β-(1→3)-Glucan-mannitol conjugates: scope and amazing results

Introduction

β-(1→3)-Glucans are amongst natural compounds that form a bridge between folkloric medicine and modern approaches of the pharmacopeia. Indeed, they were used in Chinese populations for their beneficial contributions on health which were later identified as stimulating agents of the immune system of mammals. Consequently, these polysaccharides that are found in seaweeds, fungi and yeasts have been the subject of many studies in complementary disciplines: analysis, synthetic chemistry, physicochemistry, biology and medicine (1). The main objectives of glycoscientists involved in this field are therefore to elucidate (I) the mechanisms inducing a biological answer in vivo according to the molecular weight, the branching units, and/or the hydrosolubility (2-6); and (II) the minimal structure from these polydisperse polysaccharides which is really active (7-9). The common objective is to identify and precisely characterize fine interactions between β-(1→3)-Glucans and natural macromolecular receptors (10-24). Our laboratory, in collaboration with Vetvicka’s group, is engaged in the chemical synthesis of oligo-β-(1→3)-Glucans having well defined molecular structure in order to remove any ambiguity in interpretation of biological results and so to offer greater opportunities to further develop potential drugs. We have thus demonstrated that small linear glucans, unable to generate well-defined conformations in water, are nevertheless able to stimulate the immune system in murine model. Moreover, we have studied their interaction with CR3.

β-(1→3)-Glucans extracted from brown seaweeds are composed of at least two families of polysaccharides. The first one, which accounts for one third of the mixture, is characterized by a glucose reducing end. Conversely, the second family presents a mannitol residue grafted in primary position (25,26). However, analytical data obtained in our laboratory suggest that another connection exists in Nature (V. Ferrières, unpublished data). Based on this observation, we present herein the synthesis of glucans conjugated to the 6-position or to the 3-hydroxyl of mannitol residue for their potential as bioactive oligosaccharides and analytical standards (Figure 1).

Figure 1 Structures of the molecular targets.

Results and discussion

According to structural and reaction considerations, a mannitol residue on the reducing end of an oligosaccharide can result from the reduction of a mannose entity. Such a strategy allowed us to prepare the molecular targets but also some analogues presenting one different stereogenic centre, i.e., mannose instead of glucose at the reducing end (Figure 1). Moreover, we focused our attention toward the possible elongation of the glucan chain thanks to an iterative sequence glycosylation/deprotection (8). Consequently, the first requirements for the synthesis of 1 and 3 dealt with the preparation of a mannosyl acceptor having a free primary hydroxyl, and glycosyl donors characterized by a good leaving group at the anomeric position (Figure 2). The former was prepared from mannose by first acido-catalyzed Fischer glycosylation of benzyl alcohol (27,28) followed by standard regioselective tritylation (29,30). The resulting intermediate 6 was subsequently either benzylated (31,32) or benzoylated and final detritylation afforded 7a (30,33) and 7b, respectively. The difference between both acceptors relies on nucleophilicity of the released hydroxyl which is less reactive in 7b than in 7a because of the electron withdrawing ability of the neighbouring benzoyl groups. This observation was experimentally corroborated by coupling reactions with the known thioglycoside 8 bearing a 2-naphthylmethyl (NAP) group at its 3-position, orthogonal to all other protections. Best glycosylation results from 7a and 8, followed by selective deprotection under oxido-reductive conditions, thereby giving the desired disaccharide 9 which was used as acceptor for further glycosylation with 8. Unfortunately, a trisaccharide presenting an unwanted new α-glycosidic linkage was obtained. We, and other authors, have previously observed such an amazing behavior of glycosyl donors bearing a participating protecting group, able to orientate the reaction towards the 1,2-trans anomer. Two interpretations can be given to support this particular reactivity: (I) a stereochemical mismatching between the donor and the acceptor in the transition state (34); and/or (II) a mechanism pathway involving three elemental steps: the formation of an orthoester which is opened by the nucleophilic attack of the triflate anion on the β-face finally followed by the rearrangement of the resulting intermediate into the α-compound (35).

With this information at hands, we turned out our strategy using donors already presenting the required β-configuration between the two glucosyl entities (Figure 3). Under suitable promoting conditions, the thioglycosides 11a (36) and 11b (37), as well as the trichloracetimidate 11c afforded the desired trisaccharide 12 which was chromatographically purified and isolated in 50%, 48%, and 80% yields, respectively. We observed that, in all cases, the best Lewis acid required to catalyze the reaction was trimethysilyl triflate (TMSOTf). We experimentally showed that other Lewis catalysts were not suitable to efficiently perform the rearrangement of the orthoester intermediate into the desired glycoside. Moreover, the control of the temperature during the transfer of the glycosyl part to the nucleophilic acceptor was also decisive. As a result, the more efficient conditions were obtained with the trichloroacetimidate donor 11c. Subsequent transesterification under Zemplen conditions and pallado-catalyzed debenzylation, both quantitatively, allowed the synthesis of the first target 3. The latter was finally reduced thanks to the use of sodium borohydride to give the laminaribiosyl-(1→6)-mannitol 1.

Figure 2 How protecting groups affect glycosylation mechanism.

Figure 3 Efficient synthesis of laminaribiosyl-(1→6)-mannose 3 and laminaribiosyl-(1→6)-mannitol 1.

It resulted from this first synthesis and from literature data that (I) subtle structural modulation on both acceptor and donor may affect the behavior of the glycosylation process; and (II) a 4,6-benzylidene ring on both the donor and the acceptor is still the best protection to efficiently provide β-(1→3)-Glucanic chain (17,38-40). This inspired us for the following synthesis of the laminaribiosyl-(1→3)-mannose 2 and its reduced counterpart 4. Consequently, we have revisited the iterative synthesis of linear β-(1→3)-Glucans (8) since only the 2 position of the reducing end of the targets differ from linear β-(1→3)-Glucans. However, the approach is valid provided that the OH-2 of the reducing glucosyl residue bears an orthogonal protecting group compared to that of OH-2 of the other glucosyl entities. For that purpose, we opted for a levulinoyl protection on OH-2a and benzoyl ester for OH-2b and OH-2c (Figure 4). Consequently, the synthetic scheme first began with known compound 14 that was a key building block for the preparation of both 15 by standard levulynoylation followed by anomeric protection by a benzyl group, and benzoylation that provided donor 17. Regioselective oxido-reductive deprotection of the 3 position by dicyanodichloroquinone (DDQ) in methanol into 15 yielded the acceptor 16 that was couple to 17 to give the disaccharide 18 in excellent 94% yield. Chain elongation further followed the two-step procedure: DDQ assisted deprotection, and glycosylation step starting with the resulting nucleophile 19 and the same key donor 17. At this stage of the synthesis, the iterative strategy differed from that previously described by selective deprotection of the 2a hydroxyl group by hydrazine acetate in 82% yield. After experimentation, the best oxidative reagent was the Dess-Martin periodinan that allowed production of the ketone 22 in a quantitative yield. Its conversion into the mannosyl derivative 23 was carried out quantitatively thanks to the hydride L-Selectride at –80 °C. Further Zemplen transesterfication and pallado-catalyzed debenzylation afforded the desired laminaribiosyl-(1→3)-mannose 4, that was finally reduced by sodium borohydride to yield the laminaribiosyl-(1→3)-mannitol 2. This iterative startegy complemented well the uloside approach previously describe starting from pure oligo-β-(1→3)-Glucans (41-43).

Figure 4 An iterative approach toward laminaribiosyl-(1→3)-mannose 4 and laminaribiosyl-(1→3)-mannitol 2.

On an analytical point of view, small structural modulations on the reducing end induced significant differences in NMR data, i.e., on the mannose or mannitol entity. Indeed, coupling constants between H-1 and H-2 for the glucosyl residues b and c range between 7.5 and 8.0 Hz, thus corroborating the desired β linkages. However the most interesting information has been provided from ¹³C NMR since glycosylation on a specific oxygen atom should cause a significant shift of the signal corresponding to the involved carbon centre towards the lower fields. By comparing the data, the C-6a in 1 and 3 appears at 72.2 and 69.3 ppm, respectively, while the same signal appear at only 63.4 and 61.0 in 2 and 4, respectively. A similar observation is possible considering now the signals of the C-3a centres: 69.4 ppm for 1 and 66.7 ppm for 3, but 77.7 ppm for 2 and 78.5 ppm for 4.

Conclusions

The synthesis of two mannitol derivatives, grafted by laminaribiose—a precursor of longer β-(1→3)-Glucan chain—was performed targeting the primary and the three positions that are found in nature. We have shown that the synthetic approaches were efficient by using a mannoside as acceptor, but that they must be adapted to the nature of the desired glycosidic linkage. Thus, the (1→6)-bound required direct connection of a disaccharidyl donor since further elongation was disrupted by the presence of the mannosyl residue itself. The (1→3)-linkage was efficiently achieved by revisiting the iterative strategy that was previously developed for the preparation of linear oligo-β-(1→3)-Glucans. This study resulted in the synthesis of two non-natural laminaribiosyl-mannose derivatives and two laminaribiosyl-mannitol ones, whose analytical data will be valuable for further characterization of extracted β-(1→3)-Glucans.

Analytical data of compounds 1-4

Trisaccahride 3 (α/β =1.0:0.3)

¹H RMN (D₂O, 400 MHz) δ (ppm): 5.08 (s, 1H, H-1aα), 4.83 (m, 0,3H, H-1aβ), 4,66 (d, 1,3H, H-1c, J_H1-H2 =7.7 Hz), 4.46 (d, 0,3H, H-1bβ, J_H1-H2 =7.7 Hz), 4.45 (d, 1H, H-1bα, J_H1-H2 =7.8 Hz), 4.13-4.06 (m, 1,3H, H-6a), 3.91-3.72 (m, 7.8H, H-2a, H-4a, H-5a, H-6’a, H-6b, H-6c), 3.71-3.52 (m, 5,2H, H-3a, H-3b, H-6’b, H-6’c), 3.50-3.22 (m, 9.1H, H-2b, H-4b, H-5b, H-2c, H-3c, H-4c, H-5c); ¹³C RMN (D₂O, 100 MHz) δ (ppm): 103.1 (C-1c), 102.8 (C-1b), 94.4 (C-1aα), 94.1 (C-1aβ), 84.5 (C-3b), 76.3 (C-5c), 75.8 (C-3c, C-5b), 73.8 (C-2c), 73.2 (C-2b), 71.7 (C-4aα), 71.5 (C-4aβ), 70.9 (C-2aα), 70.6 (C-2aβ), 70.4 (C-5aα, C-5aβ), 69.9 (C-4c), 
69.3 (C-6a), 68.5 (C-4b), 66.9 (C-3aα), 66.7 (C-3aβ), 61.0 (C-6b, C-6c). HRMS (ESI⁺): calcd m/z for [M+Na]⁺ C₁₈H₃₂NaO₁₆ 527.1588, found m/z 527.1580; calcd for [M+K]⁺ C₁₈H₃₂KO₁₆ 543.1327, found m/z 543.1334.

Laminaribiosyl-β-(1→6)-mannitol 1

¹H RMN (D₂O, 400 MHz) δ (ppm): 4.67 (d, 1H, H-1c, J_H1-H2 =7.8 Hz), 4.46 (d, 1H, H-1b, J_H1-H2 =8.0 Hz), 4.11 (dd, 1H, H-6a, J_H5-H6 =1.8 Hz, J_H6-H6’ =10.6 Hz), 3.88-3.56 (m, 12H, H-1a, H-1’a, H-2a, H-3a, H-3b, H-4a, H-5a, H-6b, H-6c, H-6’a, H-6’b, H-6’c), 3.48-3.37 (m, 5H, H-2b, H-3c, H-4b, H-5b, H-5c), 3.32 (t, 1H, H-4c, J_H3-H4 = J_H4-H5 =9.0 Hz), 3.27 (dd, 1H, H-2c, J_H2-H3 =9.3 Hz); ¹³C RMN (D₂O, 100 MHz) δ (ppm): 103.1 (C-1c), 103.0 (C-1b), 84.6 (C-3b), 76.3 (C-5c), 75.9, 75.8 (C-3c, C-5b), 73.8 (C-2c), 73.3 (C-2b), 72.2 (C-6a), 71.1 (C-2a), 70.0 (C-5a), 69.9 (C-4c), 69.4 (C-3a), 69.3 (C-4a), 68.5 (C-4b), 63.5 (C-1a), 61.0 (C-6b, C-6c); HRMS (ESI⁺): [M+Na]⁺ C₁₈H₃₄NaO₁₆, calcd m/z 529.1745, measured m/z 529.1739; [M+K]⁺ C₁₈H₃₄KO₁₆, calcd m/z 545.1484, measured m/z 545.1476; [M-H+2Na]⁺ C₁₈H₃₃Na₂O₁, calcd m/z 551.1564, measured m/z 551.1570.

Trisaccharide 4

¹H RMN (D₂O, 400 MHz) δ (ppm): 5.13 (d, 1H, H-1aα, J_H1a-H2a =1.8 Hz), 4.67 (d, 1H, H-1c, J_H1c-H2c =7.7 Hz), 4.53 (d, 1H, H-1b, J_H1b-H2b =8.0 Hz), 4.03 (dd, 1H, H-2a, J_H2-H3 =3.1 Hz), 3.96 (dd, 1H, H-3a, J_H3-H4 =9.5 Hz), 3.90-3.80 (m, 3H, H-6a, H-6b, H-6c), 3.80-3.73 (m, 1H, H-4a), 3.72-3.55 (m, 5H, H-5a, H-6’a, H-3b, H-6’b, H-6’c), 3.53-3.36 (m, 5H, H-2b, H-64b, H-5b, H-3c, H-5c), 3.32 (t, 1H, H-4c, J_H3-H4 = J_H4-H5 =9.7 Hz), 3.27 (dd, 1H, H-2c, J_H2-H3 =9.3 Hz); ¹³C RMN (D₂O, 100 MHz) δ (ppm): 103.1 (C-1c), 100.5 (C-1b), 94.1 (C-1aα), 84.5 (C-3b), 78.5 (C-3a), 76.3 (C-5c), 75.9 (C-3c, C-5b), 75.8 (C-2c), 73.1 (C-2b), 72.7 (C-4a), 69.9 (C-4c), 68.6 (C-2a), 68.4 (C-4b), 65.5 (C-5a), 61.2, 61.0 (C-6a, C-6b, C-6c); HRMS (ESI⁺): [M+Na]⁺ C₁₈H₃₂NaO₁₆, calcd m/z 527.1588, measured m/z 527.1587; [M+K]⁺ C₁₈H₃₂KO₁₆, calcd m/z 543.1327, measured m/z 543.1343.

Laminaribiosyl-β-(1→3)-mannitol 2

¹H RMN (D₂O, 400 MHz) δ (ppm): 4.82 (d, 1H, H-1c, J_H1-H2 =8.0 Hz), 4.65 (d, 1H, H-1b, J_H1-H2 =8.1 Hz), 4.09-3.90 (m, 7H, H-1a, H-2a, H-3a, H-5a, H-6a, H-6b, H-6c), 
3.86-3.66 (m, 6H, H-1’a, H-4a, H-6’a, H-3b, H-6’b, H-6’c), 3.64-3.38 (m, 7H, H-2b, H-4b, H-5b, H-2c, H-3c, H-4c, H-5c); ¹³C RMN (D₂O, 100 MHz) δ (ppm): 103.1 (C-1c), 102.5 (C-1b), 84.4 (C-3b), 77.3 (C-3a), 76.3 (C-5c), 75.8 (C-3c), 75.5 (C-5b), 73.7 (C-2c), 73.4 (C-2b), 70.9, 70.8 (C-2a, C-5a), 69.9 (C-4c), 69.6 (C-4a), 68.8 (C-4b), 63.4 (C-6a), 62.6 (C-1a), 61.2, 61.0 (C-6b, C-6c); HRMS (ESI⁺): calcd m/z for [M+Na]⁺ C₁₈H₃₄NaO₁₆: 529.1745, found 529.1743; calcd m/z for [M+K]⁺ C₁₈H₃₄KO₁₆: 545.1484, found 545.1480; calcd m/z for [M-H+2Na]⁺ C₁₈H₃₃Na₂O₁₆ 551.1564, found 551.1561.

Acknowledgements

K.D. acknowledges Goemar Laboratories for a PhD grant. The authors are also grateful to Cancéropole Grand Ouest, and more especially the axis Produits de la Mer en Cancérologie, for complementary financial supports.

Disclosure: This original work is exclusively proposed for publication in Annals of Translational Medicine and was not submitted elsewhere. The company Goemar has partly supported this work, so that people scientifically involved in this work are obviously coauthors of the paper.

Supplementary material

Benzyl 2,3,4-tri-O-benzoyl-α-D-mannopyranoside 7b: to a solution of 6 (3.48 g, 6.79 mmol) in pyridine (50 mL) was added benzoyl chloride (4.71 mL, 40.76 mmol). The reaction was stirred for 2 d at RT before the solvent was removed under reduced pressure. The crude was then diluted in AcOEt (50 mL) and washed with a 10% aq sol HCl, a sat aq sol of NaHCO₃, and brine. The organic layer was dried over MgSO₄ and concentrated. The resulting crude was further diluted in anhydride acetic (80 mL), warmed at 100 °C for 20 min and added of water (20 mL). After stirring for 30 min, the media was cooled to 0 °C under reduced pressure. The desired compound 7b was finally purified by chromatography [light petroleum/AcOEt (7:3; v/v)] and isolated in a 72% yield for two steps (2.47 g). Rf (light petroleum/AcOEt, 7:3) 0.3. [α]²⁰_D +73.5 (c =2.0, CHCl₃). ¹H RMN (CDCl₃, 400 MHz) δ (ppm): 8.05-8.00 (m, 2H, H arom), 7.88-7.82 (m, 2H, H arom), 7.72-7.76 (m, 2H, H arom), 7.55-7.10 (m, 14H, H arom), 5.95 (dd, 1H, H-3, J_H2-H3 =3.6 Hz, J_H3-H4 =10.2 Hz), 5.77 (t, 1H, H-4, J_H4-H5 =10.2 Hz), 5.66 (dd, 1H, H-2, J_H1-H2 =1.5 Hz), 5.13 (d, 1H, H-1), 4.75 (d, 1H, CH₂Ph, J =11.9 Hz), 4.62 (d, 1H, CH₂Ph), 4.00 (ddd, 1H, H-5, J_H5-H6 =2.3 Hz, J_H5-H6’ =3.8 Hz), 3.72 (dd, 1H, H-6, J_H6-H6’ =13.0 Hz), 3.67 (dd, 1H, H-6’). ¹³C RMN (CDCl₃, 100 MHz) δ (ppm): 165.6, 165.5, 165.5 (OCOPh), 136.4, 133.7, 133.5, 133.2 (C quat arom), 130.1, 129.9, 129.7, 129.2, 128.6, 128.5, 128.4, 128.3, 128.2, 127.9, 127.8 (C arom), 97.1 (C-1), 71.1 (C-5), 70.6 (C-2), 70.1 (CH₂Ph), 69.6 (C-3), 67.3 (C-4), 61.2 (C-6).

Ethyl 2,4,6-tri-O-acetyl-3-O-(2-methylnaphtyl)-1-thio-β-D-glucopyranoside 8: to a solution of ethyl 3-O-(2-methylnaphtyl)-1-thio-β-D-glucopyranoside (1 g, 2.46 mmol) in pyridine (10 mL) was added dropewise acetic anhydride (1.2 mL, 12.73 mmol) and the reaction was stirred at RT for 20 h. After removal of the solvent under reduced pressure, the crude was diluted in CH₂Cl₂, washed with 10% aq sol HCl, sat aq sol of NaHCO₃, and brine. The organic layer was dried (MgSO₄), concentrated, and purified by chromatography [light petroleum/AcOEt (7:3; v/v)] to yield 8 as a white solid (1.08 g, 90%). mp 134 °C. Rf (light petroleum/AcOEt, 7:3) 0.3. [α]²⁰_D –19.6 (c =1.0, CH₂Cl₂). ¹H RMN (CDCl₃, 400 MHz) δ (ppm): 7.83-7.33 (m, 7H, H arom), 5.14 (t, 1H, H-2, J_H1-H2 = J_H2-H3 =9.9 Hz), 5.12 (t, 1H, H-4, J_H3-H4 = J_H4-H5 =9.9 Hz), 4.80 (d, 1H, CH₂Ar, J =12.0 Hz), 4.75 (d, 1H, CH₂Ar), 4.40 (d, 1H, H-1), 4.19 (dd, 1H, H-6, J_H5-H6 =5.4 Hz, J_H6-H6’ =12.5 Hz), 4.11 (dd, 1H, H-6’, J_H5-H6’ =2.3 Hz), 3.76 (t, 1H, H-3), 3.60 (ddd, 1H, H-5), 2.76-2.64 (m, 2H, CH₂CH₃), 2.07, 1.98, 1.92 (3s, 9H, COCH₃), 1.26 (t, 3H, CH₃, ²J =7.4 Hz). ¹³C RMN (CDCl₃, 100 MHz) δ (ppm): 170.8, 169.4, 169.3 (3C, COCH₃), 135.2, 133.1, 132.9 (C quat arom), 128.2, 127.8, 127.6, 126.4, 126.2, 126.0, 125.6 (C arom), 83.6 (C-1), 81.4 (C-3), 76.1 (C-5), 74.2 (C-7), 71.2 (C-2), 69.6 (C-4), 62.4 (C-6), 23.9 (CH₂CH₃), 20.9, 20.8, 20.8 (COCH₃), 14.7 (CH₂CH₃). Elemental analysis: calcd for C₂₅H₃₀O₈S: C 61.21%, H 6.16%, S 6.54%; measured: C 61.64%, H 6.30%, S 6.20%.

Benzyl 2,4,6-tri-O-acetyl-β-D-glucopyranosyl-(1→6)-2,3,4-tri-O-benzyl-α-D-mannopyranoside 9: to a solution of donor 8 (100 mg, 0.204 mmol) and acceptor 7a (92 mg, 0.170 mmol) in CH₂Cl₂ were successively added N-iodosuccinimide (NIS, 58 mg, 0.258 mmol) and trimethylsilyl triflate (TMSOTf, 1.5 µL, 8 µmol). After 30 min at RT, the reaction was quenched by adding few drops of NEt₃. The mixture was then filtered, concentrated and submitted to chromatographic purification [light petroleum/AcOEt (7:3, v/v)]. This afforded a disaccharide (91 mg, 94 µmol) isolated in 55% yield and as a colorless oil. Rf (light petroleum/AcOEt, 7:3) 0.3. The product was further dissolved in a 4:1 mixture of CH₂Cl₂, CH₃OH (10 mL) before adding 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 64 mg, 0.282 mmol). After stirring overnight at RT, dilution with CH₂Cl₂was performed (40 mL). The resulting organic layer was washed with sat aq NaHCO₃, water, dried (MgSO₄), and concentrated. The residue was purified by chromatography [light petroleum/AcOEt (10:7; v/v)] to give 9 (53 mg) as a colorless oil and in 68% yield. Rf (light petroleum/AcOEt, 10:7) 0.1. ¹H RMN (CDCl₃, 400 MHz) δ (ppm): 7.29-7.16 (m, 20H, H arom), 4.88 (t, 1H, H-4b, J_H3-H4 = J_H4-H5 =9.7 Hz), 4.87 (d, 1H, OCH₂Ph, ²J =11.2 Hz), 4.83 (d, 1H, H-1a, J_H1-H2 =1.6 Hz), 4.82 (dd, 1H, H-2b, J_H1-H2 =7.6 Hz, J_H2-H3 =9.7 Hz), 4.62 (d, 1H, C-1OCH₂Ph, J =11.4 Hz), 4.62 (s, 2H, OCH₂Ph), 4.51 (s, 2H, OCH₂Ph), 4.51 (d, 1H, OCH₂Ph), 4.45 (d, 1H, H-1b), 4.35 (d, 1H, C-1OCH₂Ph), 4.16 (dd, 1H, H-6b, J_H5-H6 =4.8 Hz, J_H6-H6’ =12.2 Hz), 4.04 (dd, 1H, H-6’b, J_H5-H6’ =2.3 Hz), 4.04 (dd, 1H, H-6a, J_H5-H6 =4.0 Hz, J_H6-H6’ =11.0 Hz), 3.87 (dd, 1H, H-3a, J_H2-H3 =3.0 Hz, J_H3-H4 =8.9 Hz), 3.81-3.73 (m, 2H, H-4a, H-5a), 3.73 (dd, 1H, H-2a), 3.67 (dd, 1H, H-6’a, J_H5-H6’ =5.6 Hz), 3.59 (dt, 1H, H-3b, J_H3-OH3 =7.1 Hz), 3.48 (ddd, 1H, H-5b), 2.65 (d, 1H, OH-3b), 2.02, 1.96, 1.91 (3 s, 9H, COCH₃). ¹³C RMN (CDCl₃, 100 MHz) δ (ppm): 170.8, 170.6, 170.4 (OCOCH₃), 138.3, 138.0, 137.0 (C quat arom), 128.4, 128.3, 128.1, 127.9, 127.7, 127.6, 127.5 (C arom), 100.7 (C-1b), 96.8 (C-1a), 80.1 (C-3a), 74.9 (OCH₂Ph), 74.8 (C-4a), 74.3 (C-2a), 74.1 (C-2b), 73.9 (C-3b), 72.6 (OCH₂Ph), 72.0 (OCH₂Ph), 71.7 (C-5a), 71.6 (C-5b), 70.9 (C-4b), 68.6 (C-6a), 68.5 (C-1OCH₂Ph), 62.1 (C-6b), 20.8, 20.7 (OCOCH₃).

Benzyl 2,4,6-tri-O-acetyl-3-O-(2-methylnaphtyl)-α-D-glucopyranosyl-(1→3)-2,4,6-tri-O-acetyl-β-D-glucopyranosyl-(1→6)-2,3,4-tri-O-benzyl-α-D-mannopyranoside 10: to a solution of 8 (38 mg, 0.077 mmol) and 9 (53 mg, 0.064 mmol) in CH₂Cl₂ cooled at –10 °C were successively added NIS (22 mg, 0.098 mmol) and TMSOTf (0.6 µL, 3 μmol). After 45 min, few drops of NEt₃ were added, the mixture was filtered, the filtrate concentrated under reduced pressure, and the crude was subjected to column chromatography [light petroleum/AcOEt (3:2, v/v)]. The trisaccharide (48 mg) was isolated in 60% yield. Rf (light petroleum/AcOEt, 3:2) 0.3. ¹H RMN (CDCl₃, 400 MHz) δ (ppm): 7.76-7.70 (m, 3H, H arom), 7.62 (s, 1H, H arom), 7.38-7.35 (m, 2H, H arom), 7.30-7.17 (m, 22H, H arom), 5.19 (d, 1H, H-1c, J_H1-H2 =3.6 Hz), 5.10 (t, 1H, H-4c, J_H3-H4 = J_H4-H5 =9.7 Hz), 5.04 (dd, 1H, H-2b, J_H1-H2 =7.9 Hz, J_H2-H3 =9.7 Hz), 5.00 (t, 1H, H-4b, J_H3-H4 = J_H4-H5 =9.9 Hz), 4.87 (d, 1H, CH₂ NAP, J =10.9 Hz), 4.81 (d, 1H, H-1a, J_H1-H2 =1.5 Hz), 4.75 (d, 1H, OCH₂Ph, ²J =12.0 Hz), 4.70 (dd, 1H, H-2c, J_H2-H3 =10.2 Hz), 4.64 (d, 1H, OCH₂Ph), 4.63 (s, 2H, OCH₂Ph), 4.61 (d, 1H, C-1OCH₂Ph, J =11.4 Hz), 4.51 (s, 2H, OCH₂Ph), 4.49 (d, 1H, CH₂ NAP), 4.33 (d, 1H, H-1b), 4.32 (d, 1H, C-1OCH₂Ph), 4.12 (dd, 1H, H-6b, J_H5-H6 =4.6 Hz, J_H6-H6’ =12.5 Hz), 4.08 (dd, 1H, H-6a, J_H5-H6 =1.8 Hz, J_H6-H6’ =10.4 Hz), 4.08 (dd, 1H, H-6c, J_H5-H6 =5.0 Hz, J_H6-H6’ =10.7 Hz), 3.99 (dd, 1H, H-6’b, J_H5-H6’ =2.5 Hz), 3.96 (dd, 1H, H-6’c, J_H5-H6’ =2.0 Hz), 3.88 (dd, 1H, H-3a, J_H2-H3 =3.1 Hz, J_H3-H4 =9.7 Hz), 3.87 (dd, 1H, H-3c), 3.86 (ddd, 1H, H-5c), 3.81 (t, 1H, H-3b), 3.79 (ddd, 1H, H-5a, J_H4-H5 =9.7 Hz, J_H5-H6’ =7.2 Hz), 3.74 (dd, 1H, H-2a), 3.67 (t, 1H, H-4a), 3.58 (dd, 1H, H-6’a), 3.43 (ddd, 1H, H-5b), 1.99, 1.96, 1.86, 1.68 (4 s, 18H, COCH₃). ¹³C RMN (CDCl₃, 100 MHz) δ (ppm): 170.7, 169.4, 169.2, 168.9 (OCOCH₃), 138.3, 138.2, 138.0, 137.1, 135.5, 133.2, 132.8 (C quat arom), 128.4, 128.3, 128.1, 127.9, 127.8, 127.7, 127.6, 127.5, 126.1, 125.9, 125.5 (C arom), 101.1 (C-1b), 96.8 (C-1a), 95.8 (C-1c), 80.2 (C-3a), 76.7 (C-3c), 76.2 (C-3b), 75.2 (CH₂ NAP), 75.0 (C-4a, OCH₂Ph), 74.3 (C-2a), 73.2 (C-2c), 72.8 (OCH₂Ph), 72.1 (OCH₂Ph), 71.7 (C-5b, C-2b), 71.5 (C-5a), 70.2 (C-4c), 69.0 (C-6a, C-4b), 68.5 (C-1OCH₂Ph), 68.4 (C-5c), 61.8 (C-6b), 61.4 (C-6c), 20.9, 20.8, 20.7, 20.6 (OCOCH₃). HRMS (ESI⁺): [M+Na]⁺ C₆₉H₇₆NaO₂₂ calcd m/z 1,279.4726, found m/z 1279.4705; [M+K]⁺ C₆₉H₇₆KO₂₂ calcd m/z 1,295.4465, found m/z 1,295.4537.

2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl-(1→3)-2,4,6-tri-O-acetyl-α-D-glucopyranosyl trichloroacetimidate 11c: to a solution of peracetylated laminaribiose (2.2 g, 3.25 mmol in CH₂Cl₂ (25 mL) was added morpholine (1.4 mL, 16.25 mmol). After stirring under reflux for 4 h, the mixture was cooled at RT, diluted with CH₂Cl₂ (25 mL), washed with sat aq NaHCO₃, and water. The organic layer was then dried (MgSO₄) and concentrated to yield the desired hemiacetal. The latter (2.07 g, 3.25 mmol) was dissolved in CH₂Cl₂ (25 mL) and 1,8-diazobicyclo[5,4,0]undec-7-ene (DBU, 100 μL, 0.65 mmol) and trichloroacetonitrile (3.3 mL, 32.5 mmol) were added at RT. After 1 h, the reaction media was concentrated and the resulting crude purified by chromatography [light petroleum/AcOEt (3:2, v/v)] to afford 11c (1.86 g, 74 over 2 steps). Rf (light petroleum/AcOEt, 3:2) 0.25. [α]₂₀^D +41.6 (c =1.0, CH₂Cl₂). ¹H RMN (CDCl₃, 400 MHz) δ (ppm): 8.70 (s, 1H, NH), 6.44 (d, 1H, H-1a, J_H1-H2 =3.8 Hz), 5.15 (dd, 1H, H-3b, J_H2-H3 = J_H3b-H4b =9.5 Hz), 5.11 (dd, 1H, H-4a, J_H3-H4 = J_H4-H5=9.7 Hz), 5.08 (dd, 1H, H-2a, J_H2-H3 =9.9 Hz), 5.06 (dd, 1H, H-4b, J_H4-H5 =9.5 Hz), 4.90 (dd, 1H, H-2b, J_H1-H2 =8.1 Hz), 4.65 (d, 1H, H-1b), 4.35 (dd, 1H, H-6b, J_H5-H6 =4.6 Hz, J_H6-H6’ =12.4 Hz), 4.21-4.07 (m, 5H, H-3a, H-5a, H-6a, H-6’a, H-6’b), 3.73 (ddd, 1H, H-5b, J_H5-H6’ =2.3 Hz), 2.10, 2.08, 2.06, 2.05, 2.00, 1.97 (6s, 21H, COCH₃). ¹³C RMN (CDCl₃, 100 MHz) δ (ppm): 170.7, 170.5, 170.3, 169.6, 169.3, 169.1 (COCH₃), 160.5 (CNHCCl₃), 100.9 (C-1b), 93.1 (C-1a), 90,7 (CCl₃), 76.4 (C-3a), 72.8 (C-3b), 71.7 (C-5b), 71.6 (C-2a), 71.3 (C-2b), 70.2 (C-5a), 68.0 (C-4b), 67.1 (C-4a), 61.6 (C-6b), 61.5 (C-6a), 20.7, 20.6, 20.5, 20.3 (COCH₃). HRMS (ESI⁺): [M+Na]⁺ C₂₈H₃₆NaNO₁₈Cl₃ calcd m/z 802.0896, found m/z 802.0899.

Benzyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl-(1→3)-2,4,6-tri-O-acetyl-β-D-glucopyranosyl-(1→6)-2,3,4-tri-O-benzyl-α-D-mannopyranoside 12: a solution of 11c (1.85 g, 2.369 mmol) and 7a (1.17 g, 2.164 mmol) in CH₂Cl₂ was cooled at -50 °C before adding TMSOTf (15 µL, 0.083 mmol). The reaction was quenched after stirring for 15 min by NEt₃, concentrated and purified by chromatography [light petroleum/AcOEt (1:1, v/v)] to afford an orthoester (1.98 g) which was rearranged after dissolution (CH₂Cl₂, 10 mL) thanks to TMSOTf (20 μL, 0.111 mmol) at 0 °C. After neutralization by NEt₃, concentration and chromatography [light petroleum/AcOEt (1:1, v/v)], the expected trisaccharide 12 (1.83 g) was obtained as a white solid in 73% yield. mp 70 °C. Rf (light petroleum/AcOEt, 1:1) 0.4. [α]₂₀^D +11.6 (c =1.0, CH₂Cl₂). ¹H RMN (CDCl₃, 400 MHz) δ (ppm): 7.30-7.20 (m, 20H, H arom), 5.06 (t, 1H, H-3c, J_H2-H3 = J_H3-H4=9.4 Hz), 5.00 (t, 1H, H-4c, J_H4-H5 =9.4 Hz), 4.99 (dd, 1H, H-2b, J_H1-H2 =8.2 Hz, J_H2-H3 =9.7 Hz), 4.88 (t, 1H, H-4b, J_H3-H4 = J_H4-H5 =9.7 Hz), 4.87 (d, 1H, OCH₂Ph, ²J =10.9 Hz), 4.83 (dd, 1H, H-2c, J_H1-H2 =7.9 Hz), 4.82 (d, 1H, H-1a, J_H1-H2 =1.7 Hz), 4.64 (s, 2H, OCH₂Ph), 4.60 (d, 1H, OCH₂Ph, J =12.0 Hz), 4.53 (s, 2H, OCH₂Ph), 4.51 (d, 1H, OCH₂Ph), 4.50 (d, 1H, H1c), 4.36 (d, 1H, H1b), 4.34 (d, 1H, C-1OCH₂Ph), 4.32 (dd, 1H, H-6c, J_H5-H6 =4.3 Hz, J_H6-H6’ =12.7 Hz), 4.14-4.04 (m, 3H, H-6a, H-6b, H-6’b), 3.98 (dd, 1H, H-6’c, J_H5-H6’ =2.3 Hz), 3.88 (dd, 1H, H-3a, J_H2-H3 =3.0 Hz, J_H3-H4 =9.2 Hz), 3.82-3.77 (m, 2H, H-3b, H-5a), 3.76 (dd, 1H, H-2a), 3.68 (t, 1H, H-4a, J_H4-H5 =9.2 Hz), 3.65-3.52 (m, 3H, H-5b, H-5c, H-6’a), 2.08, 2.03, 2.00, 1.99, 1.97 (5s, 21H, COCH₃). RMN ¹³C (CDCl₃, 100 MHz) δ (ppm): 170.8, 170.5, 170.3, 169.4, 169.2, 169.1, 168.4 (COCH₃), 138.3, 138.2, 138.0, 137.1 (C quat arom), 128.4, 128.3, 127.9, 127.8, 127.7, 127.6, 127.5 (C arom), 100.9 (C-1b, C-1c), 96.7 (C-1a), 80.2 (C-3a), 78.9 (C-3b), 74.9 (C-4a, OCH₂Ph), 74.4 (C-2a), 72.9 (C-3c), 72.8 (OCH₂Ph), 72.4 (C-2b), 72.1 (OCH₂Ph), 71.7, 71.6, 71.5 (C-5a, C-5b, C-5c), 70.9 (C-2c), 68.7 (C-6a), 68.4 (C-1OCH₂Ph), 68.2 (C-4b), 67.9 (C-4c), 62.0 (C-6b), 61.6 (C-6c), 20.8, 20.7, 20.6, 20.5, 20.3 (COCH₃). Elemental analysis calcd for C₆₀H₇₀O₂₃: C 62.17%, H 6.09%; found C 62.09%, H 6.20%.

Benzyl β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→6)-2,3,4-tri-O-benzyl-α-D-mannopyranoside 13: deacylation of 12 (883 mg, 0.761 mmol) was performed in 5:1:1 CH₃OH/NEt₃/H₂O mixture (5 mL). After stirring overnight, the solvent was evaporated under reduced pressure to give 13 (655 mg) in a quantitative yield as a whithe solid. mp 95 °C. Rf (CH₂Cl₂/MeOH, 9:1) 0.2. [α]₂₀^D +13.8 (c =1.0, CH₂Cl₂). ¹H RMN (CDCl₃, 400 MHz) δ (ppm): 7.32-7.19 (m, 20H, H arom), 4.85 (d, 1H, OCH₂Ph, ²J =11.2 Hz), 4.83 (d, 1H, H-1a, J_H1-H2 <1.0 Hz), 4.63 (d, 1H, C-1OCH₂Ph, J =12.2 Hz), 4.60 (s, 2H, OCH₂Ph), 4.59 (d, 1H, OCH₂Ph), 4.52 (d, 1H, OCH₂Ph, ²J =11.4 Hz), 4.51 (d, 1H, OCH₂Ph), 4.44 (d, 1H, H-1c, J_H1-H2 =7.6 Hz), 4.38 (d, 1H, C-1OCH₂Ph), 4.28 (d, 1H, H-1b, J_H1-H2 =7.1 Hz), 4.05-4.02 (m, 1H, H-6a), 3.90-3.86 (m, 2H, H-3a, H-4a), 3.83-3.70 (m, 5H, H-2a, H-5a, H-6’a, H-6b, H-6c), 3.67 (dd, 1H, H-6’b, J_H5-H6 =4.8 Hz, J_H6-H6’ =12.2 Hz), 3.61 (dd, 1H, H-6’c, J_H5-H6 =5.3 Hz, J_H6-H6’ =11.7 Hz), 3.47-3.40 (m, 3H, H-2b, H-3b, H-4b), 3.38-3.35 (m, 1H, H-3c), 3.33-3.24 (m, 3H, H-2c, H-4c, H-5c), 3.24-3.20 (m, 1H, H-5b). ¹³C RMN (CDCl₃, 100 MHz) δ (ppm): 138.0, 137.9, 137.3, 136.7 (C quat arom), 128.2, 128.1, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 127.3 (C arom), 103.9 (C-1c), 102.9 (C-1b), 96.8 (C-1a), 87.0 (C-3b), 79.6 (C-3a), 76.3 (C-5c), 76.0 (C-3c), 75.4 (C-5b), 74.9 (OCH₂Ph), 74.3 (C-4a, C-5a), 73.8 (C-2c), 72.6 (OCH₂Ph), 72.4 (C-2b), 72.0 (OCH₂Ph), 71.3 (C-2a), 69.6 (C-4c), 68.7 (C-1OCH₂Ph), 68.6 (C-4b), 68.2 (C-6a), 61.5 (C-6b), 61.2 (C-6c). Elemental analysis: calcd for C₄₆H₅₆O₁₆ C 63.88%, H 6.53%; found C 63.33%, H 6.42%.

β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→6)-(α,β)-D-mannopyranose 3: to a solution of 13 (200 mg, 0.231 mmol) in 1:1 ethanol/water (15 mL) were successively added acetic acid (100 μL) and palladium acetate (40 mg). After vigorous stirring under hydrogen atmosphere for 48 h, the media was filtered over celite, concentrated and coevaporated with absolute ethanol. This afforded 3 (120 mg, 100%) as a white foam and as an anomeric mixture (α/β =3:1). ¹H RMN (D₂O, 400 MHz) δ (ppm): 5.08 (s, 1H, H-1aα), 4.83 (m, 0.3H, H-1aβ), 4.66 (d, 1.3H, H-1c, J_H1-H2 =7.7 Hz), 4.46 (d, 0.3H, H-1bβ, J_H1-H2 =7.7 Hz), 4.45 (d, 1H, H-1bα, J_H1-H2 =7.8 Hz), 4.13-4.06 (m, 1.3H, H-6a), 3.91-3.72 (m, 7.8H, H-2a, H-4a, H-5a, H-6’a, H-6b, H-6c), 3.71-3.52 (m, 5.2H, H-3a, H-3b, H-6’b, H-6’c), 3.50-3.22 (m, 9.1H, H-2b, H-4b, H-5b, H-2c, H-3c, H-4c, H-5c). ¹³C RMN (D₂O, 100 MHz) δ (ppm): 103.1 (C-1c), 102.8 (C-1b), 94.4 (C-1aα), 94.1 (C-1aβ), 84.5 (C-3b), 76.3 (C-5c), 75.8 (2C, C-3c, C-5b), 73.8 (C-2c), 73.2 (C-2b), 71.7 (C-4aα), 71.5 (C-4aβ), 70.9 (C-2aα), 70.6 (C-2aβ), 70.4 (2C, C-5aα, C-5aβ), 69.9 (C-4c), 69.3 (C-6a), 68.5 (C-4b), 66.9 (C-3aα), 66.7 (C-3aβ), 61.0 (2C, C-6b, C-6c). HRMS (ESI⁺): calcd m/z for [M+Na]⁺ C₁₈H₃₂NaO₁₆ 527.1588, found m/z 527.1580; calcd for [M+K]⁺ C₁₈H₃₂KO₁₆ 543.1327, found m/z 543.1334.

β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→1)-D-mannitol 1: to a solution of 3 (35.5 mg, 0.070 mmol) in 5:1 CH₃OH/H₂O (5 mL) was added sodium borohydride (27 mg, 0.714 mmol). After stirring for 6 d at RT, adding few drops of acetic acid allowed neutralization. The resulting mixture was concentrated under reduced pressure and successively coevaoprated with 5:1 CH₃OH/acetic acid, and CH₃OH. The resulting compound 1 was chemically purified after acetylation (acetic anhydride: 5 mL, 0.053 mmol, pyridine: 5 mL, 48 h, RT). At the end of the reaction, the solvent was removed under reduced pressure and the crude oil diluted in CH₂Cl₂ (20 mL). This organic layer was washed with 10% HCl aq sol, and sat aq NaHCO₃, the dried over MgSO₄, and finally concentrated. The peracetylated compound was chromatographied [light petroleum/AcOEt (2:3, v/v)]. The deprotection step involved dissolution of the resulting derivative in 5:1:1 CH₃OH/NEt₃/H₂O mixture (7 mL), stirring for 20 h at RT, and freeze drying, so that 1 was isolated as a white foam (34 mg, 100%). ¹H RMN (D₂O, 400 MHz) δ (ppm): 4.67 (d, 1H, H-1c, J_H1-H2 =7.8 Hz), 4.46 (d, 1H, H-1b, J_H1-H2 =8.0 Hz), 4.11 (dd, 1H, H-6a, J_H5-H6 =1.8 Hz, J_H6-H6’ =10.6 Hz), 3.88-3.56 (m, 12H, H-1a, H-1’a, H-2a, H-3a, H-3b, H-4a, H-5a, H-6b, H-6c, H-6’a, H-6’b, H-6’c), 3.48-3.37 (m, 5H, H-2b, H-3c, H-4b, H-5b, H-5c), 3.32 (t, 1H, H-4c, J_H3-H4 = J_H4-H5 =9.0 Hz), 3.27 (dd, 1H, H-2c, J_H2-H3 =9.3 Hz). ¹³C RMN (D₂O, 100 MHz) δ (ppm): 103.1 (C-1c), 103.0 (C-1b), 84.6 (C-3b), 76.3 (C-5c), 75.9, 75.8 (C-3c, C-5b), 73.8 (C-2c), 73.3 (C-2b), 72.2 (C-6a), 71.1 (C-2a), 70.0 (C-5a), 69.9 (C-4c), 69.4 (C-3a), 69.3 (C-4a), 68.5 (C-4b), 63.5 (C-1a), 61.0 (C-6b, C-6c). HRMS (ESI⁺): calcd m/z for [M+Na]⁺ C₁₈H₃₄NaO₁₆: 529.1745, found 529.1739; calcd m/z for [M+K]⁺ C₁₈H₃₄KO₁₆: 545.1484, found 545.1476; calcd m/z for [M-H+2Na]⁺ C₁₈H₃₃Na₂O₁₆ 551.1564, found 551.1570.

Benzyl 4,6-O-benzylidene-2-O-levulinoyl-β-D-glucopyranoside 16: to a solution of 14 (1.1 g, 1.844 mmol) in CH₂Cl₂/CH₃OH (4:1, v/v) at RT was added DDQ (810 mg, 3.568 mmol). After stirring for 5 h, the media was diluted with CH₂Cl₂ (50 mL) and washed twice by sat aq NaHCO₃. The organic layer was then dried (MgSO₄), the solvent removed under reduced pressure and the resulting crude subjected to chromatography [light petroleum/AcOEt (3:1, v/v)] to yield the desired acceptor 16 (690 mg, 82%) as a white solid. mp 122 °C. Rf (light petroleum/AcOEt, 3:1) 0.2. [α]₂₀^D –62.2 (c =1.0, CH₂Cl₂). ¹H RMN (CDCl₃, 400 MHz) δ (ppm): 7.50-7.48 (m, 2H, H arom), 7.38-7.28 (m, 8H, H arom), 5.55 (s, 1H, CHO₂Ph), 4.99 (dd, 1H, H-2, J_H1-H2 =8.0 Hz, J_H2-H3 =9.1 Hz), 4.89 (d, 1H, C-1OCH₂Ph, J =12.3Hz), 4.62 (d, 1H, C-1OCH₂Ph), 4.56 (d, 1H, H-1), 4.36 (dd, 1H, H-6a, J_H5-H6a =4.8 Hz, J_H6a-H6b =10.4 Hz), 3.86 (t, 1H, H-4, J_H3-H4 = J_H4-H5 =8.0 Hz), 3.80 (t, 1H, H-6b, J_H5-H6b =10.4 Hz), 3.61 (t, 1H, H-3), 3.42 (dt, 1H, H-5), 3.05 (s, 1H, OH-3), 2.79-2.73 (m, 2H, CH₂COCH₃), 2.59-2.51 (m, 2H, CH₂CO₂), 2,16 (s, 3H, CH₃). ¹³C RMN (CDCl₃, 100 MHz) δ (ppm): 207.1 (COCH₃), 172.0 (CO₂), 136.9 (C quat arom), 129.2, 128.4, 128.3, 127.9, 127.7, 126.3 (C arom), 101.9 (CHO₂Ph), 99.8 (C-1), 80.5 (C-4), 74.5 (C-2), 72.0 (C-3), 70.8 (CH₂Ph), 68.5 (C-6), 66.2 (C-5), 38.1 (CH₂COCH₃), 29.8 (CH₃), 28.0 (CH₂CO₂). Elemental analysis: calcd for C₂₅H₂₈O₈: C 65.78%, H 6.18%; found C 66.04%, H 6.23%.

Benzyl 2-O-benzoyl-4,6-O-benzylidene-3-O-(2-methylnaphtyl)-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-2-O-levulinoyl-β-D-glucopyranoside 18: to a suspension of molecular sieves 4Å (100 mg) in CH₂Cl₂ cooled at –60 °C were successively added the donor 17 (1.73 g, 3.108 mmol), NIS (763 mg, 3.390 mmol), and TMSOTf (50 µL, 0.283 mmol). The reaction was stirred for 3 h before neutralizing by few drops of NEt₃. After filtration and removal of the solvent, a chromatographic purification [toluene/AcOEt (9:1, v/v)] gave 18 as a white solid (2.51 g, 94%). mp 178-179 °C. [α]₂₀^D –21.0 (c =1.0, CH₂Cl₂). Rf (light petroleum/AcOEt, 3:1) 0.2. ¹H RMN (CDCl₃, 400 MHz) δ (ppm): 7.90-7.86 (m, 2H, H arom), 7.70-7.67 (m, 1H, H arom), 7.60-7.21 (m, 24H, H arom), 5.53 (s, 1H, CHbO₂Ph), 5.28 (s, 1H, CHaO₂Ph), 5.26 (t, 1H, H-2b, J_H1-H2 = J_H2-H3 =7.1 Hz), 5.06 (dd, 1H, H-2a, J_H1-H2 =7.8 Hz, J_H2-H3 =8.9 Hz), 4.94 (d, 1H, CH₂ NAP, J =12.1 Hz), 4.93 (d, 1H, H-1b), 4.84 (d, 1H, CH₂ NAP), 4.80 (d, 1H, C-1OCH₂Ph, J =12.2 Hz), 4.52 (d, 1H, C-1OCH₂Ph), 4.45 (d, 1H, H-1a), 4.35 (dd, 1H, H-6a, J_H5-H6 =4.9 Hz, J_H6-H6’ =10.4 Hz), 4.13 (dd, 1H, H-6b, J_H5-H6 =4.9 Hz, J_H6-H6’ =10.4 Hz), 3.93 (t, 1H, H-3a, J_H3-H4 =8.9 Hz), 3.95 (t, 1H, H-4b, J_H3-H4 = J_H4-H5 =9.1 Hz), 3.85 (dd, 1H, H-3b), 3.81 (t, 1H, H-6’a), 3.80 (t, 1H, H-4a, J_H4-H5 =8.9 Hz), 3.44 (dd, 1H, H-5a), 3.43 (ddd, 1H, H-5b, J_H5-H6’ =10.4 Hz), 3.68 (t, 1H, H-6’a), 2.66-2.46 (m, 2H, CH₂CO₂), 2.40-2.27 (m, 2H, CH₂COCH₃), 2.08 (s, 3H, CH₃). ¹³C RMN (CDCl₃, 100 MHz) δ (ppm): 206.4 (COCH₃), 171.1 (CO₂CH₂), 165.0 (OCOPh), 137.3, 137.0, 136.7, 135.3, 133.1, 133.0 (C quat arom), 132.8, 129.7, 129.4, 128.9, 128.3, 128.2, 127.9, 127.8, 127.7, 127.5, 126.7, 126.1, 126.0, 125.8, 125.6 (C arom), 101.7 (CbHO₂Ph), 100.9 (CaHO₂Ph), 100.1 (C-1b), 99.8 (C-1a), 80.7 (C-4b), 79.7 (C-4a), 78.1 (C-3b), 77.5 (C-3a), 74.0 (C-2b), 73.4 (CH₂ NAP), 72.9 (C-2a), 70.6 (C-1OCH₂Ph), 68.6 (C-6a, C-6b), 66.3 (C-5a), 65.9 (C-5b), 37.8 (CH₂COCH₃), 29.7 (CH₃), 27.5 (CH₂CO₂). Elemental analysis: calcd for C₅₆H₅₄O₁₄: C 70.72%, H 5.72%; found C 70.89%, H 5.72%.

Benzyl 2-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-2-O-levulinoyl-β-D-glucopyranoside 19: DDQ (820 mg, 3.6 mmol) was added to a solution of 18 (1.71 g, 1.8 mmol) in a 4:1 CH₂Cl₂/CH₃OH mixture. After stirring for 16 h at RT, the media was diluted in CH₂Cl₂, washed with sat aq NaHCO₃, and water. The organic layer was then dried (MgSO₄) and concentrated under reduced pressure. A chromatographic purification afforded 19 as a white solid. mp 214 °C. Rf (light petroleum/AcOEt, 2:1) 0.3. [α]₂₀^D –43.0 (c =1.0, CH₂Cl₂). ¹H RMN (CDCl₃, 400 MHz) δ (ppm): 8.06-8.01 (m, 2H, H arom), 7.63-7.23 (m, 19H, H arom), 5.57 (s, 1H, CHbO₂Ph), 5.31 (s, 1H, CHaO₂Ph), 5.14 (dd, 1H, H-2b, J_H1-H2 =6.8 Hz, J_H2-H3 =7.1 Hz), 5.09 (dd, 1H, H-2a, J_H1-H2 =7.5 Hz, J_H2-H3 =8.4 Hz), 5.02 (d, 1H, H-1b), 4.83 (d, 1H, C-1OCH₂Ph, J =12.2 Hz), 4.55 (d, 1H, C-1OCH₂Ph), 4.49 (d, 1H, H-1a), 4.39 (dd, 1H, H-6a, J_H5-H6 =4.8 Hz, J_H6-H6’ =10.6 Hz), 4.18 (dd, 1H, H-6b, J_H5-H6 =5.1 Hz, J_H6-H6’ =10.6 Hz), 4.04 (dd, 1H, H-3a, J_H3-H4 =9.1 Hz), 3.99 (dd, 1H, H-3b, J_H3-H4 =9.3 Hz), 3.89 (t, 1H, H-6’a, J_H5-H6’ =10.6 Hz), 3.78 (t, 1H, H-4a, J_H4-H5 =9.1 Hz), 3.75 (t, 1H, H-4b, J_H4-H5 =9.3 Hz), 3.70 (t, 1H, H-6’b, J_H5-H6’ =10.6 Hz), 3.49 (ddd, 1H, H-5a), 3.46 (ddd, 1H, H-5b), 2.72 (s, 1H, OH-3b), 2.65-2.49 (m, 2H, CH₂CO₂), 2.37-2.25 (m, 2H, CH₂COCH₃), 2.12 (s, 3H, CH₃). ¹³C RMN (CDCl₃, 100 MHz) δ (ppm): 206.4 (COCH₃), 171.1 (CO₂CH₂), 165.7 (OCOPh), 137.0, 136.9, 136.7 (C quat arom), 133.3, 129.8 (C arom), 129.5 (C quat arom), 129.3, 129.1, 128.4, 128.3, 128.2, 128.1, 127.8, 127.6, 126.2. 126,0 (C arom), 101.6 (CbHO₂Ph), 101.5 (CaHO₂Ph), 100.0 (C-1b), 99.7 (C-1a), 80.5 (C-4b), 79.6 (C-4a), 77.6 (C-3a), 75.6 (C-2b), 72.9 (C-2a), 70.6 (C-1OCH₂Ph), 68.6 (C-6a, C-6b), 66.2 (C-5a), 65.8 (C-5b), 37.7 (CH₂COCH₃), 29.7 (CH₃), 27.5 (CH₂CO₂). Elemental analysis: calcd for C₄₅H₄₆O₁₄: C =66.66%, H =5.72%; found C =66.53%, H =5.80%.

Benzyl 2-O-benzoyl-4,6-O-benzylidene-3-O-(2-methylnaphtyl)-β-D-glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-2-O-levulinoyl-β-D-glucopyranoside 20: to a solution of donor 17 (226 mg, 0.406 mmol) and acceptor 19 (300 mg, 0.370 mmol) in CH₂Cl₂ cooled at –80 °C, were successively added molecular sieves 4Å, NIS (100 mg, 0.444 mmol) and TMSOTf (6.7 µL, 0.037 mmol). The Lewis acid was neutrlized after stirring for 2 h by adding few drops of NEt₃. The media was filtered, concentrated and chromatographied eluting with toluene/AcOEt (9:1, v/v) to give 20 (328 mg, 68%) as a white solid. mp 115 °C. Rf (light petroleum/AcOEt, 2:1) 0.5. [α]₂₀^D –4.7 (c =1.0, CH₂Cl₂). ¹H RMN (CDCl₃, 400 MHz) δ (ppm): 7.95-7.86 (m, 4H, H arom), 7.70-7.13 (m, 33H, H arom), 5.51 (s, 1H, CHO₂Ph), 5.43 (s, 1H, CHO₂Ph), 4.94 (d, 1H, CH₂ NAP, J =12.2 Hz), 4.83 (d, 1H, CH₂ NAP), 4.78 (d, 1H, C-1OCH₂Ph, J =12.4 Hz), 4.50 (s, 1H, CHO₂Ph), 4.49 (d, 1H, C-1OCH₂Ph), 2.69-2.47 (m, 2H, CH₂CO₂), 2.40-2.21 (m, 2H, CH₂COCH₃), 1.95 (s, 3H, CH₃) and Tables S1 and S2. RMN ¹³C (CDCl₃, 100 MHz) δ (ppm): 206.6 (COCH₃), 171.1 (CO₂CH₂), 165.0, 164.8 (OCOPh), 137.3, 137.1, 137.0, 135.4 (C quat arom), 133.3, 133.1 (C arom), 133.0, 132.8 (C quat arom), 129.8, 129.7 (C arom), 129.4 (C quat arom), 129.4, 128.9, 128.7, 128.6, 128.3, 128.2, 128.1, 127.9, 127.8, 127.7, 127.6, 127.5, 126.6, 126.4, 126.1, 126.0, 125.7, 125.6 (C arom), 102.1 (CHO₂Ph), 101.1 (CHO₂Ph), 100.9 (CHO₂Ph), 73.8 (CH₂ NAP), 70.4 (C-1OCH₂Ph), 37.7 (CH₂COCH₃), 29.3 (CH₃), 27.6 (CH₂CO₂) and Table S3. Elemental analysis: cacld for C₇₆H₇₂O₂₀: C =69.93%, H =5.56%; found C =69.89%, H =5.63%.

Table S1 Complementary NMR data for compounds 21, 23, 24
Full table

Table S2 Complementary NMR data for compounds 21, 23, 24
Full table

Table S3 Complementary NMR data for compounds 21, 23, 24
Full table

Benzyl 2-O-benzoyl-4,6-O-benzylidene-3-O-(2-methylnaphtyl)-β-D-glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-β-D-glucopyranoside 21: to a solution of 20 (500 mg, 0.383 mmol) in pyridine (7.6 mL) was added 1M hydrazine in pyridine/acetic acid (7.6 mL, 3:2, v/v). After stirring at RT for 3 h, 2,4-pentanedione (2.1 mL) was added and the solvent removed under reduced pressure. The crude was then diluted in CH₂Cl₂ (50 mL) and washed with 10% aq sol HCl, sat aq NaHCO₃, and brine. The organic layer was finally dried (MgSO₄), concentrated, and the desired compound 21 purified by chromatography eluting with [light petroleum/AcOEt (2:1, v/v)] to afford a white solid (377 mg, 82%). mp 136 °C. Rf (light petroleum/AcOEt, 2:1) 0.5. [α]₂₀^D +13.4 (c =1.0, CH₂Cl₂). ¹H RMN (CDCl₃, 400 MHz) δ (ppm): 7.94-7.90 (m, 4H, H arom), 7.68-7.66 (m, 1H, H arom), 7.58-7.19 (m, 32H, H arom), 5.54 (s, 1H, CHO₂Ph), 5.52 (s, 1H, CHO₂Ph), 4.95 (d, 1H, CH₂ NAP, J =12.4 Hz), 4.88 (d, 1H, C-1OCH₂Ph, J =11.6 Hz), 4,83 (d, 1H, CH₂ NAP), 4.77 (s, 1H, C-1OCH₂Ph), 4.53 (d, 1H, C-1OCH₂Ph) and Tables S1 and S2. ¹³C RMN (CDCl₃, 100 MHz) δ (ppm): 165.1, 164.7 (2C, OCOPh), 137.3, 137.2, 136.9, 135.3, 133.0, 132.8, 129.2 (8C, C quat arom), 133.4, 133.0, 129.8, 129.7, 129.3 (C arom), 129.2 (C quat arom), 129.0, 128.7, 128.5, 128.4, 128.3, 128.2, 128.0, 127.9, 127.8, 127.5, 126.7, 126.4, 126.1, 126.0, 125.7, 125.6 (C arom), 102.0 (CHO₂Ph), 101.2 (CHO₂Ph), 100.5 (CHO₂Ph), 73.8 (CH₂ NAP), 71.2 (CH₂ NAP) and Table S3. Elemental analysis: calcd for C₇₁H₆₆O₁₈: C =70.64%, H =5.51%; found C =70.49%, H =5.54%.

Benzyl 2-O-benzoyl-4,6-O-benzylidene-3-O-(2-methylnaphtyl)-β-D-glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-β-D-glucopyranos-2-uloside 22: compound 21 (330 mg, 0.273 mmol) was diluted in DMSO/acetic acid (9 mL, 2:1, v/v). After stirring for 48 h at RT, the media was added of CH₂Cl₂ (50 mL) and then washed with sat aq NaHCO₃, and water. After removal of the solvent and coevaopration with toluene, the crude was subjected to column chromatography [light petroleum/AcOEt (3:1, v/v)] to give the expected trisaccharide 22 (180 mg, 55%). Rf (light petroleum/AcOEt, 2:1) 0.3. ¹H RMN (CDCl₃, 400 MHz) δ (ppm): 7.95-7.91 (m, 4H, H arom), 7.67-7.60 (m, 1H, H arom), 7.57-7.10 (m, 32H, H arom), 5.53 (s, 1H, CHO₂Ph), 5.41 (s, 1H, CHO₂Ph), 5.40 (dd, 1H, H-2c, J_H1-H2 =7.8 Hz, J_H2-H3 =8.4 Hz), 5.32 (d, 1H, H-1c), 5.27 (dd, 1H, H-2b, J_H1-H2 =2.4 Hz, J_H2-H3 =1.6 Hz), 
5.19 (d, 1H, H-1b), 4.94 (d, 1H, CH₂ NAP, J =11.7 Hz), 4.82 (d, 1H, CH₂ NAP), 4.82 (d, 1H, C-1OCH₂Ph, J =12.0 Hz), 4.64 (s, 1H, H-1a), 4.61 (d, 1H, C-1OCH₂Ph), 4.42 (s, 1H, CHO₂Ph), 4.35 (d, 1H, H-3a, J_H3-H4 =10.4 Hz), 4.35 (t, 1H, H-6a, J_H5-H6 = J_H6-H6’ =5.5 Hz), 4.28 (dd, 1H, H-6c, J_H5-H6 =4.9 Hz, J_H6-H6’ =10.4 Hz), 4.24 (dd, 1H, H-4b, J_H3-H4 =7.5 Hz, J_H4-H5 =10.6 Hz), 4.11 (dd, 1H, H-6b, J_H5-H6 =4.8 Hz, J_H6-H6’ =10.2 Hz), 4.01 (dd, 1H, H-3b), 3.95 (t, 1H, H-3c, J_H3-H4 =8.4 Hz), 3.91 (t, 1H, H-4c, J_H4-H5 =8.4 Hz), 3.77 (t, 1H, H-6’c, J_H5-H6’ =10.4 Hz), 3.72 (ddd, 1H, H-5b, J_H5-H6’ =10.2 Hz), 3.63 (ddd, 1H, H-5c), 3.56 (t, 1H, H-6’a, J_H5-H6’ =5.5 Hz), 3.55 (dt, 1H, H-5a, J_H4-H5 =9.0 Hz), 3.41 (t, 1H, H-6’b), 2.98 (dd, 1H, H-4a). ¹³C RMN (CDCl₃, 100 MHz) δ (ppm): 165.2, 165.0 (OCOPh), 137.3, 137.2, 136.8, 135.9, 135.4 (C quat arom), 133.5, 133.3 (C arom), 133.0, 132.8 (C quat arom), 129.8, 129.7, 129.3, 129.2 (C arom), 129.0 (C quat arom), 129.0, 128.9, 128.7, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.5, 126.7, 126.4, 126.2, 126.1, 126.0, 125.7, 125.6 (C arom), 101.6 (CHO₂Ph), 101.2 (CHO₂Ph), 100.6 (CHO₂Ph), 98.6 (C-1a), 97.6 (C-1c), 96.1 (C-1b), 81.6 (C-4c), 79.9 (C-4a), 78.1 (C-3c), 77.4 (C-4b), 75.9 (C-3a), 75.8 (C-3b), 73.9 (CH₂ NAP), 73.1 (C-2c), 71.8 (C-2b), 70.3 (1OCH₂Ph), 68.9 (C-6b), 68.7 (C-6c), 68.3 (C-6a), 66.4 (C-5a), 66.2 (C-5c), 64.9 (C-5b). HRMS (ESI⁺): calcd m/z for [M+Na]⁺ C₇₁H₆₄NaO₁₈ 1,227.3990, found m/z 1,227.3988; calcd m/z for [M+Na+CH₃OH]⁺ C₇₂H₆₈NaO₁₉ 1,259.4252, found m/z 1,259.4245.

Benzyl 2-O-benzoyl-4,6-O-benzylidene-3-O-(2-methylnaphtyl)-β-D-glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-β-D-mannopyranoside 23: to a solution of uloside 22 (180 mg, 0.149 mmol) in CH₂Cl₂/THF (1:1, v/v) cooled at –78 °C was added L-Selectride (1 M in THF, 150 μL, 0.15 mmol). After stirring for 45 min, few drops of acetic acid were added to neutralize the media. At RT, the mixture was diluted CH₂Cl₂ (30 mL), washed with 10% aq HCl, sat aq NaHCO₃, and brine. The resulting layer was finally dried over MgSO₄ and concentrated under reduced pressure to afford the mannoside 23 as a white solid (180 mg, 100%). mp 180 °C. Rf (light petroleum/AcOEt, 2:1) 0.2. [α]₂₀^D –4.4 (c =1.0, CH₂Cl₂). ¹H RMN (CDCl₃, 400 MHz) δ (ppm): 7.85-7.83 (m, 2H, H arom), 7.73-7.67 (m, 3H, H arom), 7.62-7.17 (m, 32H, H arom), 5.50 (s, 1H, CHO₂Ph), 5.42 (s, 1H, CHO₂Ph), 5.21 (s, 1H, CHO₂Ph), 4.90 (d, 1H, CH₂ NAP, J =12.4 Hz), 4.87 (d, 1H, C-1OCH₂Ph, J =12.2 Hz), 4.80 (d, 1H, CH₂ NAP), 4.59 (d, 1H, C-1OCH₂Ph) and Tables S1 and S2. RMN ¹³C (CDCl₃, 100 MHz) δ (ppm): 164.8, 164.8 (OCOPh), 137.2, 137.1, 136.5, 135.2 (C quat arom), 133.2 (C arom), 132.9, 132.8 (C quat arom), 132.8, 129.7, 129.6 (C arom), 129.4, 129.1 (C quat arom), 129.0, 128.9, 128.7, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.8, 127.5, 126.7, 126.4, 126.3, 126.1, 126.0, 125.7, 125.6 (C arom), 101.5 (CHO₂Ph), 101.1 (CHO₂Ph). 73.5 (CH₂ NAP), 70.4 (C-1OCH₂Ph) and Table S3.

Benzyl 4,6-O-benzylidene-3-O-(2-methylnaphtyl)-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-β-D-mannopyranoside 24: to a solution of 23 (170 mg, 0.141 mmol) in CH₃OH/CH₂Cl₂ (2:1, v/v, 15 mL) was added sodium methylate in methanol (0.1 M, 3 mL, 0.3 mmol). After stirring for 6 h at RT, neutralization by Amberlite IR-120 (H⁺-form) afforded a suspension which was then filtered and concentrated. Purification by chromatography [CH₂Cl₂/C (99:1, v/v)] gave the desired mannoside 24 as a white solid (126 mg, 72%). mp >230 °C. Rf (CH₂Cl₂/MeOH, 97:3) 0.4. [α]₂₀^D –43.5 (c =1.0, CH₂Cl₂). ¹H RMN (CDCl₃, 400 MHz) δ (ppm): 7.70-7.19 (m, 27H, H arom), 5.47 (s, 1H, CHO₂Ph), 5.44 (s, 1H, CHO₂Ph), 5.42 (s, 1H, CHO₂Ph), 4.96 (d, 1H, CH₂ NAP, J =12.0 Hz), 4.88 (d, 1H, CH₂ NAP), 4.84 (d, 1H, C-1OCH₂Ph, J =11.9 Hz), 4.56 (d, 1H, C-1OCH₂Ph) and Tables S1 and S2. RMN ¹³C (CDCl₃, 100 MHz) δ (ppm): 137.1, 137.0, 136.9, 136.2, 135.8, 133.1, 132.8 (7C, C quat arom), 129.0, 128.9, 128.8, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.6, 127.5, 127.4, 126.8, 126.5, 126.2, 126.1, 126.0, 125.9, 125.8, 125.7 (C arom), 101.6 (CHO₂Ph), 101.2 (CHO₂Ph), 100.7 (CHO₂Ph), 74.4 (CH₂ NAP), 70.7 (C-1OCH₂Ph) and Table S3. HRMS (ESI⁺): calcd m/z for [M+Na]⁺ C₅₇H₅₈NaO₁₆ 1,021.3623, found m/z 1,021.3624; calcd m/z for [M+K]⁺ C₅₇H₅₈KO₁₆ 1,037.3362, found m/z 1,037.3370; calcd m/z for [M-H+2Na]⁺ C₅₇H₅₇Na₂O₁₆ 1,043.3442, found m/z 1,043.3463.

β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→3)-(α,β)-D-mannopyranose 4: compound 24 (90 mg, 0.090 mmol)was first dissolved in AcOEt/CH₃OH/CH₂Cl₂ (2:2:1, v/v/v, 10 mL) before adding palladium acetate (60 mg, 0.267 mmol). The suspension was vigorously stirred at RT under hydrogen atmosphere for 7 d, and then filtered over a pad of celite. The hydroorganic layer was extracted with CH₂Cl₂, concentrated, coevaoprated with ethanol, and subjected to size exclusion chromatography (Sephadex G-10, water) to give 4 in a quantitative yield as a white foam. ¹H RMN (D₂O, 400 MHz) δ (ppm): 5.13 (d, 1H, H-1aα, J_H1-H2 =1.8 Hz), 4.67 (d, 1H, H-1c, J_H1-H2 =7.7 Hz), 4.53 (d, 1H, H-1b, J_H1-H2 =8.0 Hz), 4.03 (dd, 1H, H-2a, J_H2-H3 =3.1 Hz), 3.96 (dd, 1H, H-3a, J_H3-H4 =9.5 Hz), 3.90-3.80 (m, 3H, H-6a, H-6b, H-6c), 3.80-3.73 (m, 1H, H-4a), 3.72-3.55 (m, 5H, H-5a, H-6’a, H-3b, H-6’b, H-6’c), 3.53-3.36 (m, 5H, H-2b, H-4b, H-5b, H-3c, H-5c), 3.32 (t, 1H, H-4c, J_H3-H4 = J_H4-H5 =9.7 Hz), 3.27 (dd, 1H, H-2c, J_H2-H3 =9.3 Hz). ¹³C RMN (D₂O, 100 MHz) δ (ppm): 103.1 (C-1c), 100.5 (C-1b), 94.1 (C-1aα), 84.5 (C-3b), 78.5 (C-3a), 76.3 (C-5c), 75.9 (C-3c, C-5b), 75.8 (C-2c), 73.1 (C-2b), 72.7 (C-4a), 69.9 (C-4c), 68.6 (C-2a), 68.4 (C-4b), 65.5 (C-5a), 61.2, 61.0 (C-6a, C-6b, C-6c). HRMS (ESI⁺): calcd m/z for [M+Na]⁺ C₁₈H₃₂NaO₁₆ 527.1588, found m/z 527.1587; calcd for [M+K]⁺ C₁₈H₃₂KO₁₆ 543.1327, found m/z 543.1343.

β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→3)-D-mannitol 2: to a solution of trisaccharide 4 (25 mg, 0.049 mmol) in CH₃OH/H₂O (4:1, v/v, 10 mL) was added the reducing agent sodium borohydride (19 mg, 0.502 mmol). After stirring at RT for 10 d, neutralization was performed with few drop of acetic acid. The resulting solution was concentrated under reduced pressure, coevaoprated successively with CH₃OH/ aceti acid, and CH₃OH, and finally freeze-dried. Size exclusion chromatography eluting with water (Sephadex G-10) gave 2 (23 mg) as a white foam. ¹H RMN (D₂O, 400 MHz) δ (ppm): 4.82 (d, 1H, H-1c, J_H1-H2 =8.0 Hz), 4.65 (d, 1H, H-1b, J_H1-H2 =8.1 Hz), 4.09-3.90 (m, 7H, H-1a, H-2a, H-3a, H-5a, H-6a, H-6b, H-6c), 3.86-3.66 (m, 6H, H-1’a, H-4a, H-6’a, H-3b, H-6’b, H-6’c), 3.64-3.38 (m, 7H, H-2b, H-4b, H-5b, H-2c, H-3c, H-4c, H-5c). ¹³C RMN (D₂O, 100 MHz) δ (ppm): 103.1 (C-1c), 102.5 (C-1b), 84.4 (C-3b), 77.3 (C-3a), 76.3 (C-5c), 75.8 (C-3c), 75.5 (C-5b), 73.7 (C-2c), 73.4 (C-2b), 70.9, 70.8 (C-2a, C-5a), 69.9 (C-4c), 69.6 (C-4a), 68.8 (C-4b), 63.4 (C-6a), 62.6 (C-1a), 61.2, 61.0 (C-6b, C-6c). HRMS (ESI⁺): calcd m/z for [M+Na]⁺ C₁₈H₃₄NaO₁₆: 529.1745, found 529.1743; calcd m/z for [M+K]⁺ C₁₈H₃₄KO₁₆: 545.1484, found 545.1480; calcd m/z for [M-H+2Na]⁺ C₁₈H₃₃Na₂O₁₆ 551.1564, found 551.1561.

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