Responsive Albumin-Binding Prodrug of Doxorubicin[END_REF] that is degraded by β-glucuronidase which have been found at high levels in tumour tissue. [START_REF] Connors | Cure of Mice bearing Advanced Plasma Cell Tumours with Aniline Mustard : the Relationship between Glucuronidase Activity and Tumour Sensitivity[END_REF] Assets of carbohydrates are not restricted to their biocompatibility and biodegradability. They also present a large range of remarkable biological and physico-chemical properties. Carbohydrate-protein interactions play an important role for regulating biochemical processes, such as cell differentiation, proliferation and adhesion, inflammation, and immune response. The feasibility of using carbohydrate ligands to target protein receptors, termed 'glycotargeting', was first demonstrated in 1971. [START_REF] Rogers | Hepatic uptake of proteins coupled to fetuin glycopeptide[END_REF] Since then the potential of using carbohydrates to design drug-delivering systems has been made clear. However, small molecule drugs, no matter how heavily glycosylated they are, have the potential to pass into the kidneys, through glomerular filtration, and be rapidly cleared. For this reason, much effort are devoted to designing macromolecular glycosylated carriers and there is little work on the development of such glycotargeting prodrugs. [START_REF] Benjamin G Davis | Drug delivery systems based on sugarmacromolecule conjugates[END_REF] Carbohydrates also interact with other carbohydrates through the establishment of an extensive network of hydrogen bondings and of hydrophobic interactions. In particular, polysaccharidepolysaccharide interactions play an important role in the control of the architecture of animal and plant cells. Cellulose and xyloglucan (XG) assemble to form the cellulose/XG network, which is considered the dominant load-bearing structure in the growing cell walls of nongraminaceous land plants. In a biomimetic approach, Brumer et al. reported the efficient modification of cellulosic materials with different molecules including enzymes and the cell adhesion peptide RGD through anchorage of xyloglucan-based glycoconjugates at their surface. [START_REF] Zhou | Xyloglucan in cellulose modification[END_REF][START_REF] Fink | Bacterial cellulose modified with xyloglucan bearing the adhesion peptide RGD promotes endothelial cell adhesion and metabolism-a promising modification for vascular grafts[END_REF] A particular advantage of such non-covalent modification is to circumvent the limited reactivity of insoluble cellulose and to preserve the fibre's integrity. In the present work, we report the synthesis and preliminary biological evaluation of new enzymatically activated Dox glyco-prodrugs aimed at being administered in situ at the tumor site after resection. The carbohydrates, lactose and XGO, the oligosaccharide-repeating unit of xyloglucan, were chosen to address two specific roles. Lactose binds to galectins, a family of β-galactoside binding proteins associated with tumour malignancy. [START_REF] Thijssen | Galectin expression in cancer diagnosis and prognosis: A systematic review[END_REF][START_REF] Ruvolo | Galectin 3 as a guardian of the tumor microenvironment[END_REF] Interaction between lactose and galectins is expected to decrease diffusion of the glyco-prodrug and to hamper its rapid clearance thus allowing a sustained release of the Dox when administered in solution. On the other hand, the use of XGO could allow the immobilization of the Dox prodrug onto cellulosic wound dressing. The synthetic route consisted in the conjugation of furan-modified carbohydrates and maleimide-activacted Dox by a metal free Diels Alder cycloaddition (Figure 1). We have previously used thiol-maleimide Michael-type addition [START_REF] Petrelli | Oligosaccharide-based block copolymers: Metal-free thiol-maleimide click conjugation and self-assembly into nanoparticles[END_REF] coupling for conjugation of reducing-end functionalized carbohydrates. While being a metal-free reaction, this reaction requires handling sulfurylated molecules susceptible to oxidation. Another type of ligation reaction is the Diels-Alder cycloaddition. [START_REF] De Araújo | Diels-Alder Ligation and Surface Immobilization of Proteins[END_REF] The inverse electron demand Diels-Alder reaction (iEDDA) between 1,2,4,5-tetrazines and olefins has increasingly gained importance in chemical biology as a fast and metal-free bioorthogonal ligation reaction. [START_REF] Devaraj | Tetrazine-Based Cycloadditions: Application to Pretargeted Live Cell Imaging[END_REF][START_REF] Blackman | Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity[END_REF] Furan-maleimide cycloaddition is another type of popular Diels-Alder reaction having the advantage of using widely available reactants. This conjugation method recently allowed us to prepare glycosylated chromatographic matrices for the capture of carbohydrate-binding proteins. [START_REF] Petrelli | Efficient Conjugation of Oligosaccharides to Polymer Particles through Furan/Maleimide Diels-Alder Reaction: Application to the Capture of Carbohydrate-Binding Proteins[END_REF] This reaction allows catalysis-free introduction of the carbohydrate part in water or in organic solvent at a late stage, avoiding extensive carbohydrate protecting group strategies. The presence of a peptide-or ester-containing spacer arm between the maleimide group and the anthracycline should allow the enzymatic release of the Dox by endogenous peptidases or esterases.

Results and Discussion

Synthesis

We sought to obtain two types of glyco-prodrugs of Dox with different activating mechanisms. Our first strategy was to link the drug to the carbohydrate part through ester formation, permitting the release of the drug by esterases, particularly human carboxylesterase 2 that is commonly expressed in tumor tissues. [START_REF] Xu | Human carboxylesterase 2 is commonly expressed in tumor tissue and is correlated with activation of irinotecan[END_REF] The ester functionality is linked to the primary amine of Dox by a p-aminobenzyl alcohol (PABA) spacer. Hydrolysis of the ester bond will initiate a cascade reaction of this so-called self-immolative spacer, first reported by Katzenellenbogen, [START_REF] Carl | A novel connector linkage applicable in prodrug design[END_REF] ultimately resulting in the release of Dox. Our second strategy sought to enable Dox release by specific proteases. To this goal, we chose a tripeptide linker (D-Ala-Phe-Lys) [START_REF] Eisenbrand | An Approach Towards More Selective Anticancer Agents[END_REF] known to be cleaved by plasmin, which has been identified as a potential target protease for prodrug activation of cancer drugs. [START_REF] Chakravarty | Plasmin-activated prodrugs for cancer chemotherapy. 2. Synthesis and biological activity of peptidyl derivatives of doxorubicin[END_REF] This linker has previously been used in combination with the PABA self-immolative linker in the synthesis of Dox [START_REF] De Groot | Synthesis and Biological Evaluation of Novel Prodrugs of Anthracyclines for Selective Activation by the Tumor-Associated Protease Plasmin[END_REF] and Dox derivative prodrugs. [START_REF] Barthel | Synthesis and Biological Characterization of Protease-Activated Prodrugs of Doxazolidine[END_REF] The latter were shown to be stable in human plasma and poorly membrane permeable, thereby limiting activation to locally secreted proteolytic activity and reducing the likelihood of severe side effects.

Starting with the linker drug end of the prodrugs, the synthesis of the maleimidyl ester linked Dox (MalEsterDox 1) was performed starting from ω-aminocapronic acid 2 (Scheme 1). The five-carbon spacer length provided by the use of ω-aminocapronic acid has previously been reported for enzymatically cleaved prodrugs of Dox. [START_REF] Gao | Synthesis of Acid-Labile PEG and PEG-Doxorubicin-Conjugate Nanoparticles via Brush-First ROMP[END_REF] Scheme 1. Synthesis of 1. a) (Boc)2O (1.1 equiv.) in NaOH(aq.), 1,4-dioxane 1:2, 16 h, 89%. b) DCC (1 equiv.), DMAP (20 mol%), p-methoxybenzaldehyde (0.95 equiv.) in CH2Cl2, 0 ℃ to r.t., 21 h, 86%. c) NaBH4 (0.95 equiv.) in CF3CH2OH, 0 ℃, 1 h 20 min, 90%. d) TFA: CH2Cl2 1:1, r.t. 20 min, then e) 3-(N-maleimido)propionic acid (1.1 equiv.), DCC (1.1 equiv.), NMM (3 equiv.) in CH2Cl2, 0 ℃ to r.t., 17 h, 51% over 2 steps. f) Bis(4-nitrophenyl) carbonate (1.5 equiv.), DIPEA (3 equiv.) in CH2Cl2, r.t., 4 h, 70%. g) Dox*HCl (0.9 equiv.), Et3N (2 equiv.) in DMF, r.t., 22h, 59%.

tert-Butyloxycarbonyl (tBoc) protection of the amine with di-(tert-butyl)dicarbonate in the presence of sodium hydroxide (NaOH) gave carboxylic acid 3 which was coupled with phydroxybenzaldehyde by Steglich esterification to give ester 4 in 86% yield. The next step was the reduction of the aldehyde to the corresponding alcohol. Performing the reduction with NaBH4 in THF at 0°C resulted in multiple products as observed by TLC already after 15 min. Trifluoroethanol has previously been reported as a co-solvent for NaBH4 reductions. [START_REF] Tajbakhsh | Catalyst-Free One-Pot Reductive Alkylation of Primary and Secondary Amines and N,N-Dimethylation of Amino Acids Using Sodium Borohydride in 2,2,2-Trifluoroethanol[END_REF][START_REF] Setoi | Enantiospecific total synthesis of (-)swainsonine: new applications of sodium borohydride reduction[END_REF] Reduction of aldehyde 4 with NaBH4 in trifluoroethanol at 0°C resulted in full conversion after one hour and allowed us to isolate hydroxyl 5 in high yield (90%). Next, the maleimide (Mal) was introduced by tBoc deprotection with trifluoroacetic acid (TFA) followed by amidation with 3-maleimidopropionic acid in the presence of dicyclohexylcarbodiimide (DCC) and Nmethylmorpholine (NMM). MalEsterOH 6 was isolated in 51% over two steps. To introduce the carbamate-linked Dox, the hydroxyl of 6 was converted to a mixed anhydride by reaction with bis p-nitrophenoxyanhydride in the presence of N,N-diisopropylethylamine (DIPEA) in dichloromethane (DCM) (70% yield). Stirring 7 in dimethylformamide (DMF) at room temperature (r.t.) with Dox in the presence of triethylamine resulted in MalEsterDox 1 in 59% yield.

The synthesis of the peptide-linked glyco-prodrug started with the synthesis of the D-Ala-Phe-Lys tripeptide. The synthesis of this tripeptide has previously been described both in solution [START_REF] De Groot | Synthesis and Biological Evaluation of Novel Prodrugs of Anthracyclines for Selective Activation by the Tumor-Associated Protease Plasmin[END_REF] and on solid support. [START_REF] Barthel | Synthesis and Biological Characterization of Protease-Activated Prodrugs of Doxazolidine[END_REF] Our synthetic strategy for the Dox tripeptide motif started from Nα-Fmoc-Nε-Alloc-Lysine 8 which was activated by iso-butylchloroformate and functionalized with p-aminobenzylalcohol to give 9 in 72 % yield (Scheme 2). Scheme 2. Synthesis of 13. a) ibutylchloroformate (1.1 equiv.), p-aminobenzylalchohol (1.2 equiv.), NMM (2.3 equiv.) in THF, -40 ℃, 72%. b) 1,4-dioxane:MeOH: 2M NaOH (aq.), r.t., 20 min, then c) DMTMM (1 equiv.), FmocPheOH (1 equiv.), NMM (1.1 equiv.) in CH3CN, r.t., 19h, then d) 1,4-dioxane:MeOH: 2M NaOH (aq.), r.t., 20 min, then e) DMTMM (1 equiv.), Boc-D-AlaOH (1 equiv.), NMM (1.1 equiv.) in CH3CN, r.t., 14 h, 47% over 4 steps. f) TFA: CH2Cl2 0.8:1, r.t., 15 min, then g) DMTMMBF4 (1.1 equiv.), 3-(N-maleimido)propionic acid (1.2 equiv.), NMM (1.1 equiv.) in CH3CN, r.t., 22 h, 43% over two steps. h) bis(4-nitrophenyl) carbonate (5 equiv.), DIPEA (3 equiv.) in DMF, r.t., 46 h then i) Dox*HCl (0.9 equiv.), Et3N (2 equiv.) in DMF, r.t., 24h, 75%.

The tripeptide linker was synthesized by solution phase peptide synthesis with N-Fmoc protected amino acids using 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate (DMTMMBF4) [START_REF] Kamiński | A Study on the Activation of Carboxylic Acids by Means of 2-Chloro-4,6-dimethoxy-1,3,5-triazine and 2-Chloro-4,6-diphenoxy-1,3,5-triazine[END_REF][START_REF] Raw | An improved process for the synthesis of DMTMM-based coupling reagents[END_REF] as coupling reagent followed by deprotection with NaOH (aq.). The tripeptide 10 was obtained in 47% yield over 4 steps. The maleimide was introduced in a similar way as for the previously described linker by TFA deprotection of the tBoc followed by amidation to give MaltriPep 11 in 43% yield over two steps. The hygroscopic nature of the tripeptide in combination with the risk of hydrolyzing the maleimide made it necessary to thoroughly dry (high vacuum pump) and store all following intermediates below -15℃. As for the ester-based linker described above, the hydroxyl function of 11 was activated as a mixed anhydride by reaction with five equivalents of bis p-nitrophenoxy carbonate in the presence of DIPEA. The mixed anhydride 12 was isolated by precipitation in diethyl ether and was used in the following substitution without further purification. Stirring 12 with Dox and Et3N in DMF at room temperature gave MaltriPepDox 13 in 75% yield and high purity as confirmed by HPLC (see SI).

The next step was the conjugation of the drug-linker part with the carbohydrate moiety. The furfuryl-functionalized carbohydrates were obtained through a two steps method for synthesizing N-glycosylamides first introduced by Lokhoff et al. [START_REF] Lockhoff | Glycolipids as Immunomodulators: Syntheses and Properties[END_REF] and utilized in our group to synthesize a range of reducing-end functionalized oligosaccharides. [START_REF] Petrelli | Oligosaccharide-based block copolymers: Metal-free thiol-maleimide click conjugation and self-assembly into nanoparticles[END_REF][START_REF] Petrelli | Efficient Conjugation of Oligosaccharides to Polymer Particles through Furan/Maleimide Diels-Alder Reaction: Application to the Capture of Carbohydrate-Binding Proteins[END_REF] The unprotected carbohydrate was stirred in neat furfurylamine for 72 hours to afford a transient glycosylamine that was made non-hydrolyzable by acylation of the nitrogen with acetic anhydride. The synthesis of furfuryl-lactose 14 was previously published for the production of conjugatable glycosaminoglycans. [START_REF] Priem | Chemobacterial synthesis of conjugatable glycosaminoglycans[END_REF] The furfuryl-XGO 15 was synthesized for this study from the oligosaccharide-repeating unit of tamarind seed xyloglucan. First, the XGO repeating unit, which consists in a mixture of hepta-, octa-and nona-saccharides in a ratio of 15/35/50, was obtained quantitatively by enzymatic hydrolysis of the polysaccharide as reported previously. [START_REF] York | Structural analysis of xyloglucan oligosaccharides by 1H-n.m.r. spectroscopy and fast-atombombardment mass spectrometry[END_REF] The furfuryl-XGO was then obtained in 90% yield following the same protocol as for lactose. The conjugation of MalEsterDox 1 with furfuryl-lactose 14 in DMF and water (1:1) at 37 ºC gave LacEsterDox 16 in high yield (91%, Scheme 3). The XGOEsterDox prodrug 17 was prepared under similar reaction conditions but was isolated in 35% yield only after purification on reverse phase C18 cartridges. The yield is lower than typically observed in this type of cycloaddition and is partly explained by extensive chromatographic purification. The reaction conditions and the purification procedure should be optimized but sufficient quantities for drug release and cell toxicity studies were isolated and no attempt to improve the yield have been done so far. Scheme 3. Synthesis of 16 and 17. DMF:H2O 1:1, 37 ºC, 3 and 4 days for 16 and 17, respectively.

To obtain the tripeptide linked glyco-prodrug (LactriPep(Alloc)Dox) (18, Scheme 4), 13 and furfuryl-lactose 14 were stirred in a mixture of DMF and H2O (2:1) at 37℃ for 7 days. 18 (57% yield) was isolated in high purity as confirmed by HPLC (see SI) after purification on silica eluting with 10% water in acetonitrile. The last step of removal of the Alloc protecting group from the lysine side chain was more tricky. Commonly used conditions for Alloc removal include catalytic amounts of Pd 0 in the presence of an allyl scavenger such as morpholine, tributyltin hydride or silane. [START_REF] Guibé | Allylic protecting groups and their use in a complex environment part II: Allylic protecting groups and their removal through catalytic palladium π-allyl methodology[END_REF] However, using catalytic amounts of Pd(PPh3)4 (20 mol%) and morpholine as allyl scavenger, we were unable to isolate the desired product, only observing degradation on TLC. Eliminating the scavenger and running the reaction with equimolar amount of Pd(PPh3)4 in a mixture of acetic acid and dichloromethane as described by Barthel et al. [START_REF] Barthel | Synthesis and Biological Characterization of Protease-Activated Prodrugs of Doxazolidine[END_REF] gave the desired product 19 (LactriPepDox) in 59% yield after precipitation from diethyl ether.

Enzymatic release of Dox from glyco-prodrugs

To confirm the intended enzymatic release of Dox, the LactriPepDox 19 (100 µM) was incubated in vitro in the presence 15 µg/ml of human plasmin at 37 ºC in phosphate buffer (pH 7.5). The disappearance of prodrug and appearance of Dox was followed by HPLC (Figure 2). We first checked the stability of the prodrugs in these conditions in the absence of plasmin (no release after up to 25h at 37°C, data not shown). In the presence of plasmin, nearly full conversion of the prodrug into Dox was achieved after 2 h. In contrast, incubating 16 with human carboxylesterase 2 (hCE2) under identical conditions resulted in full release after 23h. The rate of hydrolysis for XGOEsterDox 17 was slightly slower than for 16 (83% after 23h), possibly owing to the steric effect of the larger oligosaccharide. Nevertheless, after 48h, 98% of 17 had been hydrolyzed. The difference in rate between hCE1 and hCE2 could be explained by the presence of a phenyl on the alcohol side since hCE2 is known to hydrolyze esters with bulkier alcohol groups at a higher rate than hCE1. [START_REF] Casey Laizure | The Role of Human Carboxylesterases in Drug Metabolism: Have We Overlooked Their Importance?[END_REF] As was observed for the tripeptide prodrug 19, incubating the ester prodrug (LacEsterDox) in the absence of esterase resulted in less than 2% Dox after 27h (data not shown). These results confirm that Dox is enzymatically released from prodrugs 16, 17 and 19 and that these three prodrugs are stable in the buffers in absence of enzyme.

Biological evaluation

As a preliminary test for verifying the biological interest of the prodrugs, the cytotoxicity of compounds 16, 17 and 19 was assayed on MCF-7 breast cancer cells and compared with that of free Dox (Table 1

Conclusion

We have synthesized a small series of enzymatically activatable glyco-prodrugs of Dox. We demonstrated the release of the drug by enzymatic cleavage in vitro and in cellulo on MCF-7 cancer cells, as the prodrugs and Dox itself display similar IC50. The late stage metal free cycloaddition used to ligate the oligosaccharide and the drug allows this methodology to be used with different oligosaccharides and potentially with different drugs. Current work in our groups includes extending this ligation to synthesize prodrugs of other drug classes.

Experimental procedures

General considerations

All commercial chemicals were used without prior purification. [START_REF] Kim | Lethal Effect of Adriamycin on the Division Cycle of HeLa Cells[END_REF] H-and [START_REF] Dubowchik | Cathepsin B-Labile Dipeptide Linkers for Lysosomal Release of Doxorubicin from Internalizing Immunoconjugates: Model Studies of Enzymatic Drug Release and Antigen-Specific In Vitro Anticancer Activity[END_REF] C-NMR spectra were obtained at 400 and 101 MHz respectively, using a Bruker Advance DRX 400 spectrometer. Solvent residual peaks were used as reference and calibrated as follows; CDCl3: 7.26ppm; MeOD-d4: 3.31ppm; DMSO-d6: 2.50 ppm. Reactions were monitored by TLC (Merck Silica gel 60 F254) and analyzed under UV (254 nm). Reactions involving carbohydrates were followed by staining TLC plates with H2SO4: MeOH : H2O (3:45:45). Column chromatography was performed by manual flash chromatography (wet-packed silica, 0.04-0.063 mm) or by automated column chromatography on a Grace Reveleris instrument using prepacked silica columns. Analytical high performance liquid chromatography (HPLC) was performed using a Waters µBondapak C18-column (3.9 x 300mm, 125Å pore size, 10 µm particle size) connected to a Waters photodiode array detector 996. HPLC method A: 10-100% MeOH in water (phosphoric acid pH 2.5) over 5 min, 2 ml/min. HPLC method B: 0-100% MeOH in water (phosphoric acid pH 2.5) over 5 min, then 100% water for 5 min, 2 ml/min. Mass analyses (ESI) were recorded on a Bruker Daltonics Esquire 3000+ instrument and HRMS analyses were obtained on a Waters Xevo G2-S QTOF instrument.

6-((tert-Butoxycarbonyl)amino)hexanoic acid (3) Following a published procedure, [START_REF] Gavande | Design, Synthesis, and Pharmacological Evaluation of Fluorescent and Biotinylated Antagonists of ρ1 GABAC Receptors[END_REF] NaOH (4.0 g, 0.1 mol) was dissolved in water (130 ml) and 1,4-dioxane (260 ml) was added. ω-Aminocapronic acid (13.1 g, 99.9 mmol) was added, allowed to dissolve and the solution was cooled to 0 °C. Di-tert-butyl dicarbonate (24.0 g, 110 mmol) was added in three portions, the reaction mixture was stirred at 0 °C for 30 min and then at r.t. for 16 h. The 1,4-dioxane was removed by rotary evaporation and the crude was diluted with water (400 ml) and washed with EtOAc (2 x 200 ml). The aqueous phase was acidified by addition of 1 M HCl (aq.) and extracted with EtOAc (3 x 300 ml). The organic phases from the second extraction were collected and dried over MgSO4. Removal of the solvents gave the expected product 3 as an oil that crystallizes to light pink crystals (20.6 g, 89% yield). Rf : 0.20 (2% MeOH in CH2Cl2). [START_REF] Kim | Lethal Effect of Adriamycin on the Division Cycle of HeLa Cells[END_REF] MalEsterDox (1) 7 (132 mg, 0.24 mmol) was dissolved in dry DMF (2.2 ml) and the solution was added to Doxorubicin hydrochloride (128 mg, 0.22 mmol). Et3N (61 µl, 0.44 mmol) was added and the dark red reaction mixture was stirred under argon. After 22 h, the reaction mixture was added dropwise to stirred DEE (75 ml) resulting in a red precipitate which was collected by filtration and further purified by flash column chromatography eluting with 2-5% MeOH in CH2Cl2. 1 (124 mg, 59% yield) was isolated as a red foam. Rf : 0.29 (5% MeOH in CH2Cl2); [START_REF] Kim | Lethal Effect of Adriamycin on the Division Cycle of HeLa Cells[END_REF] 9) Following a published procedure, [START_REF] De Groot | Synthesis and Biological Evaluation of Novel Prodrugs of Anthracyclines for Selective Activation by the Tumor-Associated Protease Plasmin[END_REF] FmocLys(Alloc)OH (1.44g, 3.18 mmol) was dissolved in dry THF (20 ml) under argon in a two-necked flask equipped with an addition funnel. The solution was cooled to -40 °C and N-methylmorpholine (0.39 ml, 3.54 mmol) and isobutyl chloroformate (0.45 ml, 3.47 mmol) were added. The turbid mixture was stirred at -40 °C for 3 h after which a solution of p-aminobenzylalcohol (470 mg, 3.82 mmol) and Nmethylmorpholine (0.42 ml, 3.82 mmol) in dry THF (6 ml) was added dropwise over 10 min. The reaction mixture was stirred at -40 °C for 2 h and then at room temperatrure for 1 h. The solvents were removed and the yellow residue was taken up in CH2Cl2 (250 ml). The organic phase was washed with 0.5 M NaHCO3 solution (100 ml), 0.5 M NaHSO4 solution (100 ml) and brine (100 ml). The organic solvents were removed under reduced pressure and the yellow residue was purified by flash column chromatography on silica (2-10% methanol in CH2Cl2) to provide 9 as a white solid (1.28 g, 72% yield). [START_REF] Kim | Lethal Effect of Adriamycin on the Division Cycle of HeLa Cells[END_REF] H NMR (400 MHz, dmso-d6) δ 9.96 (s, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.74 (dd, J = 7.5, 4.9 Hz, 2H), 7.62 (d, J = 7.9 Hz, 1H), 7.55 (d, J = 8.4 Hz, 2H), 7.41 (t, J = 7.3 Hz, 2H), 7.37-7.29 (m, 2H), 7.24 (d, J = 8.3 Hz, 2H), 7.19 (t, J = 5.8 Hz, 1H), 5.89 (ddt, J = 17.2, 10.5, 5.3 Hz, 1H), 5.30-5.20 (m, 1H), 5.18 -5.12 (m, 1H),5.10 (t, J = 5.7 Hz, 1H) 4.52-4.39 (m, 4H), 4.34 -4.06 (m, 4H), 3.07 -2.91 (m, 2H), 1.75-1.56 (m, 2H), 1.51 -1.21 (m, 4H). [START_REF] Dubowchik | Cathepsin B-Labile Dipeptide Linkers for Lysosomal Release of Doxorubicin from Internalizing Immunoconjugates: Model Studies of Enzymatic Drug Release and Antigen-Specific In Vitro Anticancer Activity[END_REF] C NMR (101 MHz, DMSO-d6) δ 171.0, 156.1, 155.9, 143.9, 143.8, 140.7, 137.5, 137.4, 133.8, 127.6, 127.0, 126.9, 125.3, 120.1, 118.9, 116.8, 65.6, 64.1, 62.6, 55. 4, 46.7, 31.6, 29.1, 22.9. HRMS m/z [M+H] + calculated for C32H36N3O6: 558.2604, found: 558.2606.

Tripep (10) Compound 9 (560 mg, 1.00 mmol) was dissolved in a premade mixture of 1,4dioxane:MeOH:2M NaOH(aq.) (14:5:1, 20 ml). [START_REF] De Groot | Design, Synthesis, and Biological Evaluation of a Dual Tumor-specific Motive Containing Integrin-targeted Plasmin-cleavable Doxorubicin Prodrug[END_REF] After 30 min (TLC, CH2Cl2:MeOH 90:10), the solution was neutralized by addition of acetic acid. The solvents were removed by evaporation and coevaporated with 1,4-dioxane (2 x 20 ml). The oily solid residue was washed with DEE (3 x 10 ml) and then with CH2Cl2 (10 x 10 ml). The CH2Cl2 fractions were collected and the solvent was removed to yield the free amine as a pale yellow oil (326 mg) which was used in the next step without further purification. A solution of DMTMMBF4 (365 mg, 1.11 mmol), FmocPheOH (390 mg, 1.01 mmol) and Nmethyl morpholine (121 µl, 1.10 mmol) in CH3CN (5 ml) was stirred for 10 min and then added to a solution of the crude form the previous step (326 mg) in CH3CN (5 ml). The reaction mixture was stirred at r.t for 19 h (TLC, CH2Cl2:MeOH 90:10). The solvents were removed by evaporation and CH2Cl2 (100 ml) was added and the organic phase was washed with water (25 ml), 0.5 M NaHCO3 (25 ml), 0.5 M NaHSO4 (3 x 25 ml) and water (50 ml). The solvents were removed from the organic phase to give an off-white solid (665 mg) which was used in the next step without further purification.

A premade mixture of 1,4-dioxane:MeOH:2M NaOH(aq.) (14:5:1, 20 ml) was added to the crude from the previous step and the reaction mixture was stirred at r.t. for 50 min (TLC, CH2Cl2:MeOH 90:10). The reaction was neutralized by addition of acetic acid. The solvents were removed by evaporation and coevaporated with 1,4-dioxane (20 ml). The resulting white residue was washed with DEE (3 x 5 ml) and then with CH2Cl2 (10 x 20 ml). The white precipitate formed in the DEE phase was collected by filtration, dissolved in CH2Cl2 and pooled with the other CH2Cl2 fractions. The CH2Cl2 fractions were collected and the solvent was removed to yield the free amine as an off white solid (463 mg) which was used in the next step without further purification. A solution of DMTMMBF4 (360 mg, 1.10 mmol), Boc-D-AlaOH (190 mg, 1.00 mmol) and Nmethyl morpholine (121 µl, 1.10 mmol) in CH3CN (5 ml) was stirred for 10 min and then added to a solution of the crude from the previous step (463 mg) in CH3CN (6 ml). The reaction mixture was stirred at r.t for 14 h (TLC, CH2Cl2:MeOH 90:10). The solvents were removed by evaporation and CH2Cl2 (70 ml) was added and the organic phase was washed with water (40 ml), 0.5 M NaHSO4 (2 x 20 ml), 0.5 M NaHCO3 (3 x 20 ml) and water (20 ml). The solvents were removed from the organic phase to give an off-white solid, which was purified by flash column chromatography on silica eluting with 2-5% methanol in CH2Cl2. The product was dryloaded on to the column after being dissolved in 10% methanol in CH2Cl2 and dried on to silica. Compound 10 was isolated as a white solid (309 mg, 47% over 4 steps). 1 H NMR (400 MHz, MeOD-d4) δ 7.61 (d, J = 8.3 Hz, 2H), 7.30 (d, J = 8.5 Hz, 2H), 7.28 -7.09 (m, 5H), 5.91 (ddt, J = 16.3, 10.7, 5.5 Hz, 1H), 5.28 (dd, J = 17.1, 1.7 Hz, 1H), 5.16 (d, J = 10.5 Hz, 1H), 4.60 (dd, J = 9.8, 4.5 Hz, 1H), 4.56 (s, 2H), 4.50 (d, J = 5.0 Hz, 2H), 4.43 (dd, J = 9.6, 4.9 Hz, 1H), 4.00 (q, J = 7.1 Hz, 1H), 3.30-3.23 (m, 1H), 3.11 (t, J = 6.7 Hz, 2H), 2.95 (dd, J = 14.2, 9.8 Hz, 1H), 2.02 -1.78 (m, 2H),1.59-1.40 (m, 4H) 1.33 (s, 9H), 1.13 (d, J = 7.1 Hz, 3H). [START_REF] Dubowchik | Cathepsin B-Labile Dipeptide Linkers for Lysosomal Release of Doxorubicin from Internalizing Immunoconjugates: Model Studies of Enzymatic Drug Release and Antigen-Specific In Vitro Anticancer Activity[END_REF] 11) 10 (150 mg, 0.23 mmol) was suspended in dry CH2Cl2 (1ml) under argon, TFA (0.8 ml) was added and the solution was stirred for 15 min. Full consumption of 10 was confirmed by TLC (10% methanol in CH2Cl2). The solvents were removed and coevaporated with CH2Cl2 (3 x 1 ml). The resulting residue was taken up in CH3CN (3.5 ml) and neutralized by addition of NMM (150 µl). DMTMMBF4 (82 mg, 0.25 mmol), 3-(maleimidyl)propanoic acid (46 mg, 0.27 mmol) and NMM (27 µl, 0.25 mmol) were stirred in CH3CN (1.5 ml) at r.t. until all material dissolved (~5 min). The solution of deprotected 10 was then added and the reaction mixture was stirred at r.t. for 22 h. The formed precipitate was filtered off and washed with ice cold CH3CN and DEE. The filtrate was collected, diluted in CH2Cl2 (100 ml) and washed with 0.5 M NaHSO4 (30 ml) and water (30 ml). The organic phases were dried over MgSO4 and the solvents were then removed to give a white solid which was pooled with the precipitated material. The crude was purified by flash column chromatography on silica eluting with 5% methanol in CH2Cl2. The crude product was dryloaded on to the column after being dissolved in methanol and dried on to silica. The expected product 11 was isolated as a white solid (72 mg, 43% over 2 steps). [START_REF] Kim | Lethal Effect of Adriamycin on the Division Cycle of HeLa Cells[END_REF] H NMR (400 MHz, MeOD-d4) δ 7.60 (app d, 2H),7.29 (app d, 2H), 7.28-7.14 ( m, 5H), 6.76 (s, 2H) 5.91 (ddt, J = 16.3, 10.6, 5.4 Hz, 1H), 5.28 (dd, J = 17.2, 1.8 Hz, 1H), 5.20 -5.12 (m, 1H), 4.62 -4.52 (m, 3H), 4.50 (dt, J = 5.6, 1.6 Hz, 2H), 4.40 (dd, J = 9.6, 5.0 Hz, 1H), 4.14 (q, J = 7.1 Hz, 1H), 3.73-3.57 (m, 2H), 3.30-3.25 (m, 1H, partly overlapping with solvent peak), 3.13 (t, J = 6.7 Hz, 2H), 2.95 (dd, J = 14.2, 10.0 Hz, 1H), 2.47-2.27 (m, 2H), 2.00 -1.84 (m, 2H), 1.62 -1.36 (m, 4H), 1.14 (d, J = 7.1 Hz, 3H). [START_REF] Dubowchik | Cathepsin B-Labile Dipeptide Linkers for Lysosomal Release of Doxorubicin from Internalizing Immunoconjugates: Model Studies of Enzymatic Drug Release and Antigen-Specific In Vitro Anticancer Activity[END_REF] 12) 11 (65 mg, 0.09 mmol) and bis(p-nitrophenyl)carbonate (82 mg, 0.27 mmol) were dissolved in dry DMF (1.5 ml) under argon. The flask was capped, evacuated and refilled with argon several short turns before DIPEA (47 µl, 0.27 mmol) was added by syringe. The reaction mixture was stirred at r.t. for 23 h at which time a second portion of bis(p-nitrophenyl)carbonate (55 mg, 0.18mmol) was added. The reaction was stirred for an additional 23 h after which all 11 was consumed as verified by TLC (5% methanol in CH2Cl2). The reaction mixture was diluted in CH2Cl2 (40 ml), washed with citric acid solution (10 wt%, 2 x 15 ml) and dried over MgSO4. The solvents were removed resulting in a yellow oil, which was dissolved in a small amount of CH2Cl2 and added dropwise to DEE (40 ml) while stirring. The product precipitated as a white solid (60 mg) containing ~15% p-nitrophenol (estimated from 1 H-NMR integrals). The identity of the expected product was confirmed by 1 H-NMR, ESI-MS and HRMS and the material was used without further purification in the following step. [START_REF] Kim | Lethal Effect of Adriamycin on the Division Cycle of HeLa Cells[END_REF] 13) 12 (53 mg, 0.061 mmol) was dissolved in dry DMF (1.7 ml) and the hydrochloride salt of doxorubicin (35 mg, 0.061 mmol) was added followed by Et3N (18 µl, 0.12 mmol). The reaction mixture was stirred at r.t. and followed by TLC (5% methanol in CH2Cl2). After 24 h, the reaction mixture was added dropwise to DEE (35 ml) under stirring which resulted in a red precipitate. The precipitate was collected by filtration and isolated by washing the filter paper with a solution of 5% methanol in CH2Cl2. The solvents were removed to give a red solid, which was purified by flash column chromatography on silica eluting with 5% methanol in CH2Cl2. The product was dryloaded on to the column after being dissolved in methanol:CH2Cl2 mixture and dried on to silica. The expected product 13 was isolated as a red solid (59 mg, 75%). HPLC method A. MS (ESI) m/z: [M+Na] + : 1296.5; HRMS m/z [M+Na] + calculated for C64H71N7O21Na: 1296.4601, found: 1296.4608. Characteristic peaks of maleimidyl (6.96 ppm, s, 2H), Vinyl proton from Alloc protecting group (5.86 ppm, m, 1H), methoxy methyl of doxorubicin (3.97 ppm, s, 3H), Alaninyl methyl (1.12 ppm, d, 3H) and doxorubicin glycon methyl (0.91ppm, d, 3H) with matching integrals found on 1 H-NMR (DMSO-d6) (see SI).

N-Acetamido-N-furfuryl-XGO (15)

A solution of XGO [START_REF] York | Structural analysis of xyloglucan oligosaccharides by 1H-n.m.r. spectroscopy and fast-atombombardment mass spectrometry[END_REF] (1.015 g, 8.3 mmol based on the octasaccharide) in furfurylamine (4 mL) was stirred for 72h at 25°C. The solution was poured into ethyl acetate (25 mL), cooled to 0°C and centrifuged for 20 min at 6000 rpm. The solid was washed with ethyl acetate and centrifuged again. The crude solid of glycosylamine was then dissolved in methanol (15 mL) and acetic anhydride (7 mL) was slowly added at 0°C to the solution. The reaction mixture was then allowed to warm up to room temperature and stirred a further 16h. The solution was then concentrated and co-evaporated with toluene. The residue was dissolved in 14% aqueous ammonia (50 mL), stirred 1h at room temperature to hydrolyze potential O-acetyl groups. After concentration of the solution to half of its initial volume under vacuum, the aqueous phase was extracted 3 times with ethyl acetate to remove free N-acetyl furfurylamine and concentrated. Nacetamido-N-furfuryl-XGO was isolated in 90% yield (998 mg) after purification by flash chromatography on silica gel (CH3CN/H2O 7:3 v/v). [START_REF] Kim | Lethal Effect of Adriamycin on the Division Cycle of HeLa Cells[END_REF] H NMR (400 MHz, D2O) δ 7.51 and 7.45 (2 x s, rotamers, 1H), 6.46-6.36 (m, 2H), 5.53 (d, J = 9.0 Hz, 0.3H, H1 GlcN rotamer), 5.18 (m, 1H), 5.13 (d, J = 9.0 Hz, 1H, H1 GlcN rotamer), 4.96 (m, 2H), 4.62-4.55 (m, 5H), 4.06-3.33 (m, 51H), 2.30 and 2.26 ( 2 x s, rotamers, 3H, NCOCH3). The presence of the amide rotamers was confirmed by their disappearance at elevated temperature (323K) as observed by VT 16) 1 (21 mg, 0.022 mmol) was dissolved in DMF (60 µl) and N-acetamido-N-furfuryl-Lactose 40 (28 mg, 0.06 mmol) was added followed by water (40µl). The reaction mixture was stirred at 37 °C for 3 days. The solvents were removed by rotary evaporation and the crude material was dissolved in CH2Cl2/MeOH, loaded on to silica and purified by flash column chromatography eluting with 10-20% H2O in CH3CN. 16 was isolated as a red solid (30 mg, 91%) after lyophilization. HPLC method A (see SI); MS (ESI) m/z: [M+Na] + : 1443.6; HR-MS m/z [M+Na] + calculated for C67H80N4O30Na 1443.4755; Found 1443.4757. XGOEsterDox ( 17) 1 (10 mg, 0.01 mmol) was dissolved in DMF (60 µl) and N-acetamido-N-furfuryl-XGO 15 (66 mg, 0.05 mmol) was added followed by water (60µl). The reaction mixture was stirred at 37 °C for 4 days. The solvents were removed by rotary evaporation and the crude was purified by two consecutive C18-columns (1g) eluting with a gradient of 0-100% MeOH in water. The expected product 17 was isolated as a red solid (8 mg, 35%) after lyophilization. HPLC method A (see SI); (ESI) m/z: [M-H] -: 2140.9 (DP7) , 2302.9 (DP8), 2464.9 (DP9). HR-MS m/z [M+Na] + calculated for DP7; C94H124N4O52Na; 2163.7029 Found 2163.7079, m/z [M+Na] + calculated for DP8; C100H134N4O57Na; 2325.7607 Found 2325.7495, m/z [M+Na] + calculated for DP9; C106H144N4O62Na; 2487.8135 Found 2487.8136. LactriPep(Alloc)Dox ( 18) 13 (15 mg, 0.012 mmol) was dissolved in DMF (80 µl) and N-acetamido-N-furfuryl-Lactose (16 mg, 0.035 mmol) was added followed by water (40µl). The reaction mixture was stirred at 37 °C for 7 days. The solvents were removed by rotary evaporation and the crude material was dissolved in CH2Cl2/MeOH, loaded on to silica and purified by flash column chromatography eluting with 10% H2O in CH3CN. The expected product 18 was isolated as a red solid (12 mg, 58%) after lyophilization. HPLC method A (see SI). MS (ESI) m/z: [M+Na] + : 1759.7; HRMS m/z [M+Na] + calculated for C83H100N8O33Na: 1759.6290, found: 1759.6302. LactriPepDox (19) Following a modified procedure for Alloc deprotection, 33 18 (7 mg, 4.0 µmol) was dissolved in degassed CH2Cl2:AcOH (5:1, 600 µl). Pd(PPh3)4 (6 mg, 5.2 µmol) was added. The reaction was stirred at rt in the dark and followed by TLC (CH2Cl2:MeOH:NH4OH 80:18:2). After 30 min, the CH2Cl2 was removed and DEE (1 ml) was added which resulted in a red precipitation which was washed with DEE (1 ml), EtOAc (2 x 1 ml) and then DEE (1 ml) again. Ultrasonication and quick centrifugation was used in each wash cycle. The resulting red pellet was dried in a dessicator under low pressure. The resulting red solid (4 mg, 59%) had a mass on ESI-MS and HRMS corresponding with the expected product 19 and consisted of one main compound with minor impurities on HPLC-UV. HPLC method A. MS (ESI) m/z: [M+H] + : 1653.6 HRMS m/z [M+H] + calculated for C79H97N8O31: 1653.6260, found: 1653.6244.

Enzymatic release of Doxorubicin

Plasmin and human carboxylesterases were purchased from Sigma-Aldrich.

Plasmin

Following a protocol from the enzyme provider, 47 a 100 µM solution of 19 in phosphate buffer (pH 7.5) containing 100mM Lysine was incubated at 37 ℃ with plasmin from human plasma (15µg/ml). The reaction was followed by reversed phase HPLC (method B) by injecting 15µl aliquots from the reaction. The ratio of released doxorubicin over prodrug was calculated from integration of the peaks at 480 nm and plotted against time.

Carboxylesterase

A 100 µM solution of ester linked prodrug (16 or 17) in Tris buffer (50 mM, pH 7.4) [START_REF] Ross | Enzymatic Activity of Human Carboxylesterases[END_REF] was incubated at 37 ℃ with either recombinant human carboxylesterase 1 isoform b (100µg/ml) or recombinant human carboxylesterase 2 (100µg/ml) expressed in baculovirus infected BTI insect cells . The reaction was followed by reversed phase HPLC (method B) by injecting 15 µl aliquots from the reaction. The ratio of released doxorubicin over prodrug was calculated from integration of the peaks at 480 nm and plotted against time.

Growth inhibition assay

The breast cancer cells MCF-7 were seeded at a density of 2×10 3 cells per well in 96-well plates. After 24 h, cells were treated with an increasing concentration of compounds, from 0 to 100 µM. Cells were incubated for 72 h, and then, 20 μL of MTS (Promega) was added per well for one hour at 37 °C. The absorbance was read at 492 nm using a spectrophotometer (SPECTRAmax, Molecular Devices). IC50 values were obtained from the cytotoxicity curves using SOFTmax PRO software.

Table of Contents graphics (TOC)

Figure 1 .

 1 Figure 1. General scheme of the enzymatically activated glyco-prodrugs of Dox. D.A.=Diels -Alder.

Scheme 4 .

 4 Scheme 4. Synthesis of 19. a) DMF:H2O 2:1, 37℃, 7 days, 58%. b) Pd(PPh3)4 (1.2 equiv.), CH2Cl2:AcOH 5:1, r.t., 30 min, 59%.

Figure 2 .

 2 Figure 2. In vitro protease mediated release of Dox (•) from LactriPepDox 19 (100µM) using 15µg/ml plasmin in phosphate buffer (pH 7.5) at 37 ºC.

Figure 3 .

 3 Figure 3. In vitro esterase mediated release of Dox using 100µg/ml esterase in 50 mM Tris buffer (pH 7.4) at 37 ºC from LacEsterDox 16 with hCE2(▲), from LacEsterDox 16 with hCE1(♦), from XGOEsterDox 17 with hCE2(■) and LacEsterDox 16 without enzyme (x).The values for hCE2 measurements are mean values of duplicate experiments.

Table 1 .

 1 ). Inhibition of MCF-7 breast cancer cell growth by the glyco-prodrugs and free Dox as reference. IC50 values were calculated as the concentration of compound needed to reduce cell growth by 50%. Values are reported as mean ± the standard deviation (SD) of triplicate measurements.The plasmin-activated prodrug 19 displayed a slightly higher (~2 fold) IC50 compared to Dox while the esterase activated prodrugs 16 and 17 showed IC50 values similar to that of the parent drug. Since it has been previously shown that protection of the primary amine of Dox results in a significant decrease in activity,[START_REF] Ryppa | In Vitro and in Vivo Evaluation of Doxorubicin Conjugates with the Divalent Peptide E-[c(RGDfK)2] that Targets Integrin αvβ3[END_REF] these results are a clear indication of Dox release from prodrugs 16, 17 and 19 in presence of the MCF-7 cells. One would have probably expected a more pronounced difference between the IC50 of the ester and amide prodrugs as plasmin (which is not over-expressed on MCF-7 cells) has not been added in the cell culture medium), and further experiments are now required to study the mode of action of the glyco-prodrugs.

	Compound	IC50 (µM)
	16	0.51 ± 0.07
	17	0.31 ± 0.15
	19	1.11 ± 0.11
	Dox	0.53

Nevertheless, the similar IC50 obtained for XGOEsterDox 17 and LacEsterDox 16 are very encouraging since they show that the release of Dox is not dependent on the size and therefore steric hindrance of the oligosaccharide moiety. In a forthcoming work, we wish to implement the use xyloglucan glycoconjugates to the development of drug-functionalized cellulosic biomaterials.

  H NMR (400 MHz, CDCl3) δ 10.85 (br s, 1H), 5.97 (br s, 0.3H, -

	29.8, 28.5, 26.3, 24.6; MS (ESI) m/z: [M+Na] + : 360.2; HRMS m/z [M+Na] + calculated for
	C18H27NO5Na: 360.1787, found: 360.1786.
	MalEsterOH (6)
	5 (400 mg, 1.19 mmol) was dissolved in dry CH2Cl2 (1.5 ml) and TFA (1.5 ml) was added. The
	reaction was stirred at r.t. for 20 min. Full consumption of 5 was observed by TLC (10% MeOH
	in CH2Cl2). Solvents were removed and coevaporated four times with CH2Cl2. The resulting
	clear oil was redissolved in dry CH2Cl2 (2 ml) and basified (pH approx. 8) by addition of N-
	methylmorpholine (400 µl). 3-(Maleimidyl)propanoic acid (220 mg, 1.30 mmol) was added,
	the reaction was cooled on ice and DCC (268 mg, 1.30 mmol) was added. The ice bath was
	removed and the reaction was stirred at r.t. for 17 h. The white precipitate that had formed was
	filtered off and the filtrate was concentrated. The yellow oil that resulted was redissolved in
	CH2Cl2 (20 ml), washed with 0.5 M HCl (5 ml) and dried over MgSO4 and filtered. Removal
	of the solvents gave a white solid, which was purified by flash column chromatography on silica
	eluting with 25% cyclohexane in EtOAc to obtain 6 as a white (236 mg, 51% yield). Rf :
	0.52 (10% MeOH in CH2Cl2); 1 H NMR (400 MHz, CDCl3) δ 7.41-7.34 (m, 2H), 7.09-7.02 (m,
	2H), 6.68 (s,2H), 5.74 (br s, 1H), 4.67 (s, 2H), 3.81 (t, J = 7.2 Hz, 2H), 3.28-3.19 (m, 2H), 2.55
	(t, J = 7.3 Hz, 2H), 2.48 (t, J = 7.2 Hz, 2H), 1.86 (br s, 1H), 1.75 (p, J = 7.4 Hz, 2H), 1.58 -
	1.48 (m, 2H), 1.46 -1.36 (m, 2H); 13 C NMR (101 MHz, CDCl3) δ172.3, 170.7, 169.6, 150.2,
	138.7, 134.3, 128.2, 121.8, 64.8, 39.4, 34.9, 34.4, 34.2, 29.2, 26.4, 24.5; MS (ESI) m/z:
	36 (2% MeOH in CH2Cl2); 1 H NMR (400 MHz, CDCl3) δ 9.98 (s, 1H), 7.95-7.86 (m, 2H), 7.30-7.20 (m, 2H), 4.57 (br s, 1H), 3.14 (q, J = 6.7 Hz, 2H), 2.59 (t, J = 7.4 Hz, 2H), 1.77 (p, J = 7.5 Hz, 2H), 1.61 -1.49 (m, 2H), 1.48 -1.38 (m, 11H); 13 C NMR (101 MHz, CDCl3) δ 191.0, 171.4, 156.1, 155.5, 134.1, 131.3, 122.5, 79.3, 40.5, 34.3, 29.9, 28.5, 26.3, 24.5; MS (ESI) m/z: [M+Na] + : 358.3 HRMS m/z [M+Na] + [M+Na] 28 (2% MeOH in CH2Cl2); 1 H NMR (400 MHz,
	calculated for C18H25NO5Na: 358.1630, found: 358.1627. CDCl3) δ 8.31-8.23 (m, 2H), 7.50-7.43 (m, 2H), 7.41-7.35 (m, 2H), 7.16-7.09 (m, 2H), 6.70 (s,
	2H), 5.64 (br s, 1H), 5.28 (s, 2H), 3.84 (t, J = 7.2 Hz, 1H), 3.31-3.20 (m, 2H), 2.58 (t, J = 7.4
	4-(Hydroxymethyl)phenyl-6-((tert-butoxycarbonyl)amino)hexanoate (5) Hz, 1H), 2.51 (t, J = 7.1 Hz, 1H), 1.82-1.72 (m, 2H), 1.58 -1.50 (m, 2H), 1.49 -1.38 (m, 2H);
	4 (1.00 g, 2.98 mmol) was dissolved in trifluoroethanol (6 ml) and the flask was cooled on ice. 13 C NMR (101 MHz, CDCl3) δ 172.0, 170.7, 169.6, 155.6, 152.6, 151.3, 145.6, 134.4, 131.9,
	NaBH4 (107 mg, 2.83 mmol) was added and the reaction was stirred on ice. Full consumption 130.2, 125.5, 122.2, 121.9, 70.4, 39.4, 34.9, 34.4, 34.3, 29.3, 26.4, 24.5; MS (ESI) m/z: [M+H] + :
	of 4 was observed after 1 h on TLC (2% MeOH in CH2Cl2). The reaction was kept on ice and 554.2 and [M+Na] + : 576.2; HRMS m/z [M+H] + calculated for C27H28N3O10: 554.1775, found:
	quenched by slow addition of sat. NH4Cl (7 ml) after 1h 20 min. The mixture was allowed to 554.1776.
	reach r.t. and stirred for 40 min. Water (5 ml) was added and the aqueous phase was extracted
	with EtOAc (3 x 30 ml). The organic phases were pooled, dried over Na2SO4 and filtered.
	Removal of the solvents gave a colorless oil, which was purified by flash column
	chromatography on silica eluting with 1% MeOH in CH2Cl2 to obtain 5 as a white solid (900
	mg, 90% yield). Rf : 0.32 (2% MeOH in CH2Cl2); 1 H NMR (400 MHz, CDCl3) δ 7.37-7.29 (m,

CONH-), 4.63 (br s, 0.7H, -CONH-),

3

.08 (br s, 2H), 2.32 (t, J = 7.4 Hz, 2H), 1.62 (p, J = 7.5 Hz, 2H), 1.53 -1.18 (m, 13H) ; ESI-MS m/z [M+Na] + : 254.2.

4-Formylphenyl-6-((tert-butoxycarbonyl)amino)hexanoate (

4

) 3 (10.0 g, 43.2 mmol) was dissolved in dry CH2Cl2 (43 ml) in a two necked flask under argon. DCC (8.9 g, 43.2 mmol) and DMAP (1.0 g, 8.2 mmol) were added, the reaction mixture was cooled to 0 °C and p-hydroxybenzaldehyde (5.0 g, 41.2 mmol) was immediately added. The reaction was stirred at 0 °C for 30 min and then at r.t. for 21 h. The white precipitate that formed was filtered off and washed twice with a small volume of CH2Cl2. The solvents were removed from the filtrate which gave an off-white solid that was redissolved in CH2Cl2 (200 ml), washed with 0.5 M HCl (50 ml), sat. NaHCO3 (50 ml) and brine (50 ml). The organic phase was dried over MgSO4 and filtered. Removal of the solvents by rotary evaporation gave the crude as an off white solid. Purification by column chromatography on silica eluting with 2% MeOH in CH2Cl2 gave 4 as a white solid (12.5 g, 86% yield). Rf : 0.2H), 7.06-6.99 (m, 2H), 4.62 (s, 3H,-CH2-and -CONH-), 3.10 (q, J = 6.6 Hz, 2H), 2.5 3 (t, J = 7.4 Hz, 2H), 2.49 (br s, 1H), 1.74 (p, J = 7.5 Hz, 2H), 1.56 -1.46 (m, 2H), 1.

45

-1.35(m, 11H) ; 13 C NMR (101 MHz, CDCl3) δ 172.2, 156.1, 150.0, 138.7, 128.0, 121.6, 79.2, 64.6, 40.4, 34.3, + : 411.3; HRMS m/z [M+H] + calculated for C20H25N2O6: 389.1713, found: 389.1714. MalEsterPNP (

7

) 6 (178 mg, 0.46 mmol) and bis(p-nitrophenyl)carbonate (210 mg, 0.69 mmol) were added to a dry flask filled with argon which was capped, evacuated and refilled with argon (repeated 3 times). Dry CH2Cl2 (4.6 ml) was added followed by diisoproylethylamine (245 µl, 1.38 mmol). The yellow reaction mixture was stirred at r.t. for 4 h in the dark. Full consumption of 6 was observed on TLC (2% MeOH in CH2Cl2). The reaction mixture was diluted with CH2Cl2 (25 ml) and washed with aq. citric acid (10 % w/v). The organic phase was dried over Na2SO4 and filtered. Removal of the solvents gave a yellow oil. DEE (25 ml) was added and brief sonication resulted in a white precipitate. The precipitate was filtered off, washed with DEE repeatedly, collected and redissolved in a minimum of CH2Cl2. The solution was added dropwise to stirred DEE (25 ml). The white precipitate that formed was collected by filtration and dried to give 7 as a white solid (178 mg, 70% yield). Rf : 0.

  H NMR (400 MHz, CDCl3) δ 13.96 (s, 1H), 13.23 (s, 1H), 8.02 (dd, J = 7.7, 1.1 Hz, 1H), 7.82 -7.73 (m, 1H), 7.39 (dd, J = 8.6, 1.1 Hz, 1H), 7.31 (d, J = 8.0 Hz, 2H), 7.01 (d, J = 8.0 Hz, 2H), 6.68 (s, 2H), 5.73 (s, 1H), 5.49 (d, J = 3.8 Hz, 1H), 5.28 (br s, 1H), 5.22 (d, J = 8.6 Hz, 1H), 5.08-4.91 (m, 2H), 4.75 (dd, J = 5.0, 1.4 Hz, 2H), 4.57 (s, 1H), 4.18 -4.08 (m, 1H), 4.07 (s, 3H), 3.85 (br s, 1H), 3.81 (t, J = 7.2 Hz, 2H), 3.66 (d, J = 7.2 Hz, 1H), 3.32 -3.16 (m, 3H), 3.08-2.94 (m, 2H), 2.53 (t, J = 7.3 Hz, 2H), 2.48 (t, J = 7.2 Hz, 2H), 2.39-2.27 (m, 1H), 2.25-2.10 (

	m, 2H), 1.91 -1.68 (m, 4H), 1.52 (p, J = 7.1 Hz, 2H), 1.46-1.33 (m, 2H), 1.28 (d, J = 6.5 Hz,
	3H); 13 C NMR (101 MHz, CDCl3) δ 214.0, 187.2, 186.8, 172.2, 170.7, 169.7, 161.2, 156.3,
	155.8, 155.6, 150.6, 135.9, 135.6, 134.3, 134.1, 133.7, 129.5, 121.8, 121.0, 120.0, 118.6, 111.7,
	111.6, 110.1, 100.8, 76.8, 69.7, 69.6, 67.5, 66.2, 65.7, 56.8, 47.2, 39.4, 35.8, 34.9, 34.4, 34.2,
	34.1, 30.3, 29.2, 26.4, 24.5, 17.0; MS (ESI) m/z: [M+Na] + : 980.3; HRMS m/z [M+H] +
	calculated for C48H52N3O18: 958.3246, found: 958.3235.
	FmocLys(Alloc)PABOH (

  C NMR (101 MHz, MeOD-d4) δ 176.9, 173.6, 172.2, 158.8, 157.9, 138.8, 138.5, 138.4, 134.5, 130.2, 129.5, 128.5, 127.8, 121.4, 117.4, 80.7, 66.3, 64.8, 56.4, 55.7, 51.8, 41.5, 37.9, 32.3, 30.5, 28.7, 24.5, 17.7. HRMS m/z [M+H] + calculated for C34H48N5O8: 654.3503, found: 654.3496.

	MaltriPep (

  C NMR (101 MHz, MeOD-d4) δ 176.2, 173.7, 173.2, 172.4, 172.1, 158.8, 138.8, 138.5, 135.4, 134.6, 130.2, 129.5, 128.5, 127.8, 121.4, 117.4, 112.9, 66.3, 64.8, 56.7, 55.8, 51.1, 41.5, 37.8, 35.2, 35.1, 32.3, 30.5, 24.5, 17.1. MS (ESI) m/z: [M+H] + : 705.4 and [M+Na] + : 727.4; HRMS m/z [M+H] + calculated for C36H45N6O9: 705.3248, found: 705.3253.

	MaltriPepOPNP (

Acknowledgments

This work was supported by the French National Research Agency (ANR) under the research grants Arcane (ANR-11-LABX-0003-01), Cellical (ANR-15-CE08-0033) and Glyco@Alps (ANR-15-IDEX-02). We acknowledge ICMG (FR 2607) platforms for granting access to the mass spectrometry and NMR facilities.

Supporting Information description

The SI contains NMR spectra and HPLC chromatograms.

Abbreviations


Flavien Labre

Sophie Mathieu

Patricia Chaud

Pierre-Yves Morvan

Romuald Vallée

William Helbert

Sébastien Fort

DMTMM-mediated amidation of alginate oligosaccharides aimed at modulating their interaction with proteins

Keywords: Alginate oligosaccharides, Amidation, Multivalency, Lectin, Alginate lyases

come

Introduction

Alginates encompass a relatively broad family of linear anionic polysaccharides composed of (1→4)-linked β-D-mannuronic acid (M) and its C-5 epimer α-L-guluronic acid (G), forming homopolymeric (MM-or GG-blocks) and heteropolymeric sequences (MG blocks). The fraction and the distribution of the two monomers vary widely depending on the source.

Although some soil bacteria of the genera Pseudomonas and Azotobacter produce alginates as exopolysaccharides, the main source of alginate is brown seaweeds in which it represents up to 40% of the organisms' dry matter, providing ∼30,000 t of alginate for industry annually [START_REF] Draget | Alginates[END_REF]. The ability of alginates to form soft hydrogels in the presence of calcium ions forms the basis for a wide variety of industrial applications, including food and pharmaceuticals. In recent years, alginate biomaterials have received increased attention for use in drug delivery systems or as biocompatible scaffolds for tissue engineering [START_REF] Lee | Alginate: properties and biomedical applications[END_REF]Szekalska, Puciłowska, Szymańska, Ciosek & Winnicka, 2016).

Alginate oligosaccharides (AOS) do not form gels, but nevertheless exhibit many important bioactivities in mammals as well as in plants. AOS promote the growth and proliferation of human cells including keratinocytes (Charruyer, Fong, Yue, Arron, Ghadially, 2016;[START_REF] Kawada | Stimulation of human keratinocyte growth by alginate oligosaccharides, a possible co-factor for epidermal growth factor in cell culture[END_REF][START_REF] Morvan | Adult human epidermal stem cells recruitment by an oligoalginate extract[END_REF] and endothelial cells [START_REF] Kawada | Alginate oligosaccharides stimulate VEGF-mediated growth and migration of human endothelial cells[END_REF]. They also potentially protect against oxidative stress in neural cells [START_REF] Tusi | Alginate oligosaccharide protects against endoplasmic reticulum-and mitochondrial-mediated apoptotic cell death and oxidative stress[END_REF]. In plants, AOS with an average degree of polymerization (DP) of 6 show elicitor activity in soybean cotyledons, stimulating the accumulation of phytoalexin and inducing phenylalanine ammonia lyase [START_REF] Draget | Alginates[END_REF]. AOS of DP 2-8 promote plant growth in rice, wheat, lettuce, carrot and tobacco, etc. by enhancing nitrogen assimilation and basal metabolism [START_REF] González | Seaweed Oligosaccharides Stimulate Plant Growth by Enhancing Carbon and Nitrogen Assimilation, Basal Metabolism, and Cell Division[END_REF] and references cited therein). Finally, AOS also have interesting properties for the fight against microbial pathogens. AOS are able to perturb multidrug-resistant (MDR) bacteria, thereby enhancing the action of selected antibiotics [START_REF] Khan | Overcoming drug resistance with alginate oligosaccharides able to potentiate the action of selected antibiotics[END_REF]. Similarly, they inhibit fungal cell growth and potentiate the activity of antifungals against Candida and Aspergillus spp. [START_REF] Tøndervik | Alginate Oligosaccharides Inhibit Fungal Cell Growth and Potentiate the Activity of Antifungals against Candida and Aspergillus spp[END_REF].

These exciting properties and possible applications in human health and wellness as well as in agronomy encourage research on the chemical modification of AOS to improve their stability and bioactivity. Acylation of the uronic groups is a prominent strategy in this regard. Firstly, the introduction of substituents at C6 can increase the stability of AOS by delaying the action of degrading enzymes, i.e. alginate lyases [START_REF] Wong | Alginate Lyase: Review of Major Sources and Enzyme Characteristics, Structure-Function Analysis, Biological Roles, and Applications[END_REF] and by reducing the spontaneous hydrolysis of alginic acid arising from intramolecular catalysis by the carboxylic acid [START_REF] Smidsrød | The influence of pH on the rate of hydrolysis of acidic polysaccharides[END_REF]. Poly(ε-caprolactone)-grafted alginates, whose degradation by Flavobacterium sp. alginate lyase can be modulated with respect to the size and grafting density of the polyester chains, have recently been investigated as enzymeresponsive vectors [START_REF] Benykhlef | Alginate grafted with poly(ε-caprolactone): effect of enzymatic degradation on physicochemical properties[END_REF]. Alginate derivatives incorporating either an aliphatic amide or ester groups have been synthesized for the development of hydrolytically stable scaffolds for nerve repair [START_REF] Morvan | Adult human epidermal stem cells recruitment by an oligoalginate extract[END_REF]. In vitro degradation studies have shown that alginamides have better stability than the ester derivatives over a two-month period.

Secondly, grafting of cell-specific ligands or signaling molecules at C6 offers the possibility to generate alginates with more targeted actions. Hydrogels made of alginates functionalized with the GRGDY cell adhesion peptide have shown a better capacity to promote the attachment and spreading of myoblast cells over those prepared with unmodified alginates [START_REF] Rowley | Alginate hydrogels as synthetic extracellular matrix materials[END_REF]. Alginate-amino acid derivatives have been designed to engineer glycocalyx-like poly(DL-lactic acid) surfaces [START_REF] Zhu | Surface engineering of poly(DL-lactic acid) by entrapment of alginate-amino acid derivatives for promotion of chondrogenesis[END_REF]. Alkaline amino acids in particular demonstrate a positive effect on the attachment and growth of chondrocytes.

Carbohydrate substituents also have a positive impact on cell adhesion and hydrogels made of galactose-modified alginates show higher recruitment of hepatocytes than those made of native alginates [START_REF] Donati | Galactose-substituted alginate: preliminary characterization and study of gelling properties[END_REF][START_REF] Tian | Preparation and characterization of galactosylated alginate-chitosan oligomer microcapsule for hepatocytes microencapsulation[END_REF][START_REF] Yang | Galactosylated alginate as a scaffold for hepatocytes entrapment[END_REF].

To the best of our knowledge, amidation reactions on alginates reported in the literature to date are mostly based on aqueous carbodiimide chemistry with N-ethyl-N′-(3dimethylaminopropyl)carbodiimide (EDC). This coupling reagent, although effective and popular, presents a major side reaction consisting of the internal rearrangement of the Oacylurea activated ester of EDC to an N-acylurea that becomes stably incorporated in the substrate [START_REF] Davidovich-Pinhas | Evaluating the mucoad-hesive properties of drug delivery systems based on hydrated thiolated alginate[END_REF]. This undesirable and impossibleto-eliminate by-product can be formed in non-negligible amounts, with reports of up to 8% of N-acylurea [START_REF] Tian | Preparation and characterization of galactosylated alginate-chitosan oligomer microcapsule for hepatocytes microencapsulation[END_REF]. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4methylmorpholinium chloride (DMTMM) is a promising alternative coupling reagent. Since its first report for the synthesis of amides in 1999 [START_REF] Kunishima | 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride: an efficient condensing agent leading to the formation of amides and esters[END_REF], the use of DMTMM has considerably increased with comparable and in some cases greater results than the most popular coupling agents. DMTMM has many advantages, including easy by-product removal, good stability in water, and high reactivity. In addition, DMTMM-activated carboxylic acids show poor reactivity with alcohols. Therefore, this reagent is particularly suitable for generating amides from hydroxy-acids without requiring protection of the OH group and without ester formation. Furthermore, DMTMM, which is cheaper and less toxic than EDC, does not generate an N-acylurea by-product [START_REF] D'este | A systematic analysis of DMTMM vs EDC/NHS for ligation of amines to hyaluronan in water[END_REF]. These features have contributed to the emergence of this reagent for the amidation of polymers and, more specifically, of polysaccharides, including heparin [START_REF] Reyes-Ortega | Smart heparin-based bioconjugates synthesized by a combination of ATRP and click chemistry[END_REF] and hyaluronic acid [START_REF] Bergman | Hyaluronic acid derivatives prepared in aqueous media by triazine-activated amidation[END_REF][START_REF] Loebel | Precise tailoring of tyramine-based hyaluronan hydrogel properties using DMTMM conjugation[END_REF]. Nevertheless, to date, there are no reports of alginate amidation with DMTMM.

In this work, we report the C6 amidation of alginate oligosaccharides in aqueous media with amino acids and amine-functionalized glycosides using DMTMM. These modifications were carried out with a view to enhancing the stability of AOS against enzymatic degradation. In addition, carbohydrate grafting was also expected to promote the establishment of multivalent interactions with specific carbohydrate-binding proteins. The synthesis of multivalent glycoarchitectures is the subject of an intense research motivated by major applications in the diagnosis and the treatment of viral or bacterial infections [START_REF] Bernardi | Multivalent glycoconjugates as anti-pathogenic agents[END_REF]. Synthetic polymers as well as polysaccharides, such as chitosan and cellulose, have been extensively used as multivalent scaffolds. Surprisingly, natural oligosaccharidic platforms, with the exception of cyclodextrins and a few mono-and disaccharides, have never really been investigated for the synthesis of multivalent glycoconjugates. Alginate oligosaccharides may thus offer new interesting scaffolds for the development of multivalent glycoarchitectures.

Materials and Methods

Material and equipment

All amino acids were purchased as methyl esters HCl salt from Sigma-Aldrich except alanine methyl ester HCl which was purchased from TCI. Hydrochloric acid, sodium hydroxide, absolute ethanol, and ammonium carbonate were purchased from Carlo ERBA and used without purification. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) was prepared according to the procedure established by [START_REF] Kunishima | 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride: an efficient condensing agent leading to the formation of amides and esters[END_REF]. In brief, equimolar quantities of 2-chloro-4,6-dimethoxy-1,3,5triazine and N-methylmorpholine were stirred for 30 min in THF at room temperature, and DMTMM, which precipitates, was then isolated by filtration and dried under vacuum. Alginate lyase from Pedobacter saltans, Pedsa-0632 (GenBank accession number ADY54336), was expressed in E. coli as recently reported in the literature [START_REF] Mathieu | Functional exploration of the polysaccharide lyase family PL6[END_REF]. Alginate lyase from Sphingomonas sp. (2500 U.mL -1 in 3.2 M ammonium sulfate) was purchased from Megazyme. Peroxidase-labeled concanavalin A (ConA-HRP) from Canavalia ensiformis was purchased from Sigma.

1 H NMR spectra were recorded on a Bruker spectrometer DPX400 at 400 MHz. Samples (10 mg) were previously freeze-dried in D2O then solubilized in D2O (500 L) and the solvent residual peak was used as an internal standard. Matrix-assisted laser desorption ionization timeof-flight (MALDI-TOF) negative-ion spectra were recorded on a Bruker Daltonics Autoflex apparatus using 2,5-dihydroxybenzoic acid (DHB) as a matrix. GPC-MALLS chromatogram was obtained by injection through three Shodex columns: OHpack SB803 HQ, OHpack SB802.5 HQ and OHpack SB802 HQ placed in series. Detection was performed with a Waters refractometer and a Wyatt multi-angle light scattering detector. UV-visible spectra were recorded on a Varian Cary 50 Bio spectrometer at 235 nm.

Characterization of native alginate oligosaccharides (AOS)

The molecular weight of AOS was determined by high-performance size-exclusion chromatography (HPSEC) using an eighteen-angle light scattering detector (DAWN™ HELEOS, Wyatt, Santa Barbara, CA USA), coupled with refractive index detector. Elution was performed on three Shodex columns: OHpack SB803 HQ, OHpack SB802.5 HQ and OHpack SB802 HQ placed in series with 0.1 M NaNO3 (±10 mM EDTA) solvent. To calculate the molecular mass, the dn/dc value used was 0.165 mL/g [START_REF] Smidsrød | A light scattering study of alginate[END_REF][START_REF] Harding | Analysis of Polysaccharides by Ultracentrifugation. Size, Conformation and Interactions in Solution[END_REF].

The molecular weight characteristics of native AOS were the followings: number-average molecular weight Mn = 3.5 kDa, weight-average molecular weight Mw = 5 kDa and polydispersity index PDI = 1.4. The M:G ratio as determined by 1 H NMR according to Grasdalen's method was 0.74 [START_REF] Grasdalen | A p.m.r. study of the composition and sequence of uronate residues in alginates[END_REF]. Infrared spectroscopy of AOS displayed characteristic absorptions at 3285 cm -1 for OH bond stretching; 1607 and 1414 cm -1 for C=O and CO bond stretching of sodium carboxylate function of the uronate unit; 1136 and 1032 cm -1 for CO and CC bond vibrations of carbohydrates. MALDI-TOF mass spectroscopy analysis in negative mode of AOS showed oligomers with a degree of polymerization (DP) ranging from 4 to 22 (see Electronic Supplementary Information for all spectra and chromatogram related to the characterization of AOS).

Determination of the Degree of Substitution.

The degree of substitution (DS) of amidated AOS was calculated using 1 H NMR according to the following formula:

DS = Is Ns 1.92 × I H1G
Is refers to the peak intensity of an isolated signal of the substituent S, and Ns is the number of protons related to this signal. IH1G refers to the signal intensity of the guluronate anomeric protons. The detailed formula for DS determination is reported in the Electronic Supplementary Information.

Determination of the grafting yield.

The reaction yield was calculated by dividing the number of moles of amidated AOS by the number of moles of native AOS. The molar mass used for native alginate oligosaccharide was 198 g.mol -1 . The molar mass of the amidated alginate oligosaccharide was defined using the following equation: Mamidated alginate = Mamidated uronate × DS + 198 × (1  DS).

General procedure for the amidation of AOS

To a solution of AOS (0.60 mmol of sodium uronate unit) in ammonium carbonate buffer (4 mL, 0.1 M, pH 9.4) is added the amine-functionalized glycoside or amino acid methyl ester (0.60 mmol), DMTMM (1.20 mmol) and the reaction mixture is stirred for 15 h at 25 °C. For the amidation with amino acids, a solution of sodium hydroxide (6 mL, 1 M, 6.00 mmol) is then added to hydrolyze the methyl esters and the reaction mixture is stirred for 1.5 h at 25 °C before being neutralized using hydrochloric acid (1M).

Isolation and purification of the amidated AOS were achieved according to the following procedure. The reaction mixture is poured into absolute ethanol (90 mL) to precipitate the amidated AOS. After 5 min, the precipitate is centrifuged (4 °C, 8500 rpm, 10 min) then solubilized in 2 mL of water and precipitated again by addition of absolute ethanol (90 mL).

The product is centrifuged (4 °C, 8500 rpm, 8 min), dried under high vacuum for 30 min to remove residual ethanol and then freeze-dried.

Enzymatic hydrolysis of amidated alginate oligosaccharides

Enzymatic hydrolysis of amidated AOS was carried out with two alginate lyases: Pedsa0632, a recombinant lyase from Pedobacter saltans expressed in E. coli [START_REF] Mathieu | Functional exploration of the polysaccharide lyase family PL6[END_REF]) and a commercial alginate lyase from Sphingomonas sp.

(Megazyme, 2500 U.mL -1 in 3.2 M ammonium sulfate). The reaction was performed by adding 5 µl of 10-fold diluted enzymes in Tris-HCl buffer (100 mM, pH 8.0, 2 mM CaCl2) to a 500 µL cuvette containing 5 mM of amidated AOS in the same buffer at 30°C. The formation of unsaturated product was monitored by measuring the absorbance at 235 nm as a function of time on a Varian Cary 50 Bio spectrometer.

Enzyme-linked lectin assay (ELLA)

The method was adapted from Köhn et al. [START_REF] Köhn | Functional Evaluation of Carbohydrate-Centred Glycoclusters by Enzyme-Linked Lectin Assay: Ligands for concanavalin A[END_REF]. Nunc MaxiSorp® plates were coated overnight with yeast mannan purchased from Sigma (100 µL per well at 10 µg.mL -1 in phosphate-buffered saline (PBS prepared by dissolution of tablets from Sigma) containing 0.1 mM Ca 2+ ) at room temperature. The wells were then washed twice with washing buffer (200 µL per well, PBS containing 0.05% (v/v) Tween 20 -PBST). The washing procedure was repeated after each incubation throughout the assay. The wells were then blocked with 3% BSA/PBS (150 µL per well) for at least 1 h at 37°C. After washing 3 times, the wells were filled with serial dilutions of peroxidase-labeled concanavalin A (ConA-HRP from Canavalia ensiformis (Sigma) previously dissolved at 0.25 mg.mL -1 in PBS; 100 µL) from 10 -1 to 10 -5 mg.ml -1 in PBS-BSA 0.3% and incubated 1 h at 37°C. The plates were then washed three times with PBST and one time with PBS and ophenylenediamine dihydrochloride (SIGMAFAST TM OPD) (50 µL per well, 0.4 mg.ml -1 ) in citrate-phosphate buffer (50 mM, pH 5.0, with 0.4 mg.ml -1 urea hydrogen peroxide) was added.

The reaction was stopped after 10 min by adding H2SO4 30% (v/v) (50 µL per well) and the absorbances were measured at 490 nm on a microtiter plate reader (Bio-Rad 680). The concentration of lectin-enzyme conjugate having an absorbance between 0.8 and 1.0 was used for inhibition experiments. The final concentration for ConA-HRP was 3 µg.mL -1 .

For inhibition experiments, the microtiter plates were coated overnight at room temperature with yeast mannan (100 µL per well, 10 µg.mL -1 ). The wells were then washed and blocked with BSA as described above. The mannose-amidated AOS (AOSMan) inhibitors were used as stock solution of 20 mM in water. During the blocking step, in 1.5 mL microtubes, 200 µL of AOSMan, at different concentrations and in a series of three-fold dilutions, was incubated with 200 µl of ConA-HRP (6 µg.mL -1 ) in 0.3% PBS-BSA for 1 h at 37°C. The above solutions (100 µL per well; 3 different wells for each concentration of inhibitors) were then transferred to the washed and the OPD substrate was added (50 µL per well). Color development was stopped after 10 min and the absorbances were measured. The percentages of inhibition were calculated as follows, where A = absorbance: % Inhibition = (A(no inhibitor) -A(with inhibitor)) / A(no inhibitor) x 100 IC50 values were reported as the concentration of inhibitors required to inhibit 50% of the ConA-yeast mannan association.

Results and Discussion

Preliminary investigation of DMTMM-mediated amidation of AOS with alanine

Alginate oligosaccharides (AOS) used in this study were prepared from Laminaria digitata alginates by controlled enzymatic depolymerization followed by an acid treatment [START_REF] Gedouin | Depolymerized sodium alginate[END_REF]. The weight average molecular weight and the polydispersity index of the AOS as determined by SEC-MALLS were 5 kDa and 1.4 kDa, respectively. A complementary analysis using MALDI-TOF mass spectrometry showed the presence of oligosaccharides with sizes in the range of DP 4 to 22. The M:G ratio as determined using 1 H NMR was 0.74. The DTMMM-mediated amidation of AOS was first investigated with alanine as model amino acid.

Preliminary reactions were performed at 25 °C in water in presence of two equivalents of the acylating agent and one equivalent of amino acid per carboxylic acid group. The use of an unprotected amino acid resulted in self-polycondensation reactions exclusively and no amidation of the alginate backbone was observed by proton NMR after precipitation of AOS in ethanol. The following reactions were therefore carried out in similar conditions but with a protected amino acid: L-alanine methyl ester hydrochloride (Scheme 1). Saponification of the methyl ester was achieved in situ by adding an aqueous solution of sodium hydroxide and then neutralizing the reaction media with hydrochloric acid. The alanine-amidated alginate oligosaccharides (AOSAla) were finally isolated by precipitation in ethanol and freeze-dried.

Scheme 1: DMTMM-mediated amidation of AOS with L-Ala-OMe in water

1 H NMR spectroscopy of the isolated product displayed a broad doublet at 1.41 ppm, which was attributed to the methyl group of the amidated alanine moieties (Fig. 1). Another piece of evidence of the alanine grafting was provided by the broadening of the peaks related to the alginate backbone in comparison with the NMR spectrum of native AOS (See Electronic Supporting Information). The presence of a well-resolved thin doublet at 1.5 ppm attributed to free alanine suggested minor contamination of the product with unreacted amino acids.

However, the very low intensity of the signal attested to the satisfactory purity of AOSAla. The absence of protons related to the DMTMM or its side products also confirmed the satisfactory purity of the amidated AOS, indicating the efficiency of the precipitation protocol. This simple procedure also eliminated the sodium chloride salts generated by the neutralization reaction after the hydrolysis of the methyl ester. No weight loss was observed after ultrafiltration of the product with a 1 kDa cut-off membrane.

The alanine grafting on AOS was also probed by infrared spectroscopy. IR spectrum of AOS displayed characteristic absorptions peaks at 1607 and 1414 cm -1 for C=O and CO stretching, respectively, in the sodium carboxylate function of the uronate unit (Fig. 2). On the IR spectrum of AOSAla, the covalent grafting of alanine was ascertained by both the shifting of the C=O band and the presence of additional peaks between 1400 cm -1 and 1663 cm -1 related to the C-N bond and the new carboxylic acid functions. In addition, MALDI-TOF mass spectroscopy confirmed the grafting by the mass gain observed on the spectrum of AOSAla compared with the AOS spectrum (See Electronic Supplementary Information).

Figure 2: IR spectra of native and alanine-amidated alginate oligosaccharides AOS and

AOSAla

Although alanine grafting proceeded with satisfactory yield (77-90%) in the above-mentioned conditions, there was a high discrepancy on the degree of substitution (DS) determined by 1 H NMR. Under similar reaction conditions and without pH adjustment, the reaction mixture was slightly acidic (pH ≈ 5) and the DS varied from 0.13 to 0.42 (Fig. 3). To circumvent this poor reproducibility, the reaction was evaluated at different pH conditions. Adjusting the pH of the reaction mixture to 7 by adding sodium hydroxide just after the addition of all the reactants significantly increased the DS and reproducibility. The addition of sodium hydroxide in the reaction media was beneficial, but it did not keep the pH constant as the reaction progressed. To control the pH more accurately, the reaction was carried out in 3-N-(morpholino)-propanesulfonic acid (MOPS) buffer at pH 7 and in ammonium carbonate buffer at pH 9.4, each presenting different advantages. The MOPS buffer, which is soluble in organic solvents, can be efficiently removed by the precipitation in ethanol. Ammonium bicarbonate, on the other hand, is volatile and can be removed by freeze-drying. The DS observed for the reaction conducted in MOPS at pH 7.0 was lower than that observed in presence of sodium hydroxide. A similar result was previously observed in 3-N-(morpholino)-ethanesulfonic acid buffer (MES) during DMTMM-mediated acylation of albumin with amino-sugars [START_REF] Farkaš | Comparison of EDC and DMTMM efficiency in glycoconjugate preparation[END_REF]. The highest grafting ratio (DS = 0.54) and the best reproducibility were obtained using ammonium carbonate buffer at pH 9.4.

DS pH conditions

Grafting of amino acids and amine-functionalized glycosides to AOS

After determining the optimal pH conditions for the amidation of AOS with alanine, we evaluated the applicability of this reaction to other amino acids and amine-functionalized glycosides (Scheme 2) as well as the possibility to reach the targeted DS.

Scheme 2: DMTMM-mediated amidation of AOS with amino acids and aminefunctionalized glycosides

First, the number of alanine and DMTMM equivalents was modified and the resulting products were characterized by NMR. The highest DS reached with alanine was 0.54 in presence of 1 amino acid equivalent and 2 DMTMM equivalents (Table 1). Using more equivalents of the acylating reagent did not help to exceed this limit, because above a certain DS (≈ 0.50), the reaction was most likely inhibited by the precipitation of the methyl ester intermediate.

Conversely, using fewer equivalents of alanine (0.35 and 0.15) led to the production of amidated AOS at the targeted DS with extremely satisfactory yields. The degradation of alginates is very slow in the region between pH 5 and 10 (Haug, Larsen, Smirsröd, 1963); however, depolymerization of AOS may occur during the amidation reaction. A SEC-MALLS analysis of AOSAla-0.13 revealed an average molecular weight of 5.2 kDa and a polydispersity index of 1.4, which is in good agreement with the expected theoretical values. Although the chromatogram suggests the presence of low molecular weight compounds in the product, their low amount (about 10%) confirms that no significant degradation occurred (See Electronic Supporting Information). To assess the general applicability of the protocol, hydrophobic leucine and hydrophilic serine were also amidated. Once again, the yields of the reactions were excellent and ranged from 70% to 90%. A good correlation was observed between the number of amino acid equivalents and the DS of the product below 0.5. Beyond 0.5 equivalents, the reaction was again limited by the solubility of the product. Nonetheless, the higher hydrophilicity of the serine made possible to attain a higher DS (0.83) than for alanine or leucine.

Amino In light of the versatility and efficiency of amidation with amino acids, we then attempted to introduce carbohydrate ligands onto the alginate backbone. Mannose and rhamnose were selected as model carbohydrates as well as for their attractive biological properties. In particular, both have demonstrated beneficial actions on keratinocytes [START_REF] Labský | Mannosides as crucial part of bioactive supports for cultivation of human epidermal keratinocytes without feeder cells[END_REF][START_REF] Andrès | Pharmacological properties of rhamnose-rich polysaccharides, potential interest in age-dependent alterations of connectives tissues[END_REF] and they may have a synergistic effect with alginate oligosaccharides in skin protection or repair. 3-(2-Aminoethylthio)propyl-α-D-mannopyranoside (Roy, Pagé, Figueroa Perez, & Verez Bencomo, 1998) and 3-(2-aminoethylthio)propyl-α-Lrhamnopyranoside [START_REF] Roy | Synthesis of Antigenic Carbohydrate Polymers Recognized by Lectins and Antibodies[END_REF] were prepared according to procedures found in the literature (See Electronic Supplementary Information). Allyl glycosides were synthesized by Fischer glycosylation and they were next engaged in a UV-promoted thiol-ene addition with cysteamine. In addition to allowing the functionalization of carbohydrates in two steps and without the use of protecting group, this synthetic sequence was carried out in water and the isolation of the product was easily performed on an ionic exchange resin. Amine-functionalized rhamnose and mannose glycosides (Rha-NH2, Man-NH2) underwent DMTMM-mediated amidation with AOS (Table 1). The reactions were conducted in ammonium carbonate buffer in the presence of two equivalents of DMTMM and with a variable amount of the carbohydrates. As observed with the amino acids, the yields of amidation for aminefunctionalized glycosides were about 80% and were little influenced by the number of glycoside equivalents. No solubility issue was encountered in this reaction, but the DS never exceeded 0.38 for the highest glycoside concentration. The limiting factor is likely due to steric hindrance.

For the reactions that were conducted with fewer than five amine-functionalized glycoside equivalents, the DS was fairly well correlated to the amount of monosaccharide introduced but in a relatively less precise way than for amino acids.

Enzymatic hydrolysis of amino acid-and carbohydrate-amidated AOS

A recent report on the enzymatic behavior of alginates grafted with poly(ε-caprolactone) (PCL) of various lengths and DS [START_REF] Benykhlef | Alginate grafted with poly(ε-caprolactone): effect of enzymatic degradation on physicochemical properties[END_REF] shows that degradation by Flavobacterium sp. alginate lyase is incomplete and is slowed down by the presence of PCL. However, to the best of our knowledge, no studies have been conducted on amidated alginate oligosaccharides.

The stability of amino acid-and carbohydrate-amidated AOS against enzymatic degradation was assessed with two alginate lyases. The first one, having an endo-M specificity, is a commercially available recombinant alginate lyase from Sphingomonas sp. (Megazyme, Bray, Ireland). The second one (Pedsa-0632 from Pedobacter saltans), has been expressed in E. coli [START_REF] Mathieu | Functional exploration of the polysaccharide lyase family PL6[END_REF], and displays an endo-MG and endo-G specificity. Regardless of their specificity, alginates lyases release unsaturated compounds via a β-elimination mechanism and their activity can be monitored using UV-visible spectroscopy at 235 nm. Native AOS and AOS amidated with different DS of leucine, serine and mannose thus underwent enzymatic hydrolyses with both alginate lyases under the same conditions and the reactions were monitored using UV spectroscopy (See Electronic Supplementary Information). Figure 4 illustrates the hydrolysis of mannose-amidated AOS by Pedsa-0632 according to their DS. All the amidated AOS, regardless of the nature of their substituent, showed similar behavior in presence of the lyases. Hydrolysis was reduced by 60-70% for the amidated AOS with a DS of only 0.1 and it was completely inhibited for grafting densities above 0.3, except in the leucine series for which no hydrolysis was observed for a DS of 0.6. As expected, enzymatic hydrolysis was hampered by steric hindrance on the alginate backbone. Interestingly, both alginates lyases were able to act on the modified oligosaccharides. This activity highlights the fact that the amidation reaction is likely to occur either on the mannuronic units or on guluronic units and that biodegradation of amidated AOS is still possible.

Multivalent binding of mannose-amidated AOS (AOSMan) with concanavalin A lectin

Alginate oligosaccharides show interesting intrinsic biological properties. Nevertheless, grafting of biomolecules such as amino acids or sugars along the alginate backbone can serve multiple purposes. The ligand can be used for site-specific delivery of the alginate oligosaccharide. Alternatively, the alginate backbone can be seen as a multivalent molecular scaffold. The binding capability of the mannosylated AOS toward concanavalin A (ConA) lectin was thus assessed using a solid-phase competitive enzyme-linked lectin assay (ELLA).

ConA is a multimeric protein with a specificity for -mannosides and its use in several studies makes it a good lectin model [START_REF] Roy | Effect of shape, size, and valency of multivalent mannosides on their binding properties to phytohemagglutinins[END_REF]. Yeast mannan was used as ligand for lectin and was immobilized on a microtiter plate. Lectin binding was assessed in the absence and in the presence of the competitive AOSMan inhibitors using a horseradish peroxidase-labeled ConA (ConA-HRP) producing a colorimetric reaction with o-phenylenediamine. IC50 values, corresponding to the concentration of inhibitors necessary to inhibit 50% of the interaction of lectin with the surface, was measured for each mannosylated alginate oligosaccharide (AOSMan-0.1, AOSMan-0.24 and AOSMan-0.38) and compared with native AOS and -Me-mannoside monosaccharide used as control (Fig. 5). First, no interaction was observed between AOS and ConA, revealing that the sodium mannuronate residue of alginate does not bind to the lectin. Regarding α-methyl-mannoside and AOSMan-0.1, similar inhibition degrees were observed with IC50 values of the order of 1 mM, meaning that the alginate oligosaccharide with a DS of 0.1 interacts with the lectin as a monosaccharide. These values are in agreement with those found in the literature [START_REF] Euzen | Synthesis of glycopeptide dendrimers, dimerization and affinity for Concanavalin A[END_REF]. For mannose-amidated alginate oligosaccharides of higher DS, the inhibition increased drastically. IC50 values down to 56.3 M and 5.6 M were determined for AOSMan-0.24 and AOSMan-0.38, respectively. For AOSMan-0.38, the affinity to ConA was 178-fold higher than for methyl -mannoside. This result is extremely encouraging because a rapid survey of the literature shows that most inhibitors displaying similar efficiency either present a higher valency or are obtained through a much more complex synthesis process. Low micromolar inhibitors of ConA include glycopolymers carrying about 50 pendant sugars [START_REF] Gou | A detailed study on understanding glycopolymer library and con a interactions[END_REF], mannose-coated single walled carbon nanotubes [START_REF] Cid Martin | Tuning of glyconanomaterial shape and size for selective bacterial cell agglutination[END_REF] or "click" high mannose oligosaccharide mimics [START_REF] Cendret | Design and synthesis of a ''click'' high-mannose oligosaccharide mimic emulating Man8 binding affinity towards Con A[END_REF]. Considering the ease of our amidation procedure, we believe that alginate oligosaccharides offer interesting perspectives for the development of a wide range multivalent glycoconjugates.

Conclusion

Here, we developed an efficient procedure for the amidation of alginate oligosaccharides based on the use of DMTMM. The reaction features easy implementation: it is carried out in a volatile buffered medium and the purification of the product is achieved by precipitation. Amino acids and carbohydrates were introduced in high yield along the alginate backbone at targeted degrees of substitution. The amidated alginate oligosaccharides showed improved resistance to enzymatic hydrolysis by alginate lyases regardless of their specificity. This stability may be related to grafting density. Carbohydrate-amidated alginate oligosaccharides were shown to be efficient multivalent inhibitors of concanavalin A lectin. These findings have exciting perspectives for the design of new anti-infection agents or the improvement of current cosmetic ingredients.

  Characterization of amidated AOS (Distinctive signals used to calculate DS are labeled with * in NMR peak lists):AOSAla: 1 H NMR (ppm, D2O, 298 K) δ 5. H1Gα,.97 * (m, 1.00 H, H1G -inner alginate chain), 4. H1Gβ, end; H1M, H5GM, H5GG -inner alginate chain), 4.41-3.62 (m, H2-4G, H2-5M -alginate; CH3-CH(NH)-COONa -alanine), 1.42 * (d, J = 7.1 Hz, 3.13 H, CH3-CH(NH)-COONa -alanine); FTIR (ATER, cm -1 ): ν 1032 and 1108 (CO and CC carbohydrates); 1407 (CO sodium carboxylate); 1560 (CNH amide II); 1594 (C=O sodium carboxylate); 1663 (C=O amide); 2933 (CH -alanine); 3285 (OH -carbohydrate). AOSSer: 1 H NMR (ppm, D2O, 298 K) δ 5.33-5.24 (m, H1Gα, H1Mα -reducing end), 5.24-5.00 * (m, 1.00 H, H1G -inner alginate chain), 5.00-4.44 (m, H1Gβ, H1Mβ -reducing end; H1M, H5GM, H5GG -inner alginate chain), 4.44-4.30 * (m, 1.60 H, HO-CH2-CH(NH)-COONaserine), 4.30-3.62 (m, H2-4G, H2-5M -alginate; HO-CH2-CH(NH)-COONa -serine); FTIR (ATER, cm -1 ): ν 1033 and 1103 (CO and CC carbohydrates); 1124 (COH -serine); 1409 (CO sodium carboxylate); 1558 (CNH amide II); 1601 (C=O sodium carboxylate); 1664 (C=O amide); 2936 (CH -serine); 3321 (OH -carbohydrate). AOSLeu: 1 H NMR (ppm, D2O, 298 K) δ 5.32-5.23 (m, H1Gα, H1Mα -reducing end), 5.23-4.99 * (m, 1.00 H, H1G -inner alginate chain), 4.99-4.61 (m, H1Gβ, H1Mβ -reducing end; H1M, H5GM, H5GG -inner alginate chain), 4.41-3.61 (m, H2-4G, H2-5M -alginate; (CH3)2-CH-CH2-CH(NH)-COONa -leucine), 1.80-1.57 (m, 3.867 H, (CH3)2-CH-CH2-CH(NH)-COONaleucine), 1.04-0.88 * (m, 7.636 H, (CH3)2-CH-CH2-CH(NH)-COONa -leucine); FTIR (ATER, cm -1 ): ν 1035 and 1134 (CO and CC carbohydrates); 1402 (CO sodium carboxylate); 1555 (CNH amide II); 1593 (C=O sodium carboxylate); 1664 (C=O amide); 2957 (CH -leucine); 3318 (OH -carbohydrate). AOSMan: 1 H NMR (ppm, D2O, 298 K) δ 5.28-5.21 (m, H1Gα, H1Mα -reducing end), 5.21-5.00 * (m, 1.00 H, H1G -inner alginate chain), 5.00-4.59 (m, H1Gβ, H1Mβ -reducing end; H1M, H5GM, H5GG -inner alginate chain), 4.90 (s, H1 -Man), 4.33-3.36 (m, H2-4G, H2-5M -alginate; H2-6 -Man, Man-O-CH2-CH2-CH2-S-CH2-CH2-NH), 2.88-2.60 (m, 2.602 H, Man-O-CH2-CH2-CH2-S-CH2-CH2-NH), 2.04-1.81 * (m, 1.309 H, Man-O-CH2-CH2-CH2-S-CH2-CH2-NH); FTIR (ATER, cm -1 ): ν 1030 and 1126 (CO and CC carbohydrates); 1417 (CO sodium carboxylate); 1584 (broad, CNH amide II and C=O sodium carboxylate); 1651 (C=O amide); 2929 (CH -Man-NH2); 3348 (OH -carbohydrate). AOSRha: 1 H NMR (ppm, D2O, 298 K) δ 5.29-5.19 (m, H1Gα, H1Mα -reducing end), 5.19-4.99 * (m, 1.00 H, H1G -inner alginate chain), 4.99-4.56 (m, H1Gβ, H1Mβ -reducing end; H1M, H5GM, H5GG -inner alginate chain), 4.82 (s, H1 -Rha), 4.30-3.38 (m, H2-4G, H2-5M -alginate; H2-5 -Rha, Rha-O-CH2-CH2-CH2-S-CH2-CH2-NH), 2.87-2.61 (m, 2.59 H, Rha-O-CH2-CH2-CH2-S-CH2-CH2-NH), 2.04-1.82 (m, 1.262 H, Rha-O-CH2-CH2-CH2-S-CH2-CH2-NH), 1.41-1.22 * (m, 2.071 H, CH3 -Rha); FTIR (ATER, cm -1 ): ν 1034 and 1127 (CO and CC carbohydrates); 1415 (CO sodium carboxylate); 1571 (CNH amide II); 1608 (C=O sodium carboxylate); 1657 (C=O amide); 2925 (CH -Rha-NH2); 3363 (OH -carbohydrate).

Figure

  Figure 1: 1 H NMR spectrum of alanine-amidated alginate oligosaccharides AOSAla

Figure 3 :

 3 Figure 3: Evolution of the DS of alanine-amidated AOSAla as determined by 1 H NMR

Figure 4 :

 4 Figure 4: UV-visible monitoring of mannose-amidated alginate oligosaccharide

Figure 5 :

 5 Figure 5: Inhibition of concanavalin A by mannose-amidated alginate oligosaccharides (AOSMan)

Table 1 : Amidation of AOS

 1

	acid/	Number of	DS	Product	Yield (%)
	Amine-functionalized	equivalents per
	glycoside	carboxylic
		function
	L-alanine	1	0.54	AOSAla-0.54	93
	L-alanine	0.35	0.35	AOSAla-0.35	74
	L-alanine	0.15	0.13	AOSAla-0.13	82
	L-serine	1	0.83	AOSSer-0.83	70
	L-serine	0.35	0.33	AOSSer -0.33	75
	L-serine	0.15	0.10	AOSSer -0.10	87
	L-leucine	1	0.65	AOSLeu-0.65	73
	L-leucine	0.35	0.34	AOSLeu-0.34	79
	L-leucine	0.15	0.14	AOSLeu-0.14	84
	Man-NH2	2	0.38	AOSMan-0.38	77
	Man-NH2	1	0.35	AOSMan-0.35	85
	Man-NH2	0.33	0.24	AOSMan-0.24	87
	Man-NH2	0.2	0.1	AOSMan-0.1	83
	Rha-NH2	1	0.35	AOSRha-0.35	85
	Rha-NH2	0.5	0.38	AOSRha-0.38	70

Acknowledgments

The research leading to these results received funding from Brittany Regional Council and OSEO as part of the FUI-AAP14"Poly-mer"project. We also gratefully acknowledge the mass spectrometry and NMR analytical facilities at ICMG (FR 2607), the chromatography facility at CERMAV as well as Carnot Institut PolyNat and Labex Arcane (ANR-11-LABX-0003-01).