Chemical Synthesis of Fucosylated Chondroitin Sulfate Tetrasaccharide with Fucosyl Branch at the 6-OH of GalNAc Residue

Fucosylated chondroitin sulfate is a unique glycosaminoglycan isolated from sea cucumbers, with excellent anticoagulant activity. The fucosyl branch in FCS is generally located at the 3-OH of D-glucuronic acid but, recently, a novel structure with α-L-fucose linked to the 6-OH of N-acetyl-galactosamine has been found. Here, using functionalized monosaccharide building blocks, we prepared novel FCS tetrasaccharides with fucosyl branches both at the 6-OH of GalNAc and 3-OH of GlcA. In the synthesis, the protective group strategy of selective O-sulfation, as well as stereoselective glycosylation, was established, which enabled the efficient synthesis of the specific tetrasaccharide compounds. This research enriches knowledge on the structural types of FCS oligosaccharides and facilitates the exploration of the structure–activity relationship in the future.

FCS and its oligosaccharides possess a variety of pharmacological properties, including anti-inflammatory [7], antitumor [8], anti-hyperglycemic actions [9], and antiviral [10] sas well as regulating immunity and cell proliferation [11].It has aroused a lot of interest because of its notable antithrombotic and anticoagulant activities [12].FCS oligosaccharides can selectively inhibit intrinsic tenase (FXase, factor IXa-VIIIa complex) in the endogenous coagulation pathway [13] with low bleeding risk.Thus, it has high potential to be developed as a novel anticoagulant and antithrombotic drug candidate [13].
Due to the complexity of the FCS polysaccharide structure, it is impossible to elucidate the structure-activity relationship and accurately identify the core fragment responsible for biological activity.Therefore, various methods are employed to obtain homogeneous FCS oligosaccharides.In 2023, Jinhua Zhao's group [14] employed copper ion-catalyzed peroxidative depolymerization [15] and β-eliminative depolymerization [16] methods to obtain fourteen FCS oligosaccharides from the sea cucumber Phyllophorella kohkutiensis (PkFCS), and they found that octasaccharide (Pk4b) with sulfated fucosebased side chains was the smallest fragment responsible for its anticoagulant activity associated with anti-FXase [14].In addition, several research groups employed chemical synthesis methods to produce FCS oligosaccharides with well-defined diverse and flexible structures.
Herein, we report the synthesis of two FCS tetrasaccharide compounds with fucosyl branches both at the 6-OH of GalNAc and the 3-OH of GlcA (FCS-1, 2, Scheme 1), based on the FCS structural types isolated from the sea cucumbers Actinopyga mauritiana [4] and Cucumaria frondose [5].glucuronic acid, while fucosyl branches at the 6-OH of GalNAc have not been synthesized yet.
Selective O-sulfation and stereoselective control of glycosidic bonds were achieved using the orthogonal protection strategy.The sulfation sites in fucose building blocks (18,20) were selectively protected with isopropylidene and 2-naphthyl methyl ether (Nap), respectively.The isopropylidene could be removed under mild acidic conditions while Scheme 1. Retrosynthetic analysis of tetrasaccharides of FCS-1 and FCS-2.
Selective O-sulfation and stereoselective control of glycosidic bonds were achieved using the orthogonal protection strategy.The sulfation sites in fucose building blocks (18,20) were selectively protected with isopropylidene and 2-naphthyl methyl ether (Nap), respectively.The isopropylidene could be removed under mild acidic conditions while the Nap could be oxidatively cleaved using 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) in DCM/H 2 O.The sulfation site 4-OH in GalNAc was protected with allyloxycarbonyl (Alloc) group.For building block 7 of GlcA, a benzoyl group (Bz) was introduced at the 2-OH of glucose to construct a 1,2-trans-glycosidic bond through neighbouring group participation, and levulinoyl (Lev) was introduced at the 3-OH as a temporary protecting group to facilitate the removal for coupling with fucose.Finally, amino-linker was introduced at the reducing end of the compound.
in DCM/H2O.The sulfation site 4-OH in GalNAc was protected with allyloxycarbonyl (Alloc) group.For building block 7 of GlcA, a benzoyl group (Bz) was introduced at the 2-OH of glucose to construct a 1,2-trans-glycosidic bond through neighbouring group participation, and levulinoyl (Lev) was introduced at the 3-OH as a temporary protecting group to facilitate the removal for coupling with fucose.Finally, amino-linker was introduced at the reducing end of the compound.
Based on the structural features of the target compounds and retrosynthetic analysis (Scheme 1), we firstly synthesized the monosaccharide building blocks of Nacetylaminogalactose (GalNAc), D-glucuronic acid (GlcA), and L-fucose (Fuc) (compounds 7, 13, 18, and 20).Using peracetylated glucose as the starting material, a seven-step reaction procedure was carried out to obtain glucosyl thioglycoside donor 7 (Scheme 2).Peracetylated glucoside was converted to β-thioglycoside in the presence of boron trifluoride diethyl etherate (BF3•Et2O) and p-tolylthiophenol, followed by deacetylation, and then the 4,6-O-benzylidene glucoside 3 [18] was obtained in the presence of camphorsulphonic acid and benzaldehyde dimethyl acetal with 75% yield for three steps.The glucoside 3 was selectively benzoylated at 2-OH with 63% yield using the Ag2O-mediated site-selective benzoylation method [20], and then Lev group was introduced at the C-3 hydroxyl to give compound 5 [21].Finally, the 4,6-O-benzylidene was selectively cleaved under the condition of trimethylsilyl trifluoromethanesulfonate (TMSOTf) and borane-tetrahydrofuran complex (BH3•THF), and the exposed C-6 hydroxyl group was protected with tert-butyldiphenylsilyl group (TBDPS) to give glucosyl thioglycoside donor 7 [22] in 89% yield.In order to construct a 1,2-trans-β-glycosidic bond of 2-deoxy-β-Dgalactopyranoside, phthaloyl (Phth) and trichloroethoxycarbonyl (Troc), through neighbouring group participation, are often used as amino-protecting groups [23].The conditions for the removal of Phth are more drastic, and reduce the activity of the glycosyl donor as an acyl-protecting group.On the other hand, the conditions for introducing and removing the Troc group are relatively milder, and enhance the activity of the Glycosyl donor.Therefore, Troc was chosen as an amino-protecting group for Nacetylaminogalactose.The compound 10 was obtained in a three-step reaction with aminogalactose hydrochloride as the starting material in 71% yield [23].Subsequently, 10 was coupled with N-benzyl-benzylcarbamate protected aminopentyl linker L-1 [24] under N-iodosuccinimide (NIS) and TMSOTf conditions to give β-linked product 11 [25] in 91% yield.Then the acetyl groups were removed and a benzylidene group was formed between the C-4/C-6 hydroxyl groups to give the 3-OH unprotected aminogalactose acceptor 13 [25] (Scheme 3).In order to construct a 1,2-trans-β-glycosidic bond of 2-deoxy-β-D-galactopyranoside, phthaloyl (Phth) and trichloroethoxycarbonyl (Troc), through neighbouring group participation, are often used as amino-protecting groups [23].The conditions for the removal of Phth are more drastic, and reduce the activity of the glycosyl donor as an acyl-protecting group.On the other hand, the conditions for introducing and removing the Troc group are relatively milder, and enhance the activity of the Glycosyl donor.Therefore, Troc was chosen as an amino-protecting group for N-acetylaminogalactose.The compound 10 was obtained in a three-step reaction with aminogalactose hydrochloride as the starting material in 71% yield [23].Subsequently, 10 was coupled with N-benzyl-benzylcarbamate protected aminopentyl linker L-1 [24] under N-iodosuccinimide (NIS) and TMSOTf conditions to give β-linked product 11 [25] in 91% yield.Then the acetyl groups were removed and a benzylidene group was formed between the C-4/C-6 hydroxyl groups to give the 3-OH unprotected aminogalactose acceptor 13 [25] (Scheme 3).In order to synthesize FCS tetrasaccharides with different sulfation modes in Fuc, two different fucose thioglycosides, 18 and 20, were designed (Scheme 4).The compound 18 was obtained through a 5-step reaction, according to the reported methods, in 69% yield [26,27].For thioglycoside 20, we first utilized dibutyltin oxide (Bu2SnO) to selectively introduce a Bn group at the 3-OH of compound 16 [28].Then C-2 and C-4 hydroxyls were protected with Nap to afford the fucose thioglycoside donor 20.In order to synthesize FCS tetrasaccharides with different sulfation modes in Fuc, two different fucose thioglycosides, 18 and 20, were designed (Scheme 4).The compound 18 was obtained through a 5-step reaction, according to the reported methods, in 69% yield [26,27].For thioglycoside 20, we first utilized dibutyltin oxide (Bu 2 SnO) to selectively introduce a Bn group at the 3-OH of compound 16 [28].Then C-2 and C-4 hydroxyls were protected with Nap to afford the fucose thioglycoside donor 20.In order to synthesize FCS tetrasaccharides with different sulfation modes in Fuc, two different fucose thioglycosides, 18 and 20, were designed (Scheme 4).The compound 18 was obtained through a 5-step reaction, according to the reported methods, in 69% yield [26,27].For thioglycoside 20, we first utilized dibutyltin oxide (Bu2SnO) to selectively introduce a Bn group at the 3-OH of compound 16 [28].Then C-2 and C-4 hydroxyls were protected with Nap to afford the fucose thioglycoside donor 20.After obtaining the three kinds of monosaccharide blocks, the synthesis of chondroitin sulfate disaccharide blocks was carried out (Scheme 5).Under the condition of NIS and trifluoromethanesulfonic acid (TfOH), the aminogalactose acceptor 13 (1 eq) and glucose donor 7 (1.3 eq) were coupled at −25 °C.The glycosylation reaction was performed in a more favorable yield of 94%, and only β-linked product 21 was found (J1,2 = 7.9 Hz).
According to the retrosynthetic analysis, the differentiation of the C-4 and C-6 hydroxyl groups of GalNAc and the conversion of glucose to glucuronic acid need to be accomplished in the disaccharide block.The 6-OTBDPS of glucoside 21 was removed to give disaccharide compound 22 in 85% yield.Selective oxidation of the primary hydroxyl group in glucose unit under 2,2,6,6-Tetramethylpiperidinooxy (TEMPO) and (diacetoxyiodo) benzene (BAIB) conditions and then methyl ester protection afforded compound 24 in 74% yield for two steps [29].Then the 4,6-O-benzylidene group of GalNAc in compound 24 was removed under acetic acid (AcOH) conditions to afford compound 25 in 89% yield.Finally, selective protection of the C-6 OH with TBDPS and the C-4 OH by Alloc gave compound 27 in two-step yield of 76%.Next, the trisaccharide backbone was constructed by removing the 6-TBDPS of GalNAc and coupling with fucose blocks, as shown in Scheme 5.After obtaining the three kinds of monosaccharide blocks, the synthesis of chondroitin sulfate disaccharide blocks was carried out (Scheme 5).Under the condition of NIS and trifluoromethanesulfonic acid (TfOH), the aminogalactose acceptor 13 (1 eq) and glucose donor 7 (1.3 eq) were coupled at −25 • C. The glycosylation reaction was performed in a more favorable yield of 94%, and only β-linked product 21 was found (J 1,2 = 7.9 Hz).Then, compound 27 was stripped of the TBDPS protecting group to give 28 for glycosylation.Unfortunately, when compound 28 was coupled with fucose donor 18 catalyzed by NIS and TfOH, the glycosylation product was α/β-mixture (Entry 1) and the α-linked trisaccharide 29 was isolated in 41% yield (H1 = 4.79 ppm, C1 = 97.9ppm for fucosyl unit) with a β-isomer of 37%.In order to improve the stereoselectivity, we optimized this glycosylation condition in terms of catalyst type, dosage and reaction temperature, and the experimental results are shown in Table 1.According to the retrosynthetic analysis, the differentiation of the C-4 and C-6 hydroxyl groups of GalNAc and the conversion of glucose to glucuronic acid need to be accomplished in the disaccharide block.The 6-OTBDPS of glucoside 21 was removed to give disaccharide compound 22 in 85% yield.Selective oxidation of the primary hydroxyl group in glucose unit under 2,2,6,6-Tetramethylpiperidinooxy (TEMPO) and (diacetoxyiodo) benzene (BAIB) conditions and then methyl ester protection afforded compound 24 in 74% yield for two steps [29].Then the 4,6-O-benzylidene group of GalNAc in compound 24 was removed under acetic acid (AcOH) conditions to afford compound 25 in 89% yield.Finally, selective protection of the C-6 OH with TBDPS and the C-4 OH by Alloc gave compound 27 in two-step yield of 76%.Next, the trisaccharide backbone was constructed by removing the 6-TBDPS of GalNAc and coupling with fucose blocks, as shown in Scheme 5.
Then, compound 27 was stripped of the TBDPS protecting group to give 28 for glycosylation.Unfortunately, when compound 28 was coupled with fucose donor 18 catalyzed by NIS and TfOH, the glycosylation product was α/β-mixture (Entry 1) and the α-linked trisaccharide 29 was isolated in 41% yield (H 1 = 4.79 ppm, C 1 = 97.9ppm for fucosyl unit) with a β-isomer of 37%.In order to improve the stereoselectivity, we optimized this glycosylation condition in terms of catalyst type, dosage and reaction temperature, and the experimental results are shown in Table 1.Then, compound 27 was stripped of the TBDPS protecting group to give 28 for glycosylation.Unfortunately, when compound 28 was coupled with fucose donor 18 catalyzed by NIS and TfOH, the glycosylation product was α/β-mixture (Entry 1) and the α-linked trisaccharide 29 was isolated in 41% yield (H1 = 4.79 ppm, C1 = 97.9ppm for fucosyl unit) with a β-isomer of 37%.In order to improve the stereoselectivity, we optimized this glycosylation condition in terms of catalyst type, dosage and reaction temperature, and the experimental results are shown in Table 1.

Entry
Using the same glycosylation conditions, trisaccharide 30 was obtained using the disaccharide acceptor 28 with another fucose donor 20.The α/β ratio was 2.75:1.After purification, the α-linked trisaccharide compound 30 was obtained in two steps with 67% yield (Figure S14).Comparing the stereoselectivity of two glycosylation products (29,30), it was found that the 2-Nap-protected fucose donor 20 was superior to that of the isopropylidene-protected fucose donor 18, which might be due to the spatial effect of 2-Nap.
Next, the assembly of the tetrasaccharide compounds was performed (Scheme 6).Under the condition of hydrazine acetate, the Lev at the C-3 of 29 and 30 was removed to afford the two trisaccharide acceptors 31 and 32.Subsequently, fucose donors 18 and 20 were coupled with 31 and 32, respectively, under the glycosylation conditions described above.To our surprise, only the α-linked tetrasaccharide products 33 and 34 (J 1 ′′′ ,2 ′′′ = 3.3 Hz) were found (Figure S15), while the β-isomer was not detected.
Next, the assembly of the tetrasaccharide compounds was performed (Scheme 6).Under the condition of hydrazine acetate, the Lev at the C-3 of 29 and 30 was removed to afford the two trisaccharide acceptors 31 and 32.Subsequently, fucose donors 18 and 20 were coupled with 31 and 32, respectively, under the glycosylation conditions described above.To our surprise, only the α-linked tetrasaccharide products 33 and 34 (J1‴,2‴ = 3.3 Hz) were found (Figure S15), while the β-isomer was not detected.In the fucosylation reaction, the same fucose donor showed better glycosylation stereoselectivity with GlcA 3-OH than with GalNAc 6-OH.We hypothesize that the difference in reactivity between the primary and secondary hydroxyl groups of the acceptor may be a contributing factor.Additionally, spatial orientation likely influences the formation of the α-isomer during the coupling of the fucose donor with GlcA 3-OH.
Due to the strong electron-withdrawing nature of the carboxyl group in glucuronic acid, the glycosylation reaction was inadequate under the same glycosylation conditions (N-iodosuccinimide (1.5 eq), TfOH (0.3 eq), 4 Å MS, CH 2 Cl 2 , −15 • C), resulting in low yields, which were 59% and 51% for tetrasaccharides 34 and 33, respectively.In order to improve the yield, the equivalent of TfOH in the glycosylation reaction was explored.It was found that increasing the TfOH equivalent could improve the yield.When the TfOH equivalent was increased from 0.3 eq to 0.6 eq, the glycosylation yield of compound 34 was increased from 51% to 83%.Similarly, the yield of compound 33 was improved from 59% to 72%.
Subsequently, a series of functional group transformations were carried out (Scheme 6).Firstly, the NHTroc in 33 and 34 was reduced to amino group using Zn powder [30] and acetylated to obtain compounds 35 and 36 in 80% and 74% yields, respectively.Then the isopropylidene at 3,4-OH and the Nap in fucose were removed, followed by removal of Alloc [18] to give pentol 37 and 38 as sulfated precursor in 78% and 60% yields, respectively.
Finally, the FCS tetrasaccharide compounds FCS-1 and FCS-2 were obtained with three steps in one pot.Firstly, compounds 37 and 38 were sulfated using sulfur trioxide trimethylamine complex (SO 3 •Me 3 N) in a microwave reactor [19] with DMF as the solvent to afford the corresponding sulfated tetrasaccharide derivatives; then, the methyl group of COOMe and benzoyl group were removed under alkaline conditions and, finally, the Bn and Cbz groups were removed through Pd/C-catalyzed hydrogenolysis to afford the target compounds FCS-1 and FCS-2 in 63% and 65% yields with three steps, respectively.FCS-1 and FCS-2 were identified by 1 H-NMR, 13 C-NMR, 2D-NMR, and HR-ESI-MS.
The FCS tetrasaccharides with fucosyl branches both at the 6-OH of GalNAc and the 3-OH of GlcA were synthesized for the first time through consecutive fucosylation using the linear synthesis method.FCS-1 and FCS-2 exhibit different fucose sulfation patterns, with FCS-1 featuring 3,4-OH sulfation and FCS-2 featuring 2,4-OH sulfation.Additionally, they both contain sulfate groups at the 4-OH of GalNAc.In the synthesis process, the 1,2-trans-β-glycosidic bond was successfully constructed by introducing the Troc protecting group in aminogalactose.The conversion of glucuronic acid was achieved at the disaccharide blocks, which reduced the reaction steps and established an efficient and concise synthesis strategy.
To a solution of disaccharide acceptor 28 (232 mg, 0.19 mmol) and fucose donor 18 [26,27] (96 mg, 0.28 mmol) in dry DCM/Et 2 O (1 mL/1 mL), dried 4 Å molecular sieves were added under a nitrogen atmosphere at room temperature.The mixture was stirred at room temperature for 1 h and then cooled to −15 • C. NIS (64 mg, 0.28 mmol) and TfOH (5.7 µL, 0.06 mmol) were added to the reaction solution and stirred for 30 min.The reaction was quenched with Et 3 N and gradually warmed to room temperature.The mixture was filtered through celite and extracted with DCM.The organic phase was washed with saturated NaHCO 3 and brine, dried with anhydrous Na 2 SO 4 , and filtered and concentrated in vacuo.The residue was purified using flash chromatography (PE/EtOAc = 1.5:1, v/v) to afford white solid compounds 29alpha (164 mg, 58% for two steps) and 29beta (91 mg, 32% for two steps), R f = 0.33 (PE/EtOAc = 1.5:1, v/v).Data for alpha anomer: 1        To a solution of 37 (22 mg, 0.02 mmol) in dry DMF (1.5 mL), SO 3 •Me 3 N (221 mg, 1.59 mmol) was added at room temperature.The reaction mixture was heated to 70 • C in a microwave synthesizer and stirred for 2 h.Et 3 N and MeOH quenched the reaction, and it was concentrated in vacuo to give crude product as yellow oil.This product could be used in the next step without purification.
This product was dissolved in THF/H 2 O (1.6 mL/0.2 mL), and LiOH aqueous solution (1 M, 1 mL) was added.The reaction mixture was stirred overnight.After being concentrated in vacuo, it was dissolved in MeOH/DCM (1.1 mL/0.2 mL), and a NaOH aqueous solution (0.5 M, 2 mL) was added.The reaction was stirred for 8 h and the pH was adjusted to neutral by the addition of IR-120 H + cation exchange resin.It was concentrated in vacuo to give crude product as yellow oil.This product could be used in the next step without purification.

1 a
Combined yield of α-and β-isomers.b Determined from the masses of the isolated and purified αand β-isomer products.

1 a
Combined yield of αand β-isomers.b Determined from the masses of the isolated and purified αand β-isomer products.