Synthesis of the Oligosaccharides Related to Branching Sites of Fucosylated Chondroitin Sulfates from Sea Cucumbers

Natural anionic polysaccharides fucosylated chondroitin sulfates (FCS) from sea cucumbers attract great attention nowadays due to their ability to influence various biological processes, such as blood coagulation, thrombosis, angiogenesis, inflammation, bacterial and viral adhesion. To determine pharmacophore fragments in FCS we have started systematic synthesis of oligosaccharides with well-defined structure related to various fragments of these polysaccharides. In this communication, the synthesis of non-sulfated and selectively O-sulfated di- and trisaccharides structurally related to branching sites of FCS is described. The target compounds are built up of propyl β-d-glucuronic acid residue bearing at O-3 α-l-fucosyl or α-l-fucosyl-(1→3)-α-l-fucosyl substituents. O-Sulfation pattern in the fucose units of the synthetic targets was selected according to the known to date holothurian FCS structures. Stereospecific α-glycoside bond formation was achieved using 2-O-benzyl-3,4-di-O-chloroacetyl-α-l-fucosyl trichloroacetimidate as a donor. Stereochemical outcome of the glycosylation was explained by the remote participation of the chloroacetyl groups with the formation of the stabilized glycosyl cations, which could be attacked by the glycosyl acceptor only from the α-side. The experimental results were in good agreement with the SCF/MP2 calculated energies of such participation. The synthesized oligosaccharides are regarded as model compounds for the determination of a structure-activity relationship in FCS.


Introduction
Different types of natural anionic polysaccharides attract increasing attention nowadays due to their biological activity of different types that makes possible their use as pharmacological regulators of several diseases related to biological processes, such as blood coagulation, thrombosis, angiogenesis, inflammation, bacterial and viral adhesion and some others. The most famous biopolymer of the discussed type is glycosaminoglycan heparin, which was found to be a leader on anticoagulant and antithrombotic market for decades [1,2]. Besides, this biopolymer was shown to attenuate metastasis and inflammation in a way of inhibition of P-and L-selectins binding to their cellular ligands [3,4].
Due to several side effects of heparin treatment, new biologically active compounds are intensively searched for and are under development as potential alternative drugs. Among them are fucosylated chondroitin sulfates (FCS) isolated from different sea cucumber species. These polysaccharides demonstrated a wide spectrum of biological activities including anticoagulant, antithrombotic, antitumor, immunostimulatory, anti-hyperglycemia, antiangiogenic, antibacterial, antiviral and some others [5][6][7][8][9][10].
A number of studied to date FCSs are glycosaminoglycans built up of alternating →4)-linked β-D-glucuronic acid and →3)-linked N-acetyl β-D-galactosamine residues in a backbone. Unlike mammalian chondroitin sulfates, these polysaccharides bear side chains containing O-sulfated fucosyl residues attached to O-3 of glucuronic acid units ( Figure 1) [5,11]. It should be noted that the presence of side chains is essential for biological properties of FCS [12]. The structures of fucosyl-branches vary accordingly to the type of sea cucumber species and determine in many respects the level and the character of its biological activity [5,11]. Moreover, the degree of O-sulfation and fucose content in FCS also depend on the geographic range and season of harvesting [13,14]. The known structures of non-sulfated and selectively O-sulfated α-L-fucosyl and α-L-fucosyl-(1→3)-α-L-fucosyl fragments which were discovered as the side chains in FCS from sea cucumbers [5,11,15] are shown in Figure 1.  [16]. But to determine pharmacophore fragments of FCS, a series of compounds with well-defined structure are required. We have started the systematic synthesis of oligosaccharides related to various fragments of these types of natural polysaccharides. In this communication, the first synthesis of non-sulfated and selectively O-sulfated di-and trisaccharides 1-8 related to the known structures of branching sites of FCS is described (Figure 2). The target compounds are built up of the propyl β-D-glucuronic acid residue bearing at O-3 the α-L-fucosyl or α-L-fucosyl-(1→3)-α-L-fucosyl substituents. The sulfation pattern of the fucosyl units was selected according to the data of holothurian FCS structures described in literature (see above for citations).

Results and Discussion
Two monosaccharides 9 and 10 ( Figure 3) bearing free hydroxyl group at C-3 were studied as glycosyl acceptors for assembling of target compounds 1-8. These compounds were prepared from 2,4-di-O-acylated derivatives of 3,6-lactone of allyl glucuronide as described previously [17].
Since the target compounds contain α-L-fucosyl residues, an efficient method for α-L-fucosylation should be applied in their synthesis. Earlier we have shown that the presence of acyl groups at O-3 and O-4 of the fucosyl donor was essential for α-glycoside formation [18][19][20]. Thus, the use of 2-O-benzyl-3-O-acetyl-4-O-benzoyl-L-fucosyl trichloroacetimidate 13 gave the only α-isomeric glycosylation product [20]. The stereochemical result of the reaction was explained by the remote participation of acyl groups with the formation of the stabilized glycosyl cation (similar to cations II and III in Figure 4 below), which could be attacked by an acceptor only from the α-side. This approach was quite different then what was used by Tamura et al., where fucosyl fluoride with non-participating allyl and benzyl groups was applied [16].  An additional requirement to the structure of the fucosyl donor is the presence of selectively removable protective groups at O-3 and O-4, which are necessary for the preparation of the partially O-sulfated oligosaccharides 3-5 and 8. It is known that chloroacetyl group (CA) could be selectively removed in the presence of benzoyl and acetyl groups. Thus, we investigated first whether the CA group could stabilize glycosyl cation in the same way as had been observed for other acyls and could thus direct the glycosylation towards the formation of the α-isomer. Following our synthetic strategy, 2-O-benzyl-3,4-di-O-chloroacetyl trichloroacetimidate 12 was regarded as a donor of choice ( Figure 3).
We conducted theoretical calculations to investigate a possibility of the discussed stereocontrolling effect of remote CA groups. At the primary level, as very coarse estimate, we performed molecular mechanics calculations following the technique described in [21] using the evaluation version of HyperChem 1.0 for Linux (HyperChem is the trademark of Hypercube, Inc. (Gainesville, FL, USA) [22]). The MM+ force field (HyperChem version of MM2 [23]) was employed with the electrostatic term using charge-charge interactions. Atomic charges were obtained from single point calculations using semiempirical PM3 approximation [24,25]. Geometry optimizations of a non-stabilized and stabilized forms of the oxocarbenium ion ( Figure 4) were conducted and the corresponding "stabilization energies" were calculated as energy differences in pairs E(I)-E(II) and E(I)-E(III). At this very low level of theory, it was found that the CA group (especially that at O-3) might have the ability to stabilize efficiently the oxocarbenium ion. Stabilization energies were computed as 11.1 and 6.2 kcal/mol for the CA groups at O-3 and O-4, respectively. Calculated values were only slightly lower than those found for the stabilizing groups in donor 13 (Table 1).
Then the study was carried out at the ab initio level with the account for electron correlation. Geometries of all the cations were optimized using the SCF/MP2 approximation with the 6-31+G* basis set as provided along with the NWChem 6.3 software package [26]. This calculation resulted in an increase in the difference in stabilization energies between 12 and 13 (Table 1), but the stabilization energies for the CA groups at O-3 and O-4 of 12 still remained rather high, suggesting their ability to interact with the cationic center. Compound 12 was prepared from diol 11 [27] by per-O-chloroacetylation followed by deallylation and subsequent trichloroacetimidation in a yield of 78% over three steps. The efficiency of this donor was then studied in direct experiments.
An attempt to involve the 2,4-di-O-benzoylated glucuronyl acceptor 9 into glycosylation with donor 12 in the presence of TMSOTf failed. The main product of the reaction was the respective N-glycosylated trichloroacetamide, while compound 9 was recovered unchanged. On the contrary, the 2,4-di-O-acetylated glucuronyl acceptor 10 under the same conditions reacted rapidly with the formation of the desired α-linked disaccharide 14 exclusively in a yield of 92% (Scheme 1). The difference in the reactivity of the acceptors 9 and 10 could be explained by the different steric availability of the hydroxyl group at C-3. Bulk benzoyl groups at O-2 and O-4 in 9 hindered the access of glycosyl donor to hydroxyl group. It should be noted that the stereochemical result of the glycosylation was still in good agreement with the theoretical prediction.
Selective removal of CA-groups in 14 using thiourea in the presence of collidine gave diol 15 in a yield of 94% (Scheme 1). This compound was used as the precursor of all target disaccharides 1-5. Thus, hydrogenolysis of 15 followed by saponification gave the non-sulfated propyl glycoside 1. The synthesis of trisaccharides 6-8 was performed using [2 + 1] strategy for the assembling of the carbohydrate chain. Allyl glucuronide 10 was used as an acceptor, and difucoside 18 was chosen as a donor. Compound 18 was prepared by stereo-and regioselective glycosylation of diol 11 with monosaccharide 12 followed by 4-O-chloroacetylation, deallylation and trichloacetimidation steps (Scheme 2). Finally, the anomeric mixture of trichloroacetimidates 18 was obtained in a yield of 66%.

Scheme 2.
Preparation of the difucoside donor 18. Reagents and conditions: Coupling of thus obtained compounds 18 and 10 in the presence of TMSOTf gave exclusively trisaccharide 19 in a yield of 90% (Scheme 3). Removal of chloroacetyl groups in 19 gave corresponding triol 20, which was further transformed into target trisaccharides 6-8 using the procedure sequences applied for the synthesis of the disaccharides 1-3, respectively. All the target compounds were characterized with 1 H and 13 C NMR spectroscopy (Tables 2 and 3). The presence of the O-sulfate group was confirmed by the downfield shift of the signal of the neighbor proton and carbon atoms in the 1 H and 13 C NMR spectra. Introduction of the sulfate group at O-2 also influenced the shift of the signals of anomeric proton and carbon atoms.   The synthesized oligosaccharides are regarded as model compounds for the investigation of a structure-activity relationship in FCS. The results of conformational analysis and biological studies of the compounds 1-8 will be published elsewhere. Synthesis and interdisciplinary studies of larger oligosaccharides related to structural fragments of FCS are in progress at this laboratory.

Characterization of Compounds
1 H and 13 C NMR spectra were recorded on Bruker DRX500 and AV600 spectrometers at 303 K in CDCl3 and D2O. Chemical shifts were reported in ppm referenced to the residual CHCl3 peak (δ 7.27) for substituted compounds and to acetone peak (0.05% as internal standard, 1 H δ 2.22 and 13 C δ 30.9) for the oligosaccharides 1-8. Signal assignment in 1 H and 13 C NMR spectra were made using COSY, TOCSY, ROESY and 1 H-13 C HSQC techniques. High-resolution mass spectra were acquired by electrospray ionization on a Bruker Daltonics micrOTOF II instrument [28]. Optical rotation values were measured using a JASCO DIP-360 polarimeter at the ambient temperature in solvents specified.

Chemical Synthesis
All glycosylation reactions were carried out under dry argon. Molecular sieves for glycosylation reactions were activated prior to application at 180 °C in vacuum of an oil pump during 2 h. (12) To a solution of the monosaccharide 11 (800 mg, 2.72 mmol) in CH2Cl2 (20 mL), Py (2 mL) and (ClCH2C(O))2O (1 g, 5.9 mmol) were added. The reaction mixture was kept at room temperature (rt) for 1 h, then diluted with EtOAc (50 mL) and washed with HCl (0.1 M) (20 mL) and distilled water (2 × 30 mL). Organic layer was separated and concentrated in vacuo. Chromatography of the residue on a silica gel column gave the totally protected monosaccharide as an amorphous solid. The product was dissolved in MeOH (30 mL), and PdCl2 (127 mg, 0.8 mmol) was added. The mixture was stirred for 3 h at rt, then it was filtered through a celite pad and the filtrate was concentrated in vacuo. Flash column chromatography of the residue on silica gel gave the respective semiacetales as an amorphous solid, which was then dissolved in CH2Cl2 (15 mL), and CCl3CN (300 µL, 3.0 mmol) together with Cs2CO3 (50 mg, 0.15 mmol) were added. The reaction mixture was stirred for 1 h at rt, then it was filtered through a celite pad and the filtrate was concentrated in vacuo. Column chromatography of the residue on a silica gel inactivated by Et3N (0.1%) gave the compound 12 as a mixture of α and β isomers in a ratio of 1:1 (1.

Allyl 2-O-benzyl-3,4-di-O-chloroacetyl-α-L-fucopyranosyl-(1→3)-2-O-benzyl-4-O-cloroacetylα-L-fucopyranoside (17)
A solution of the monosaccharide 12 (300 mg, 0.55 mmol) and the monosaccharide 11 (162 mg, 0.55 mmol) in CH2Cl2 (5 mL) was stirred at rt under argon atmosphere with molecular sieves 4 Å (500 mg) for 1 h. The mixture was cooled to −30 °С and TMSOTf of (5 µL) was added. The mixture was stirred for 15 min −30 °С, then Et3N (0.05 mL) was added. The mixture was filtered through a celite pad, and the filtrate was concentrated in vacuo. Column chromatography of the residue on a silica gel gave the disaccharide, which was dissolved in CH2Cl2 (5 mL (18) To a solution of the disaccharide 17 (230 mg, 0.30 mmol) in MeOH (5 mL) PdCl2 (22 mg, 0.12 mmol) was added. The mixture was stirred for 3 h at rt, then it was filtered through a celite pad and the filtrate was concentrated in vacuo. Flash column chromatography of the residue on a silica gel gave the respective semiacetales as an amorphous solid, which was then dissolved in CH2Cl2 (3 mL), and CCl3CN (50 µL, 0.49 mmol) together with Cs2CO3 (10 mg, 0.03 mmol) were added. The reaction mixture was stirred for 1 h at rt, then it was filtered through a celite pad and the filtrate was concentrated in vacuo. Column chromatography of the residue on a silica gel inactivated by Et3N  (1) To a solution of the disaccharide 15 (57 mg, 0.10 mmol) in MeOH (1.5 mL) and EtOAc (1.5 mL) Pd(OH)2/C (15 mg) was added. The mixture was stirred for 1 h at rt under hydrogen atmosphere, then it was filtered through a celite pad, and the filtrate was concentrated in vacuo. Column chromatography of the residue on a silica gel (Toluene-EtOAc) gave an amorphous solid, which was dissolved in THF (1.0 mL), and 0.1N(aq) LiOH (0.5 mL) was added. The mixture was kept for 1 h at rt and then 0.1N(aq) NaOH (0.5 mL) was added, and the solution was kept at 40 °C for 1 h. After the mixture was filtered through the Whatman paper filter, the filtrate was concentrated in vacuo to a volume of 1 mL. Column chromatography of the residue on the Sephadex G-15 gel in water gave the disaccharide 1 (32 mg, 0.08 mmol, 79%). To a solution of the disaccharide 15 (57 mg, 0.10 mmol) in CH2Cl2 (1 mL) Py (0.1 mL) and AcCl (0.1 mL) were added. The mixture was kept for 2 h at rt, then it was diluted with EtOAc (20 mL) and washed with HCl (0.1 M) (10 mL) and distilled water (2 × 15 mL). Organic layer was separated and concentrated in vacuo. Chromatography of the residue on a silica gel column (Toluene-EtOAc) gave the totally protected disaccharide, which was dissolved in a 1:1 mixture MeOH-EtOAc (3 mL) and Pd(OH)2/C (15 mg) was added. The mixture was stirred for 1 h at rt under hydrogen atmosphere, then it was filtered through a celite pad, and the filtrate was concentrated in vacuo. Flash chromatography of the residue on a silica gel column (Toluene-EtOAc) gave an amorphous solid, which was dissolved in DMF (1 mL) and PySO3 (80 mg, 0.5 mmol) was added. The reaction mixture was kept for 1 h at rt and then quenched with NaHCO3 (200 mg). The resin Amberlite IR-120 (Na + ) and MeOH (1 mL) were added, and the mixture was stirred for 1 h. Then the resin was filtered off, and the filtrate was concentrated in vacuo to a volume of 1 mL. Chromatography of the residue on a silica gel column (CH2Cl2-MeOH) gave the sulfated disaccharide, which was dissolved in THF (1.0 mL), and 0.1N(aq) LiOH (0.5 mL) was added. The mixture was kept for 1 h at rt and then 0.1N(aq) NaOH (0.5 mL) was added, and the solution was kept at 40 °C for 1 h. After the mixture was filtered through the Whatman paper filter, the filtrate was concentrated in vacuo to a volume of 1 mL. Column chromatography of the residue on the Sephadex G-15 gel in water gave the disaccharide 2 (33 mg, 0.065 mmol, 64%). To a solution of the disaccharide 15 (57 mg, 0.10 mmol) in DMF (1 mL) PySO3 (80 mg, 0.5 mmol) was added. The reaction mixture was kept for 1 h at rt and then quenched with NaHCO3 (200 mg). The resin Amberlite IR-120 (Na + ) and MeOH (1 mL) were added, and the mixture was stirred for 1 h. Then the resin was filtered off, and the filtrate was concentrated in vacuo to a volume of 1 mL. Chromatography of the residue on a silica gel column (CH2Cl2-MeOH) gave the sulfated disaccharide, which was dissolved in THF (1.0 mL) and H2O (50 µL), and Pd(OH)2/C (15 mg) was added. The mixture was stirred for 30 min at rt under hydrogen atmosphere, then it was filtered through a celite pad. To the filtrate 0.1N(aq) LiOH (0.5 mL) was added. The mixture was kept for 1 h at rt and then 0.1N(aq) NaOH (0.5 mL) was added, and the solution was kept at 40 C for 1 h. Then the mixture was filtered through the Whatman paper filter, and the filtrate was concentrated in vacuo to a volume of 1 mL. Column chromatography of the residue on the Sephadex G-15 gel in water gave the disaccharide 3 (37 mg, 0.062 mmol, 62%).  (4) To a solution of the disaccharide 16 (55 mg, 0.08 mmol) in DMF (1 mL) PySO3 (65 mg, 0.4 mmol) was added. The reaction mixture was kept for 1 h at rt and then quenched with NaHCO3 (200 mg). The resin Amberlite IR-120 (Na + ) and MeOH (1 mL) were added, and the mixture was stirred for 1 h. Then the resin was filtered off, and the filtrate was concentrated in vacuo to a volume of 1 mL. Chromatography of the residue on a silica gel column (CH2Cl2-MeOH) gave the sulfated disaccharide, which was dissolved in THF (1.0 mL) and H2O (50 µL), and Pd(OH)2/C (15 mg) was added. The mixture was stirred for 30 min at rt under hydrogen atmosphere, then it was filtered through a celite pad. To the filtrate 0.1N(aq) LiOH (0.5 mL) was added. The mixture was kept for 1 h at rt and then 0.1N(aq) NaOH (0.5 mL) was added, and the solution was kept at 40 C for 1 h. Then the mixture was filtered through the Whatman paper filter, and the filtrate was concentrated in vacuo to a volume of 1 mL. Column chromatography of the residue on the Sephadex G-15 gel in water gave the disaccharide 4 (24 mg, 0.048 mmol, 62%).  (5) To a solution of the disaccharide 16 (47 mg, 0.07 mmol) in a 1:1 mixture MeOH-EtOAc (3 mL) Pd(OH)2/C (15 mg) was added. The mixture was stirred for 1 h at rt under hydrogen atmosphere, then it was filtered through a celite pad, and the filtrate was concentrated in vacuo. Flash chromatography of the residue on a silica gel column (Toluene-EtOAc) gave an amorphous solid, which was dissolved in DMF (1 mL) and PySO3 (110 mg, 0.7 mmol) was added. The reaction mixture was kept for 1 h at rt and then quenched with NaHCO3 (200 mg). The resin Amberlite IR-120 (Na + ) and MeOH (1 mL) were added, and the mixture was stirred for 1 h. Then the resin was filtered off, and the filtrate was concentrated in vacuo to a volume of 1 mL. Chromatography of the residue on a silica gel column (CH2Cl2-MeOH) gave the sulfated disaccharide, which was dissolved in THF (1.0 mL), and 0.1N(aq) LiOH (0.5 mL) was added. The mixture was kept for 1 h at rt and then 0.1N(aq) NaOH (0.5 mL) was added, and the solution was kept at 40 C for 1 h. Then the mixture was filtered through the Whatman paper filter, and the filtrate was concentrated in vacuo to a volume of 1 mL. Column chromatography of the residue on the Sephadex G-15 gel in water gave the disaccharide 5 (25 mg, 0.04 mmol, 58%).
MM+ optimizations were carried out as described in the text until the RMS gradient attained 0.01 kcal/mol· Å.
Ab initio calculations used the SCF/MP2 approach with frozen core orbitals: 1s were frozen for all carbons and oxygens, 1s, 2s and 2p were frozen for chlorines. Optimizations were carried out until the RMS gradient attained the value of 0.0001. After that, vibrational analysis was performed to check that no negative frequencies were present.

Conclusions
The efficient synthesis of a series of non-sulfated and selectively O-sulfated di-and trisaccharides related to branching sites of fucosylated chondroitin sulfates from holothurias has been performed. The synthesized compounds are built up of the propyl β-D-glucuronic acid residue bearing at O-3 α-L-fucosyl or α-L-fucosyl-(1→3)-α-L-fucosyl substituents. The sulfation pattern in the fucosyl units was selected according to the known to date holothurian FCS structures. Stereospecific formation of the α-glycoside bond was achieved using 2-O-benzyl-3,4-di-O-chloroacetyl-α-L-fucosyl trichloroacetimidate as a donor. Stereochemical outcome of the glycosylation was explained by the possible remote participation of the chloroacetyl groups at O-3 and O-4 with the intermediate formation of the stabilized glycosyl cations, which are hindered from the β-side, and thus could only be attacked by the glycosyl acceptor from the α-side. The experimental results in glycosylation reactions were in good agreement with the SCF/MP2 calculated energies of such participation. Obtained NMR characteristics of synthesized oligosaccharides can be used as model in further analyses of natural FCSs.