Semisynthesis of Chondroitin Sulfate Oligosaccharides Based on the

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Semi-synthesis of chondroitin sulfate oligosaccharides based on the enzymatic degradation of chondroitin Xiao Zhang, Huiying Liu, Wang Yao, Xiangbao Meng, and Zhongjun Li J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b00112 • Publication Date (Web): 08 May 2019 Downloaded from http://pubs.acs.org on May 10, 2019

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The Journal of Organic Chemistry

Semi-synthesis of chondroitin sulfate oligosaccharides based on the enzymatic degradation of chondroitin Xiao Zhang, Huiying Liu, Wang Yao, Xiangbao Meng and Zhongjun Li * State Key Laboratory of Natural and Biomimetic Drugs; Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, P. R. China

Abstract: Chondroitin sulfate (CS) is a structurally complex polyanionic glycosaminoglycan that plays essential roles in the physiological processes. Here we report a facile approach to a library of CS tetra- and hexasaccharides based on the enzymatic degradation of chondroitin over 10~11 steps, which is the shortest synthetic route toward size-defined CS oligosaccharides reported to date. Subsequent biotinylation enabled the investigation of their interactions with growth factors, filling in the gaps of the existing research and providing probes for further exploration of the biological functions of CS.

Chondroitin sulfate (CS) is a major component of the extracellular matrix (ECM), which is known to be involved in cell−cell recognition,1 cancer metastasis,2 parasitic infections,3 and neuron growth regulation.4 As an important member of the glycosaminoglycan family, CS contains the repeating disaccharide unit of Dglucuronic acid (GlcA) and 2-acetamido-2-deoxy-D-galactose (GalNAc) arranged in the sequence of GlcAβ(1→3)-GalNAc-β(1→4). In addition, both the GlcA and GalNAc residues carry sulfate groups, giving rise to the structural and functional diversity of CS. CS has been found to be closely related to the occurrence and development of CNS diseases, the in-depth

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exploration and understanding of the relationship between CS and nervous system function is of great importance. Previous studies have shown that CS plays critical roles in modulating diverse neurological processes by regulating various growth factors, and their specific sequences with particular sulfation patterns are responsible for protein recognition.5 However, the existing studies have been established on the basis of CS tetrasaccharides and discrepancy in different binding experiments have been observed.5a, 6 Further structureactivity relationship studies are hindered due to the limited access to enough pure CS material with defined sulfation patterns and longer sequences, thus the availability of structurally explicit CS is the key to solving the current bottlenecks in related research. Despite recent advances in the synthesis of CS oligosaccharides,5a, 7 the extremely long routes, low yields and small scale limit the supply of enough pure CS materials for biological research, which stagnates current studies at tetrasaccharide level, so the development of new synthetic strategies for CS oligomers, especially for hexamer and longer sequences is of great importance but still challenging. To reduce protecting-group manipulations and the iterative glycosylation steps, our research group has recently reported the semi-synthesis of CS-E tetrasaccharide from hyaluronan with enzymatic degradation and C4 hydroxyl group conversion as the key steps7h (Scheme 1). However, this approach could only afford CS tetrasaccharides and the C4-conversion necessitated extremely strict manipulations. In order to further simplify the semi-synthetic route and access longer CS sequences, here we report a facile approach to a library of CS tetra- and hexasaccharides based on the enzymatic degradation of chondroitin over 10~11 steps. To the best of our knowledge, this is the shortest synthetic route toward structurally defined CS oligosaccharides reported to date. Furthermore, we have also biotinylated these CS variants to make them useful glycoprobes, and subsequently explored their interactions with growth factors to determine the structure-activity relationships.


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Scheme 1. Semi-synthesis of CS oligosaccharides based on the enzymatic degradation of hyaluronan or chondroitin

Previously our research group reported the enzymatic degradation of chondroitin in the presence of Bovine testicular hyaluronidase (BTH),8 which recognizes chondroitin and catalyzes both the hydrolysis and the transglycosylation processes probably due to the structural similarity between chondroitin and hyaluronan.9 Briefly, chondroitin sulfate was firstly desulfated with acidic methanol, and then degraded in the presence of BTH for 7 days, which afforded chondroitin tetra- and hexasaccharides as expected in 38% and 35% yield, respectively, from chondroitin (Scheme 2). To obtain a chemically stable and stereochemically homogeneous product and for the later attachment of an analytical probe, the conversion of the hemiacetal into β-azide derivatives was carried out.10 DMC (2-chloro-N, N’-1, 3dimethylimidazolium chloride) mediated direct nucleophilic attack by NaN3 without the use of protecting groups afforded 3 and 4 in 78% and 75% yield, respectively, from 1 and 2, as previously reported.8 Subsequent methyl esterification, benzylidene formation and acetylation gave tetramer 5 and hexamer 6 in respective overall yields of 64% and 55 % over three steps.

Scheme 2. Enzymatic degradation of chondroitin and synthesis of intermediates 5 and 6



Reagents and conditions: a) HCl/MeOH, RT, 7 days; b) 0.1 M NaOH (aq), RT, 24 h, 77% for 2 steps; c) 2.5% hyaluronidase, NaOAc/NaCl buffer, pH 5.0, 37 °C, 7 days, 1, 38%, 2, 35%; d) DMC, Nmethylmorpholine, NaN3, H2O, 0 °C-RT, 3, 78%, 4, 75%; e) HCl/MeOH, 4 °C; f) PhCH(OMe)2, CSA, DMF, 45 °C, 0.05 MPa; g) Ac2O, pyridine, 0 °C-RT, 5, 64% for 3 steps, 6, 55% for 3 steps.

Next, acid hydrolysis of the benzylidene acetal released the 4, 6-OH of the GalNAc and afforded the key intermediates 7 and 8, which were transformed into the corresponding CS-A, C and E variants as shown in Scheme 3. Selective benzoylation at O-6 of 7 and 8 with benzoyl cyanide7c gave the crystalline 6-O-benzoylated derivatives 9 and 10 in 86% and 89% yield, respectively, which were further treated with an excess of sulfur trioxide/trimethylamine complex in DMF at 60 °C followed by a two-step saponification process to provide the CS-A tetra- and hexasaccharides 11 and 12, respectively, in 91% and 87% overall yields. Controlled regioselective 6-O-sulfonation of 7 and 8 at 40 °C followed by the standard global deprotection gave the CS-C oligosaccharides 13 and 14, respectively, in 94% and 88% yields. Finally, exhaustive O-sulfonation and successive global deprotection provided the CS-E derivatives 15 and 16 in 90% and 84% yields, respectively.

Scheme 3. Synthesis of CS tetra- and hexasaccharides

Reagents and conditions: a) AcOH (aq), 80 °C, 7, 79%, 8, 73%; b) BzCN, pyridine, RT, 9, 86%, 10, 89%; c) SO3·NMe3 (6 eq/OH), DMF, 60 °C; d) SO3·NMe3 (2.5 eq/OH), DMF, 40 °C; e) SO3·NMe3 (10 eq/OH), DMF,


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60 °C; f) LiOH/H2O2, THF/H2O, -5 °C-RT, then 4 M NaOH, MeOH, 0°C-RT, 11, 91% for 2 steps, 12, 87% for 2 steps, 13, 94% for 2 steps, 14, 88% for 2 steps, 15, 90% for 2 steps, 16, 84% for 2 steps.

With these structurally defined oligosaccharides in hand, analytical labels such as biotin and fluorophores could be introduced using the copper catalyzed azido-alkyne cycloaddition (CuAAC) reaction to make them useful tools for the preparation of microarrays, or as probes for the study of the biological fuctions of CS. As an example, we biotinylated chondroitin oligosaccharides 3, 4 and CS- A, C, E variants 11-16 to afford 17-24, respectively11 (Scheme 4), and quantitatively immobilized them onto streptavidin-coated sensors to investigate their interactions with growth factors by biolayer interferometry (BLI).

Scheme 4. Biotinylation of the synthetic oligosaccharides

Reagents and conditions: a) ABio, CuSO4, Na ascorbate, H2O, RT, 17, 77%, 18, 74%, 19, 78%, 20, 75%, 21, 80%, 22, 73%, 23, 72%, 24, 69%.

Key growth factors of midkine (MDK)5a, 6 and neurotrophic growth factor (NGF)12 were evaluated. When the sensors were saturated by biotinylated oligosaccharides (0.24~0.28 nm, Figure S1) during the loading procedure, all of the CS derivatives 19~24 showed high binding affinity to MDK, with KD values all in nanomolar range (Table 1, Figure S3A). No obvious binding was detected for NGF, and nonsulfated derivatives 17~18 did not interact with these growth factors. However, upon decreasing the density of immobilized oligosaccharides (0.11~0.12 nm, Figure S2), we found MDK selectively bound



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with CS-C and CS-E hexasaccharides 22, 24 with high affinity and no significant binding was observed for the other CS variants (Table 1, Figure S3B), indicating 6-O-sulfonation and longer sequence played a vital role while eliminating the possible nonspecific interactions (Figure S4). This study involved CS hexamers into the interactions with growth factors for the first time, which was a good supplement to previous studies. Table 1. KD values of Ch and CS variants with midkine and NGFa MDK

NGF

0.24~0.28 nmb

0.11~0.12 nmb

0.24~0.28 nmb

Ch-4 (17)

--c

--c

--c

Ch-6 (18)

--

c

c

--c

CS-A-4 (19)

25.2 ±0.64 nM

--c

--c

CS-A-6 (20)

27.4 ±0.73 nM

--c

--c

CS-C-4 (21)

27.1 ±0.62 nM

--c

--c

CS-C-6 (22)

12.9 ±0.25 nM

20.9 ±0.46 nM

--c

CS-E-4 (23)

13.5 ±0.25 nM

--c

--c

CS-E-6 (24)

12.2 ±0.23 nM

28.0 ±0.71 nM

--c

--

a. Gradient concentrations of growth factors (3.9 nM~125 nM); b. The enhancement of response in the loading procedure; c. No obvious binding was detected.

In summary, we have developed a step economy method for the synthesis of structurally homogeneous CS tetra- and hexasaccharides in 10~11 steps from commercially available CS polymer. Benefiting from the efficient enzymatic degradation of chondroitin, this strategy offers a simple but efficient approach to bioactive CS oligosaccharides, with short synthetic route, scalable reaction, high overall yield and low cost, providing not only the well-studied CS tetramers, but also the largely unexplored hexamers, which will fill in the gaps in the existing research and significantly contribute to the biological studies of CS.

Experimental section General. Chondroitin 4-sulfate sodium salt (CS-A, Sigma-Aldrich), hyaluronidase from bovine tests (BTH, Sigma-Aldrich, Catalog #H3506), acetyl chloride (AcCl, 98%, Beijing chemical works, analytical grade), 2-


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chloro-1,3-dimethylimidazolidinium chloride (DMC, 98%, TCI Co.), sodium azide (NaN3, 99%, SigmaAldrich), acetyl anhydride (Ac2O, 98%, Beijing chemical works, analytical grade), PhCH(OMe)2 (97%, Acros Organics), (+)-camphorsulfonic acid (CSA, 98%, TCI Co.), Et3N (97%, Beijing chemical works, analytical grade), benzoyl cyanide (BzCN, 98%, TCI Co.), SO3·NMe3 (98%, Sigma-Aldrich), H2O2 aqueous solution (35% wt, Alfa aesar), LiOH·H2O (98%, Sino pharm chemical reagent Co.), L-sodium ascorbate (99%, Alfa aesar), CuSO4 (99%, Sigma-Aldrich), biotin (99%, TCI Co.) were used without further purification. Reaction solvents were purchased in HPLC grade (Fisher), dry solvents were purchased from Sigma-Aldrich (Dry DMF: 99.8%, Catalog #227058; Dry pyridine: 99.8%, Catalog #270970). Flash chromatography was performed on silica gel 200-300 mesh (Qingdao Haiyang). The reactions were monitored by TLC on coated aluminum sheets (silica gel 60 F254, Merck), and spots were detected under UV light (254 nm) then charring with 5 % (v/v) sulfuric acid in EtOH or cerium molybdate stain (CAM) followed by heating at 150 ºC. 1H and

13

C NMR spectra were

recorded at 298K on Bruker Advance III 400 instrument. 1H and 13C spectra were referenced to residual DMSOd5H in DMSO-d6 (2.50 ppm, 1H; 39.52 ppm, 13C) and residual HOD in D2O (4.79 ppm, 1H). Chemical shifts are reported in ppm and multiplicities are indicated by s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). Coupling constants (J) are reported in Hertz. High-resolution mass spectra (HRMS) were recorded on a Bruker Solarix XR spectrometer or Shimadzu LCMS-IT-TOF spectrometer using ESI-TOF (electrospray ionization-time of flight) and data are reported in the form of (m/z). Method A: General procedure for global deprotection: A solution of sulfated derivatives (~45 μmol for tetramer, ~26 μmol for hexamer) in a mixed solvent of THF/H2O = 3/1 (2.0 mL) with a stir bar was cooled to -5 °C. A freshly prepared mixture of 1 M LiOH aq./H2O2 (35 % wt) = 2/1 (v/v, 750 μL) was then added. The colorless solution was stirred at -5 °C for 1 h then warmed to RT and stirred for another 16 h. Without monitoring by TLC, the mixture was cooled to 0 °C (ice-water bath), followed



by addition of MeOH (700 μL) and 4 M NaOH aq. (1.3 mL). The mixture was slowly warmed to RT and vigorously stirred overnight. The reaction solution was neutralized by Amberlite IR-120 [H+] resin, filtered and concentrated, then purified by Sephadex LH-20 eluent with H2O. The fractions containing the desired product (TLC monitored) were combined then lyophilized to afford the corresponding oligosaccharides as the sodium salts. Method B: General procedure for biotinylation via CuAAC. Sulfated oligosaccharide (~ 10 mg) and biotinylated alkyne Abio11 (2.0 eq) were dissolved in H2O (200 μL). CuSO4 (0.2 eq) and Na ascorbate (2.0 eq) were added. The mixture was stirred at room temperature for 48 h, and directly eluted from a column of Sephadex LH-20 with H2O to give the biotinylated derivative, the structure was confirmed by 1H NMR spectrum which showed the characteristic signal for the triazole proton at δ = 8.2~8.3 ppm. (Methyl 2, 3, 4-tri-O-acetyl-β-D-glucopyranosyluronate)-(1→3)-(2-acetamido-4, 6-O-benzylidene-2-deoxy-βD-galactopyranoside)-(1→4)-(Methyl 2, 3-di-O-acetyl-β-D-glucopyranosyluronate)-(1→3)-2-acetamido-4, 6-O-benzylidene-2-deoxy-β-D-galactopyranosyl azide (5). Tetramer 38 (430 mg, 0.51 mmol) was stirred in methanol (250 mL) containing acetyl chloride (final HCl concentration = 0.06 N) at 4 ºC for 96 h then neutralized by NaHCO3 (aq). The mixture was concentrated in vacuo and desalted by Sephadex LH-20 (MeOH) to afford the crude methyl ester as colorless amorphous solid. To the crude methyl ester in a 25 mL round-bottom flask was added dry DMF (7.5 mL), (+)-CSA (118 mg, 0.51 mmol, 1.0 equiv.) followed by addition of PhCH(OMe)2 (1.16 mL, 15 equiv.). The mixture was stirred at 45 ºC under reduced pressure overnight. After the completion of the reaction, the mixture was cooled, followed by addition of pyridine (7.5 mL) and Ac2O (5.0 mL) successively, then stirred overnight at room temperature. The reaction was quenched by addition of MeOH (5.0 mL) at 0 ºC and then concentrated. The resulting mixture was


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diluted with EtOAc (200 mL) and transferred into a separation funnel. The organic phase was washed with 1 N HCl aq. solution (40 mL×2), sat. NaHCO3 aq. solution (40 mL), brine (40 mL) and water (40 mL). The aqueous phases were combined and extracted 3 times with CH2Cl2. The combined organic phase was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by Flash silica chromatography (CH2Cl2/EtOAc/MeOH 1:1:0.08, 0.1% Et3N) to afford 5 (395 mg, 64% for 3 steps) as a white solid. Rf = 0.45 (CH2Cl2/EtOAc/MeOH =1:1:0.17); mp 212-214 °C (EtOH/Et2O); [α] 25 D= −26 ˚ (c 1.0 CHCl3); 1H NMR (400 MHz, DMSO-d6) δ 7.80 (1H, d, NH, J = 9.3 Hz), 7.43-7.37 (11H, m, NH, Ar-H), 5.49 (2H, s, PhCH), 5.29 (1H, dd, HD-3, J = 9.6 Hz, 9.6 Hz), 5.13 (1H, dd, HB-3, J = 9.4 Hz, 9.4 Hz), 5.02 (1H, d, HB-1, J = 8.1 Hz), 4.97-4.92 (2H, m, HD-1, HD-4), 4.76 (1H, dd, HD-2, J = 8.3 Hz, 8.3 Hz), 4.68 (1H, dd, HB-2, J = 9.0 Hz, 9.0 Hz), 4.55 (1H, d, HA-1, J = 9.1 Hz), 4.50-4.39 (2H, m, HC-1, HD-5), 4.26-4.25 (1H, m, HC-4), 4.20-4.19 (1H, m, HA-4), 4.164.08 (3H, m, HB-5, HA-6, HC-6), 4.06-3.92 (5H, m, HB-4, HA-2, HC-3, HC-6’, HA-6’), 3.86-3.82 (1H, m, HC-5), 3.78-3.74 (4H, m, HA-5, COOCH3), 3.66-3.63 (4H, m, HC-2, COOCH3), 3.53-3.51 (1H, m, HA-3), 1.98 (3H, s, CH3CO), 1.96 (9H, s, CH3CO), 1.94 (3H, s, CH3CO), 1.84 (3H, s, CH3CO), 1.78 (3H, s, CH3CO); 13C {1H} NMR (100 MHz, DMSO-d6) δ 170.0, 169.9, 169.8, 169.4, 169.3, 168.2, 167.8, 138.9, 138.7, 129.3, 128.5, 128.4, 126.8, 126.6, 101.0, 100.9, 100.4, 100.3, 88.6, 77.7, 77.6, 76.6, 74.7, 74.4, 73.8, 72.6, 71.9, 71.4, 70.9, 70.8, 69.5, 68.7, 68.6, 68.0, 65.9, 53.3, 53.0, 23.4, 23.2, 20.8, 20.7, 20.6; HRMS (ESI-MS) calcd for C54H65N5NaO27 +

[M+Na] + m/z 1238.3759, found 1238.3749.

(Methyl 2, 3, 4-tri-O-acetyl-β-D-glucopyranosyluronate)-(1→3)-(2-acetamido-4, 6-O-benzylidene-2-deoxy-βD-galactopyranoside)-(1→4)-(Methyl 2, 3-di-O-acetyl-β-D-glucopyranosyluronate)-(1→3)-(2-acetamido-4, 6-O-benzylidene-2-deoxy-β-D-galactopyranoside)-(1→4)-(Methyl

2,

3-di-O-acetyl-β-D-

glucopyranosyluronate)-(1→3)-2-acetamido-4, 6-O-benzylidene-2-deoxy-β-D-galactopyranosyl azide (6). Hexamer 48 (400 mg, 0.32 mmol) was stirred in methanol (250 mL) containing acetyl chloride (final HCl



concentration = 0.06 N) at 4 ºC for 96 h then neutralized by NaHCO3 (aq). The mixture was concentrated in vacuo and desalted by Sephadex LH-20 (MeOH) to afford the crude methyl ester as colorless amorphous solid. To the crude methyl ester in a 25 mL round-bottom flask was added dry DMF (7.5 mL), (+)-CSA (111 mg, 0.48 mmol, 1.5 equiv.) followed by addition of PhCH(OMe)2 (0.97 mL, 20 equiv.). The mixture was stirred at 45 ºC under reduced pressure overnight. After the completion of the reaction, the mixture was cooled, followed by addition of pyridine (7.5 mL) and Ac2O (5.0 mL) successively, then stirred overnight at room temperature. The reaction was quenched by addition of MeOH (5.0 mL) at 0 ºC and then concentrated. The resulting mixture was diluted with EtOAc (200 mL) and transferred into a separation funnel. The organic phase was washed with 1 N HCl aq. solution (40 mL×2), sat. NaHCO3 aq. solution (40 mL), brine (40 mL) and water (40 mL). The aqueous phases were combined and extracted 3 times with CH2Cl2. The combined organic phase was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by Flash silica chromatography (CH2Cl2/EtOAc/MeOH 1:1:0.10, 0.1% Et3N) to afford 6 (315 mg, 55% for 3 steps) as a white solid. Rf = 0.30 (CH2Cl2/EtOAc/MeOH =1:1:0.17); mp 225-227 °C (EtOH/Et2O); [α] 25 D= −30 ˚ (c 1.0 CHCl3); 1H NMR (400 MHz, DMSO-d6) δ 7.80 (1H, d, NH, J = 9.4 Hz), 7.43-7.37 (17H, m, NH, Ar-H), 5.49-5.45 (3H, m, PhCH), 5.28 (1H, dd, HF-3, J = 9.6 Hz, 9.6 Hz), 5.13-5.01 (3H, m, HB-3, HD-3, HB-1 or HD-1), 4.97-4.91 (3H, m, HB-1 or HD-1, HF-1, HF-4), 4.75 (1H, dd, HF-2, J = 8.6 Hz, 8.6 Hz), 4.69-4.61 (2H, m, HB-2, HD-2), 4.55 (1H, d, HA1, J = 9.1 Hz), 4.46-4.32 (3H, m, HC-1, HE-1, HF-5), 4.26-4.25 (1H, m), 4.19-4.07 (6H, m), 4.06-4.01 (5H, m), 3.96-3.91 (4H, m), 3.88-3.82 (2H, m), 3.78-3.74 (7H, m), 3.66-3.63 (4H, m), 3.53-3.51 (2H, m), 1.98-1.94 (21H, m, CH3CO), 1.84 (3H, s, CH3CO), 1.78 (3H, s, CH3CO), 1.77 (3H, s, CH3CO); 13C {1H} NMR (100 MHz, DMSO-d6) δ 170.0, 169.9, 169.7, 169.4, 169.3, 168.2, 167.8, 150.1, 138.9, 138.8, 138.7, 129.3, 128.5, 128.4, 126.8, 126.7, 126.6, 101.0, 100.9, 100.3, 88.6, 77.7, 77.5, 76.6, 76.5, 74.5, 74.4, 73.9, 72.8, 72.6, 72.0, 71.4, 70.9, 70.9, 69.5, 68.6, 68.0, 65.9, 53.3, 53.0, 29.5, 23.4, 23.3, 23.2, 22.6, 20.8, 20.7, 20.6; HRMS (ESI-MS)


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calcd for C80H96N6Na2O40 2+ [M+2Na] 2+ m/z 913.2723, found 913.2696. (Methyl

2,

3,

4-tri-O-acetyl-β-D-glucopyranosyluronate)-(1→3)-(2-acetamido-2-deoxy-β-D-

galactopyranoside)-(1→4)-(Methyl

2,

3-di-O-acetyl-β-D-glucopyranosyluronate)-(1→3)-2-acetamido-2-

deoxy-β-D-galactopyranosyl azide (7). To compound 5 (100 mg, 0.082 mmol) and a magnetic stir bar in a 100 mL round-bottom flask was added 75% AcOH aqueous solution (4 mL). The turbid colorless solution was stirred at 80 ºC for 40 min then was cooled to RT in a cold water bath. Toluene (4 mL) was added and the solvent was evaporated under reduced pressure. Flash silica chromatography (CH2Cl2/MeOH 10:1) afforded 7 (67.0 mg, 79%) as a white solid. Rf = 0.20 (CH2Cl2/MeOH =10:1); mp 205-207 °C (EtOH); [α] 25 D= +18 ˚ (c 1.0 MeOH); 1H NMR (400 MHz, DMSO-d6) δ 7.71 (1H, d, NH, J = 9.3 Hz), 7.37 (1H, d, NH, J = 5.4 Hz), 5.26 (1H, dd, J = 9.5 Hz, 9.5 Hz), 5.03-4.89 (4H, m), 4.82-4.77 (3H, m), 4.68 (1H, d, J = 8.5 Hz), 4.61-4.60 (2H, m), 4.38-4.32 (2H, m), 4.27-4.25 (1H, m), 4.064.01 (2H, m), 3.90 (1H, d, J = 9.4 Hz), 3.81-3.78 (4H, m), 3.75-3.73 (1H, m), 3.68-3.59 (6H, m), 3.53-3.48 (4H, m), 3.41-3.37 (4H, m), 1.97-1.94 (15H, m, CH3CO), 1.86 (3H, s, CH3CO), 1.80 (3H, s, CH3CO); 13C {1H} NMR (100 MHz, DMSO-d6) δ 170.0, 169.9, 169.8, 169.6, 169.5, 168.3, 167.8, 101.2, 100.8, 89.0, 79.4, 79.2, 77.8, 76.3, 75.1, 73.8, 73.1, 72.1, 71.4, 71.2, 71.0, 69.6, 67.0, 66.5, 60.5, 60.0, 53.3, 53.0, 50.1, 23.5, 23.4, 20.9, 20.7, 20.6; HRMS (ESI-MS) calcd for C40H57N5NaO27 + [M+Na] + m/z 1062.3133, found 1062.3120. (Methyl

2,

3,

4-tri-O-acetyl-β-D-glucopyranosyluronate)-(1→3)-(2-acetamido-2-deoxy-β-D-

galactopyranoside)-(1→4)-(Methyl 2, 3-di-O-acetyl-β-D-glucopyranosyluronate)-(1→3)-(2-acetamido-2deoxy-β-D-galactopyranoside)-(1→4)-(Methyl

2,

3-di-O-acetyl-β-D-glucopyranosyluronate)-(1→3)-2-

acetamido-2-deoxy-β-D-galactopyranosyl azide (8). From compound 6 (100 mg, 0.056 mmol) as described for the preparation of 7 to give 8 (62.1 mg, 73%) as a white solid. Rf = 0.20 (CH2Cl2/MeOH =7:1); mp 213-214 °C (EtOH); [α] 25 D= +23 ˚ (c 1.0 MeOH); 1H NMR



(400 MHz, DMSO-d6) δ 7.72 (1H, d, NH, J = 9.1 Hz), 7.40-7.38 (1H, m, NH), 7.29-7.27 (1H, m, NH), 5.26 (1H, dd, J = 9.5 Hz, 9.5 Hz), 5.02-4.88 (6H, m), 4.80-4.76 (3H, m), 4.69-4.61 (5H, m), 4.61-4.60 (2H, m), 4.374.31 (2H, m), 4.23-4.18 (2H, m), 4.04-3.99 (3H, m), 3.91-3.86 (2H, m), 3.82-3.77 (6H, m), 3.75-3.73 (2H, m), 3.70-3.68 (2H, m), 3.67-3.58 (6H, m), 3.53-3.46 (5H, m), 3.44-3.34 (4H, m), 1.97-1.93 (21H, m, CH3CO), 1.85 (3H, s, CH3CO), 1.82-1.78 (6H, m, CH3CO); 13C {1H} NMR (100 MHz, DMSO-d6) δ 170.0, 169.9, 169.8, 169.7, 169.6, 169.5, 168.3, 167.8, 101.2, 100.8, 89.0, 79.2, 77.8, 76.5, 76.3, 75.1, 73.8, 73.1, 73.0, 72.0, 71.4, 71.2, 71.0, 69.6, 67.0, 66.5, 60.5, 60.0, 53.4, 53.0, 50.1, 23.5, 23.4, 23.3, 20.9, 20.7; HRMS (ESI-MS) calcd for C59H84N6NaO40 + [M+Na] + m/z 1539.4616, found 1539.4575. (Methyl 2, 3, 4-tri-O-acetyl-β-D-glucopyranosyluronate)-(1→3)-(2-acetamido-6-O-benzoyl-2-deoxy-β-Dgalactopyranoside)-(1→4)-(Methyl 2, 3-di-O-acetyl-β-D-glucopyranosyluronate)-(1→3)-2-acetamido-6-Obenzoyl-2-deoxy-β-D-galactopyranosyl azide (9). To compound 7 (55 mg, 0.053 mmol, 1.0 equiv.) in a 25 ml flame-dried round bottom flask was added BzCN (42 mg, 0.32 mmol, 6.0 equiv.) and dry pyridine (1.5 mL). The mixture was stirred at RT under Ar for 6 h. After the completed of the reaction (TLC), the reaction was quenched by MeOH at 0 ºC, then evaporated in vacuo. The residue was diluted with EtOAc (75 mL), washed by 1 N HCl aq. (20 mL), sat. NaHCO3 aq. (20 mL) and brine (20 mL). The aqueous phase was extracted by CH2Cl2 (30 mL×3). The organic phase was combined, dried over Na2SO4, filtered, and then concentrated in vacuo. The crude product was purified by flash chromatography (CH2Cl2/MeOH = 25:1) to afford 9 as a white solid (56.8 mg, 86%). Rf = 0.30 (CH2Cl2/MeOH =20:1); mp 203204 °C; [α] 25 D= +12 ˚ (c 1.0 MeOH); 1H NMR (400 MHz, DMSO-d6) δ 8.02-7.47 (12H, m, NH, Ar-H), 5.29 (1H, dd, J = 9.6 Hz, 9.6 Hz), 5.17 (1H, d, J = 5.5 Hz), 5.11 (1H, dd, J = 9.3 Hz, 9.3 Hz), 5.04-5.01 (2H, m), 5.00-4.91 (2H, m), 4.84 (1H, dd, J = 8.8 Hz, 8.8 Hz), 4.75 (1H, dd, J = 8.9 Hz, 8.9 Hz), 4.49 (1H, d, J = 9.2 Hz), 4.41-4.36 (5H, m), 4.27-4.23 (1H, m), 4.15-4.06 (2H, m), 3.99-3.97 (1H, m), 3.94-3.89 (2H, m), 3.87-3.85 (1H,


Page 12 of 23

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


m), 3.76-3.68 (6H, m), 3.58 (3H, s), 1.99 (3H, s, CH3CO), 1.97 (3H, s, CH3CO), 1.96 (3H, s, CH3CO), 1.95 (3H, s, CH3CO), 1.88 (3H, s, CH3CO), 1.82 (3H, s, CH3CO); 13C {1H} NMR (100 MHz, DMSO-d6) δ 170.0, 169.8, 169.7, 169.6, 169.5, 168.2, 167.8, 167.7, 166.0, 133.9, 133.3, 131.2, 130.1, 130.0, 129.7, 129.2, 129.0, 101.3, 100.9, 88.8, 79.0, 78.9, 76.6, 75.1, 74.6, 73.8, 72.9, 72.1, 71.9, 71.4, 71.2, 71.0, 69.6, 67.4, 67.0, 64.1, 63.2, 53.3, 52.9, 50.0, 46.1, 23.4, 20.8, 20.7, 20.6; HRMS (ESI-MS) calcd for C54H65N5NaO29 + [M+Na] + m/z 1270.3657, found 1270.3650. (Methyl 2, 3, 4-tri-O-acetyl-β-D-glucopyranosyluronate)-(1→3)-(2-acetamido-6-O-benzoyl-2-deoxy-β-Dgalactopyranoside)-(1→4)-(Methyl 2, 3-di-O-acetyl-β-D-glucopyranosyluronate)-(1→3)-(2-acetamido-6-Obenzoyl-2-deoxy-β-D-galactopyranoside)-(1→4)-(Methyl

2,

3-di-O-acetyl-β-D-glucopyranosyluronate)-

(1→3)-2-acetamido-6-O-benzoyl-2-deoxy-β-D-galactopyranosyl azide (10). From compound 8 (45 mg, 0.029 mmol) as described for the preparation of 9 to give 10 (48.3 mg, 89%) as a white solid. Rf = 0.60 (CH2Cl2/MeOH =10:1); mp 205-207 °C; [α]

25

D=

+14 ˚ (c 1.0 MeOH); 1H NMR (400

MHz, DMSO-d6) δ 8.02-7.95, 7.77, 7.70-7.68, 7.60-7.52, 7.37, 7.32 (18H, m, NH, Ar-H), 5.29 (1H, dd, J = 9.7 Hz, 9.7 Hz), 5.16 (1H, d, J = 5.5 Hz), 5.12-4.91 (8H, m), 4.83 (1H, dd, J = 8.7 Hz, 8.7 Hz), 4.77-4.70 (2H, m), 4.49 (1H, d, J = 9.3 Hz), 4.39-4.23 (9H, m), 4.13-4.05 (3H, m), 4.00-3.98 (1H, m), 3.92-3.89 (3H, m), 3.82-3.64 (13H, m), 3.58 (3H, s), 1.98-1.94 (21H, m, CH3CO), 1.88 (3H, s, CH3CO), 1.82-1.81 (6H, m, CH3CO); 13C {1H} NMR (100 MHz, DMSO-d6) δ 170.0, 169.9, 169.8, 169.7, 169.6, 169.5, 168.2, 167.7, 166.0, 133.9, 130.1, 130.0, 129.7, 129.6, 129.2, 101.3, 100.9, 88.8, 79.0, 76.6, 76.5, 74.6, 73.8, 72.9, 72.7, 72.1, 71.9, 71.4, 71.2, 71.0, 69.5, 67.4, 67.0, 64.1, 63.4, 63.2, 53.3, 53.2, 52.9, 50.0, 23.4, 23.3, 20.8, 20.7; HRMS (ESI-MS) calcd for C80H96N6Na2O43 2+ [M+2Na] 2+ m/z 937.2647, found 937.2670. (β-D-glucopyranosyluronic

acid)-(1→3)-(2-acetamido-2-deoxy-4-O-sulfo-β-D-galactopyranoside)-(1→4)-

(β-D-glucopyranosyluronic acid)-(1→3)-2-acetamido-2-deoxy-4-O-sulfo-β-D-galactopyranosyl azide (11).



A mixture of the diol 9 (56.8 mg, 0.045 mmol) and SO3·NMe3 complex (76 mg, 6 eq/OH) in anhydrous DMF (1 mL) was stirred for 72 h at 60 °C, then was cooled. Et3N (150 µL) and Methanol (150 µL) was successively added, and the mixture was directly eluted from a column of Sephadex LH-20 (CH2Cl2/MeOH = 1:1) to give the sulfated intermediate as a white solid. Rf = 0.60 (CHCl3/MeOH/H2O =1.4:0.7:0.15); Compound 11 was obtained from the sulfated intermediate according to Method A, yield 42.9 mg, 91% for two steps (calculated from sodium salt form). Rf = 0.20 (EtOAc/EtOH/H2O =2:1:1); [α] 25 D= -29 ˚ (c 1.0 H2O); 1H NMR (400 MHz, D2O) δ 4.75-4.73 (2H, m), 4.51-4.49 (1H, m), 4.43-4.39 (2H, m), 4.03-3.93 (3H, m), 3.85-3.82 (1H, m), 3.783.69 (5H, m), 3.63-3.60 (2H, m), 3.51 (1H, dd, J = 9.0 Hz, 9.0 Hz), 3.46 (1H, dd, J = 9.4 Hz, 9.4 Hz), 3.40 (1H, dd, J = 9.0 Hz, 9.0 Hz), 3.32 (1H, dd, J = 9.4 Hz, 9.4 Hz), 3.29-3.25 (1H, m), 1.98-1.96 (6H, m, CH3CO); 13C {1H} NMR (100 MHz, D2O) δ 175.6, 175.0, 174.2, 103.6, 103.5, 101.0, 88.8, 80.4, 76.6, 76.4, 76.2, 75.2, 75.1, 75.0, 74.6, 73.6, 72.5, 72.1, 71.8, 61.0, 51.6, 51.0, 46.7, 44.7, 30.3, 22.6, 22.2; ESI-Q-TOF (negative mode) calcd for C28H40N5O28S2 3 [M-4Na+H]

3

m/z 319.3773, found 319.3765; calcd for C28H41N5O28S2 2 [M-

4Na+2H] 2 m/z 479.5695, found 479.5690. (β-D-glucopyranosyluronic


(β-D-glucopyranosyluronic


(β-D-glucopyranosyluronic acid)-(1→3)-2-acetamido-2-deoxy-4-O-sulfo-β-D-galactopyranosyl azide (12). A mixture of the triol 10 (48.3 mg, 0.026 mmol) and SO3·NMe3 complex (74 mg, 6 eq/OH) in anhydrous DMF (1 mL) was stirred for 72 h at 60 °C, then was cooled. Et3N (150 µL) and Methanol (150 µL) was successively added, and the mixture was directly eluted from a column of Sephadex LH-20 (CH2Cl2/MeOH = 1:1) to give the sulfated intermediate as a white solid. Rf = 0.50 (CHCl3/MeOH/H2O =1.4:0.7:0.15); Compound 12 was obtained from the sulfated intermediate according to Method A, yield 35.0 mg, 87% for two steps (calculated from sodium salt form). Rf = 0.15 (EtOAc/EtOH/H2O =2:1:1); [α] 25 D= -37 ˚ (c 1.0 H2O); 1H NMR (400 MHz,


Page 14 of 23

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D2O) δ 4.76-4.73 (2H, m), 4.50-4.49 (2H, m), 4.43-4.38 (3H, m), 4.00-3.94 (6H, m), 3.86-3.82 (1H, m), 3.783.69 (10H, m), 3.62-3.59 (3H, m), 3.53-3.49 (2H, m), 3.48-3.37 (3H, m), 3.34-3.27 (3H, m), 1.97-1.96 (9H, m, CH3CO); 13C {1H} NMR (100 MHz, D2O) δ 175.7, 175.0, 174.3, 103.7, 103.6, 103.5, 101.0, 88.9, 80.4, 76.6, 76.5, 76.2, 75.5, 75.2, 75.0, 74.6, 73.6, 72.5, 72.2, 72.1, 71.8, 61.0, 51.6, 51.5, 51.0, 46.7, 44.8, 30.3, 22.6, 22.5, 22.2; ESI-Q-TOF (negative mode) calcd for C42H61N6O42S3 3 [M-6Na+3H] 3 m/z 472.4000, found 472.4005; calcd for C42H62N6O42S3 2 [M-6Na+4H] 2 m/z 709.1037, found 709.1049. (β-D-glucopyranosyluronic


(β-D-glucopyranosyluronic acid)-(1→3)-2-acetamido-2-deoxy-6-O-sulfo-β-D-galactopyranosyl azide (13). To compound 7 (47 mg, 0.045 mmol, 1.0 equiv.) and a magnetic stir bar in a 25 mL flame-dried Schlenk vessel charged with Ar was added SO3·NMe3 complex (31 mg, 0.226 mmol, 5.0 equiv.) and dry DMF (1.5 mL). The mixture was stirred at RT until a clear colorless solution was formed, then was moved to a pre-heated 40 ºC oil bath and stirred for 2 h. The reaction was cooled and quenched by successive addition of Et3N (80 µL) and Methanol (50 µL). The mixture was directly eluted from a column of Sephadex LH-20 (CH2Cl2/MeOH = 1:1) to give the sulfated intermediate as a white solid. Rf = 0.40 (CHCl3/MeOH/H2O =1.2:0.8:0.2); Compound 13 was obtained from the sulfated intermediate according to Method A, yield 44.3 mg, 94% for two steps (calculated from sodium salt form). Rf = 0.20 (EtOAc/EtOH/H2O =2:1:1); [α] 25 D= -23 ˚ (c 1.0 H2O); 1H NMR (400 MHz, D2O) δ 4.68-4.65 (2H, m), 4.46-4.39 (3H, m), 4.15-4.11 (5H, m), 3.97-3.90 (3H, m), 3.88 (1H, dd, J = 6.0 Hz, 6.0 Hz), 3.81 (1H, dd, J = 2.9 Hz, 10.8 Hz), 3.75 (1H, dd, J = 2.9 Hz, 10.9 Hz), 3.65-3.58 (3H, m), 3.50 (1H, dd, J = 8.0 Hz, 8.0 Hz), 3.42-3.36 (2H, m), 3.40 (1H, dd, J = 9.0 Hz, 9.0 Hz), 3.29 (1H, dd, J = 8.0 Hz, 8.0 Hz), 3.26-3.21 (1H, m), 1.94 (3H, s, CH3CO), 1.93 (3H, s, CH3CO); 13C {1H} NMR (100 MHz, D2O) δ 176.0, 175.0, 174.3, 167.7, 104.1, 104.0, 101.3, 88.8, 81.1, 80.0, 79.5, 76.4, 76.1, 75.3, 74.6, 73.8, 72.7, 72.6, 72.3, 71.8, 67.6, 67.5, 67.4, 50.8, 50.3, 22.6, 22.2; ESI-Q-TOF (negative mode) calcd for C28H41N5O28S2 2 [M-4Na+2H] 2m/z



479.5695, found 479.5704. (β-D-glucopyranosyluronic


(β-D-glucopyranosyluronic


(β-D-glucopyranosyluronic acid)-(1→3)-2-acetamido-2-deoxy-6-O-sulfo-β-D-galactopyranosyl azide (14). To compound 8 (40 mg, 0.026 mmol, 1.0 equiv.) and a magnetic stir bar in a 25 mL flame-dried Schlenk vessel charged with Ar was added SO3·NMe3 complex (29.4 mg, 0.211 mmol, 8.0 equiv.) and dry DMF (1.5 mL). The mixture was stirred at RT until a clear colorless solution was formed, then was moved to a pre-heated 40 ºC oil bath and stirred for 2 h. The reaction was cooled and quenched by successive addition of Et3N (80 µL) and Methanol (50 µL). The mixture was directly eluted from a column of Sephadex LH-20 (CH2Cl2/MeOH = 1:1) to give the sulfated intermediate as a white solid. Rf = 0.60 (CHCl3/MeOH/H2O =1.0:1.0:0.3); Compound 14 was obtained from the sulfated intermediate according to Method A, yield 35.5 mg, 88% for two steps (calculated from sodium salt form). Rf = 0.15 (EtOAc/EtOH/H2O =2:1:1); [α] 25 D= -20 ˚ (c 1.0 H2O); 1H NMR (400 MHz, D2O) δ 4.68-4.65 (3H, m), 4.47-4.41 (5H, m), 4.17-4.11 (7H, m), 4.10-4.09 (1H, m), 3.99-3.87 (6H, m), 3.83 (1H, dd, J = 2.8 Hz, 10.8 Hz), 3.78-3.74 (2H, m), 3.65-3.64 (5H, m), 3.54-3.49 (2H, m), 3.43-3.38 (2H, m), 3.32-3.26 (3H, m), 1.95 (3H, s, CH3CO), 1.94 (3H, s, CH3CO), 1.93 (3H, s, CH3CO); 13C {1H} NMR (100 MHz, D2O) δ 176.0, 175.0, 174.5, 104.3, 104.2, 104.1, 101.4, 88.9, 81.2, 80.7, 80.0, 79.9, 79.7, 79.5, 78.9, 75.4, 74.7, 73.9, 72.8, 72.6, 72.4, 72.3, 71.8, 67.7, 67.6, 67.5, 67.4, 50.9, 50.4, 22.6, 22.2; ESI-Q-TOF (negative mode) calcd for C42H60N6O42S3 4 [M-6Na+2H] 4 m/z 354.0482, found 354.0478; calcd for C42H60N6NaO42S3 3 [M5Na+2H] 3 m/z 479.7273, found 479.7273. (β-D-glucopyranosyluronic

acid)-(1→3)-(2-acetamido-2-deoxy-4,

6-di-O-sulfo-β-D-galactopyranoside)-

(1→4)-(β-D-glucopyranosyluronic acid)-(1→3)-2-acetamido-2-deoxy-4, 6-di-O-sulfo-β-D-galactopyranosyl azide (15).


Page 16 of 23

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To compound 7 (47 mg, 0.045mmol, 1.0 eq.) and SO3·NMe3 complex (252 mg, 10 eq/OH) in anhydrous DMF (1 mL) was stirred for 72 h at 60 °C, then was cooled. Et3N (500 µL) and Methanol (200 µL) was successively added, and the mixture was directly eluted from a column of Sephadex LH-20 (CH2Cl2/MeOH = 1:1) to give the sulfated intermediate as a white solid. Rf = 0.50 (CHCl3/MeOH/H2O =1.0:1.0:0.3); Compound 15 was obtained from the sulfated intermediate according to Method A, yield 50.7 mg, 90% for two steps (calculated from sodium salt form). Rf = 0.30 (n-BuOH/H2O/EtOH/AcOH = 1/1/1/0.05); [α]

25

D=

-22 ˚ (c 1.0 H2O); 1H

NMR (400 MHz, D2O) δ 4.78-4.75 (3H, m), 4.55-4.53 (1H, m), 4.44 (1H, d, J = 7.8 Hz), 4.40 (1H, d, J = 7.8 Hz), 4.24-4.20 (2H, m), 4.18-4.12 (2H, m), 4.11-3.98 (6H, m), 3.71 (1H, dd, J = 9.1 Hz, 9.1 Hz), 3.63-3.58 (2H, m), 3.53 (1H, dd, J = 8.8 Hz, 8.8 Hz), 3.47-3.38 (2H, m), 3.34 (1H, dd, J = 9.1 Hz, 9.1 Hz), 3.29-3.25 (1H, m), 1.98-1.96 (6H, m, CH3CO); 13C {1H} NMR (100 MHz, D2O) δ 175.9, 175.0, 174.1, 167.0, 103.5, 101.5, 88.8, 82.0, 76.6, 76.3, 75.9, 75.0, 74.9, 74.2, 73.7, 72.5, 72.3, 71.9, 71.8, 67.8, 67.6, 51.3, 50.8, 22.6, 22.2; ESI-QTOF (negative mode) calcd for C28H41N5O34S4 2 [M-6Na+4H] 2 m/z 559.5263, found 559.5286. (β-D-glucopyranosyluronic


(1→4)-(β-D-glucopyranosyluronic

6-di-O-sulfo-β-D-galactopyranoside)-


6-di-O-sulfo-β-D-

galactopyranoside)-(1→4)-(β-D-glucopyranosyluronic acid)-(1→3)-2-acetamido-2-deoxy-4, 6-di-O-sulfo-βD-galactopyranosyl azide (16). To compound 8 (49 mg, 0.032mmol, 1.0 eq.) and SO3·NMe3 complex (270 mg, 10 eq/OH) in anhydrous DMF (1 mL) was stirred for 72 h at 60 °C, then was cooled. Et3N (500 µL) and Methanol (200 µL) was successively added, and the mixture was directly eluted from a column of Sephadex LH-20 (CH2Cl2/MeOH = 1:1) to give the sulfated intermediate as a white solid. Rf = 0.45 (CHCl3/MeOH/H2O =1.0:1.0:0.3); Compound 16 was obtained from the sulfated intermediate according to Method A, yield 49.8 mg, 84% for two steps (calculated from sodium salt form). Rf = 0.25 (n-BuOH/H2O/EtOH/AcOH = 1/1/1/0.05); [α]


25

D=

-24 ˚ (c 1.0 H2O); 1H


Page 18 of 23

NMR (400 MHz, D2O) δ 4.78-4.73 (4H, m), 4.56-4.54 (2H, m), 4.46-4.39 (3H, m), 4.25-4.11 (7H, m), 4.074.04 (3H, m), 4.02-3.98 (5H, m), 3.73-3.68 (2H, m), 3.63-3.58 (3H, m), 3.56-3.52 (2H, m), 3.46 (1H, dd, J = 9.5 Hz, 9.5 Hz), 3.41 (1H, dd, J = 9.0 Hz, 9.0 Hz), 3.37-3.30 (2H, m), 3.29-3.25 (1H, m), 1.97-1.96 (9H, m, CH3CO); 13C {1H} NMR (100 MHz, D2O) δ 175.9, 175.0, 174.1, 103.7, 103.4, 101.5, 88.8, 82.1, 76.6, 76.5, 76.3, 76.0, 75.9, 75.3, 75.0, 74.9, 74.8, 74.3, 73.7, 72.5, 72.3, 72.0, 71.9, 71.8, 67.8, 67.6, 51.4, 51.3, 50.8, 22.6, 22.5, 22.1; ESI-Q-TOF (negative mode) calcd for C42H62N6O51S6

2

[M-9Na+7H]

2

m/z 829.0389, found

829.0436. Ch-tetra+Biotin (17). Compound 17 was obtained from tetrasaccharide 3 (10.2 mg) according to Method B, yield 11.7 mg, 77% (calculated from sodium salt form). Rf = 0.25 (n-BuOH/H2O/EtOH/AcOH = 1/1/1/0.05). ESI-Q-TOF (negative mode) calcd for C47H72N8O27S 2 [M-2Na] 2 m/z 606.2119, found 606.2133. Ch-hexa+Biotin (18). Compound 18 was obtained from hexasaccharide 4 (10.0 mg) according to Method B, yield 10.1 mg, 74% (calculated from sodium salt form). Rf = 0.22 (n-BuOH/H2O/EtOH/AcOH = 1/1/1/0.05). ESI-Q-TOF (negative mode) calcd for C61H92N9O38S 3 [M-3Na] 3 m/z 530.1760, found 530.1753. CS-A-tetra+Biotin (19). Compound 19 was obtained from CS-A tetramer 11 (10.0 mg) according to Method B, yield 10.8 mg, 78% (calculated from sodium salt form). Rf = 0.35 (n-BuOH/H2O/EtOH/AcOH = 1/1/1/0.05). ESI-Q-TOF (negative mode) calcd for C47H72N8O33S3 2 [M-4Na+2H] 2 m/z 686.1687, found 686.1670. CS-A-hexa+Biotin (20). Compound 20 was obtained from CS-A hexamer 12 (9.8 mg) according to Method B, yield 9.3 mg, 75% (calculated from sodium salt form). Rf = 0.30 (n-BuOH/H2O/EtOH/AcOH = 1/1/1/0.05). ESI-Q-TOF (negative


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mode) calcd for C61H93N9O47S4 2 [M-6Na+4H] 2 m/z 915.7029, found 915.6839. CS-C-tetra+Biotin (21). Compound 21 was obtained from CS-C tetramer 13 (9.6 mg) according to Method B, yield 10.7 mg, 80% (calculated from sodium salt form). Rf = 0.30 (n-BuOH/H2O/EtOH/AcOH = 1/1/1/0.05). ESI-Q-TOF (negative mode) calcd for C47H72N8O33S3 2 [M-4Na+2H] 2 m/z 686.1687, found 686.1548. CS-C-hexa+Biotin (22). Compound 22 was obtained from CS-C hexamer 14 (9.8 mg) according to Method B, yield 9.0 mg, 73% (calculated from sodium salt form). Rf = 0.25 (n-BuOH/H2O/EtOH/AcOH = 1/1/1/0.05). ESI-Q-TOF (negative mode) calcd for C61H91N9O47S4 4 [M-6Na+2H] 4 m/z 457.3478, found 457.3470. CS-E-tetra+Biotin (23). Compound 23 was obtained from CS-E tetramer 15 (10.0 mg) according to Method B, yield 9.5 mg, 72% (calculated from sodium salt form). Rf = 0.28 (n-BuOH/H2O/EtOH/AcOH = 1/1/1/0.05). ESI-Q-TOF (negative mode) calcd for C47H72N8O39S5 2 [M-6Na+4H] 2 m/z 766.1256, found 766.1113. CS-E-hexa+Biotin (24). Compound 24 was obtained from CS-E hexamer 16 (9.3 mg) according to Method B, yield 7.8 mg, 69% (calculated from sodium salt form). Rf = 0.22 (n-BuOH/H2O/EtOH/AcOH = 1/1/1/0.05). ESI-Q-TOF (negative mode) calcd for C61H92N9O56S7 3 [M-9Na+6H] 3 m/z 690.0896, found 690.0915. BLI kinetic measurements of growth factors binding to immobilized oligosaccharides. The interaction experiments were conducted at 30 °C in HEPES buffer (0.15 M NaCl, 20 mM HEPES, pH 7.4, 0.05% Tween 20, and 0.1% BSA) using an Octet Red 96 instrument (Fortebio, USA) 8. Final volume for all the solutions was 200 µL. Assays were performed in black solid 96-well flat bottom plates with agitation set to 1000 rpm. For the loading procedure, incubation with 2 µg/mL oligosaccharide solutions for 300 s was conducted to



prepare the saturated sensors and the enhancement of response was 0.24~0.28 nm; lower oligosaccharide concentration (0.3 µg/mL) and shorter incubation time were carried out to prepare the diluted sensors with 0.11~0.12 nm enhancement of response. Increasing concentrations of growth factors were allowed to interact with the immobilized oligosaccharides. A 200 s sensor washing step was applied prior to the analysis of the association of the ligand on the sensor to the analyte in solution for 240 s. Finally the dissociation was followed for 300 s. After dissociation, the sensor surface was regenerated in 5 M NaCl aqueous solution. Correction of any systematic baseline drift was done by subtracting the shift recorded for a sensor loaded with ligand but incubated with no analyte. BLI kinetic data were analyzed using the Octet software version 7.0 and the binding curves were globally fitted using a 1:1 model.13

Associated content Supporting Information: supplementary figures for BLI sensorgram and 1H, 13C, MS spectra and selected 2D NMR for all new compounds. Author information Corresponding Author: *E-mail: [email protected] ORCID Zhongjun Li: 0000-0003-1642-7773 Notes The authors declare no competing financial interest.

Acknowledgements This work was supported by National Natural Science Foundation of China (Grants 21472007 and 21232002). The authors also thank Dr. J. Wang (State Key Laboratory of Natural and Biomimetic Drugs) for the support in


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BLI measurement.

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Semisynthesis of Chondroitin Sulfate Oligosaccharides Based on the

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