An Approach to Synthesize Chondroitin Sulfate-E (CSâE

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An Approach to Synthesize Chondroitin Sulfate-E (CS–E) Oligosaccharides Precursors Shuang Yang, Qi Liu, Guangyan Zhang, Xiaoxi Zhang, Zhehui Zhao, and Pingsheng Lei J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b00157 • Publication Date (Web): 14 May 2018 Downloaded from http://pubs.acs.org on May 14, 2018

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The Journal of Organic Chemistry

An

Approach

to

Synthesize

Chondroitin

Sulfate-E

(CS–E)

Oligosaccharides Precursors Shuang Yang, Qi Liu, Guangyan Zhang, Xiaoxi Zhang, Zhehui Zhao∗, Pingsheng Lei∗ State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, 100050, P. R. China *E-mail: [email protected]

ABSTRACT: An approach was developed to synthesize chondroitin sulfate-E (CS–E) oligosaccharides by adopting a postglycosylation-transformation strategy: different from all the traditional approaches, the characteristic groups of CS-E were introduced following the assembly of the oligosaccharides. The adjusted strategy rendered an easy chain elongation strategy. All the elongation steps generated high yields with excellent glycosylation outcomes. An orthogonally protected disaccharide was used as the building block to provide flexibility for the group transformation and derivatization at the N-2 position of the GalNAc residue and the O-1,5 positions of the GlcA residue, thereby providing ready access for the further examination of the structure-activity relationship (SAR) of CS-E molecules.

KEY WORDS: Chondroitin sulfate E, postglycosylation-transformation strategy, oligosaccharides, glycosylation, modification

INTRODUCTION

Chondroitin sulfate (CS) is a class of sulfated glycosaminoglycans made of dimeric units

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GlcA-β(1→3)-GalNAc-β(1→4). Variable sulfation gives a collection of CS subtypes, among which CS-E is characterized with sulfate motifs located at the 4 and 6 positions of the GalNAc residue. CS-E has been found to play crucial roles in many biological events, such as the development of the central nervous system, viral attachment, and growth factor signaling.1-5 CS-E has a wide range of biological activities, thereby making it an attractive target for synthetic organic chemists for the development of drug candidates. However it is not easy to obtain significant amounts of structurally defined CS-E oligosaccharides from natural sources, due to the low natural content of CS-E and the heterogeneity of the CS polymer. Without a precise molecular structure, accurate pharmacological results are difficult to achieve. In addition, the structure-activity relationship of CS-E has barely been explored systematically, and the mechanisms underlying its biofunction have remained largely elusive. To address these problems, well-defined CS-E oligomers and analogues are required. Although several total syntheses of CS-E oligosaccharides have been developed,6-17 new and efficient approaches available for diverse derivatization are still required. The present study described such an approach, to provide easy access to CS-E tetrasaccharide and hexasaccharide as well as their derivatives.

Figure 1. Previously synthesized CS-E oligosaccharides.

CS-E oligosaccharides have been mainly synthesized by four groups who have adopted different glycosyl sequences (Figure 1). Both sequences have exhibited attractive bioactivities, though the superiority of a specific sequence has not been examined. All the synthetic routes were derived from


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the construction of a suitable repeating disaccharide unit, after which the repeating unit was assembled by stepwise glycosylation. As reported, the yields of these glycosylations were usually raised by increasing the consumption of disaccharide donors, thereby there is still room to improve the route economy in CS-E oligosaccharides synthesis. Very potent donors are necessary to efficiently utilize the disaccharide repeating unit in such syntheses. We previously investigated the construction of CS-E disaccharide repeating unit 1 by the postglycosylation-oxidation strategy which exhibited excellent performance in contrast with the traditional preglycosylation-oxidation strategy, on both the glycosylation efficiency and stereoselectivity, this is likely because it benefited from the high reactivity of the glucosyl monosaccharide donors as compared to the widely used uronic acid donors (Scheme 1).18 With this acquired knowledge, we attempted to employ a new disaccharide repeating unit containing glucosyl residue rather than uronic acid residue at the reducing terminal, and apply the postglycosylation-oxidation strategy at the oligosaccharide stage, to improve the glycosylation efficiency in the synthesis of CS-E oligomers (Scheme 1).

Scheme 1. Retrosynthetic Analysis of the CS-E Oligosaccharides



To target a facile approach to derivatization, the repeating unit must be equipped with protecting groups that can be chemically selectively removed or modified. For this purpose, an orthogonal protective strategy was designed for the disaccharide unit (Scheme 1). Particularly, azido was selected as the masked amino group in the GalNAc unit. In spite of its non-participatory property, good β-selectivity can also be observed in the glycosylation of 2-azido donor under proper reaction conditions.19-23 In addition, smooth glycosylations can also be expected due to the absence of disturbance of the N-protecting group.7-8 Guided by these ideas, the synthetic route for CS-E oligosaccharides was arranged in the postglycosylation-transformation sequence (Scheme 1). In this approach, the oligosaccharide backbone was assembled with orthogonally protected disaccharide repeating unit 2, after which introduction of two characteristic CS-E groups, namely a carboxyl moiety and an acetamide group, took place at the oligosaccharide stage.

RESULTS AND DISCUSSION

Synthesis of the building blocks. Monomers 7 and 8 were prepared individually as glycosyldonor and acceptor , respectively, to give CS-E disaccharide repeating unit by the coupling reaction (Scheme 2). The synthesis of glycosyl acceptor 8 began with D-glucose monohydrate through a straightforward six-step sequence that generated a total yield of 36%.18 The synthesis of glycosyl donor 7 began from the previously described peracetylated derivative 3 of commercially available D-galactosamine hydrochloride.18 3 was reacted with p-methylbenzothiol and BF3·Et2O to afford thioglycoside 4a,b as a pair of isomers with a yield of 75%. 6a,b was prepared from a mixture of 4a,b via de-O-acetylation, 4,6-O-benzylidenation and 3-O-levulination, which produced a yield of 87% over the three steps. The anomeric thioether groups of 6a,b were then removed by the NIS/trifluoroacetic acid, and a


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corresponding hydrolysis product was produced at a yield of 76%. Subsequently the hemiacetal was converted to trichloroacetimidate 7 following the treatment of trichloroacetonitrile and a catalytic amount of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), which produced a yield of 86%.

Scheme 2. Synthesis of D-GalN-Based Donor 7 and ᴅ-Glc-Based Acceptor 8.

Reagents and conditions: a. p-Toluenethiol, BF3·Et2O, DCM, 40 °C, overnight, 75%; b. NaOMe, MeOH, r.t., 30 min; c. benzaldehyde dimethyl acetal, cat. PTSA, DMF, 40 °C, reduce pressure, 2 h; d. Levulinic acid, EDCI, DMAP, DCM, r.t., 2 h, 87% for three steps; e. NIS, TFA, DCM/H2O, 3 h, 0 °C, 76%; f. Cl3CCN, DBU, DCM, 0 °C, 4 h, 86%.

Disaccharide unit preparation. Although donor 7 was failed to be glycosylated in acetonitrile after several attempts, the BF3·Et2O/toluene system was successfully applied to the condensation of donor 7 and acceptor 8 (Table 1), exclusively affording disaccharide 2 in the desired β-configuration. After optimization, the disaccharide product was quantitatively obtained with an excess (1.8 equiv.) of imidate at -60 °C for 30 min. It should be noted that, when treated with 0.4 equiv. of BF3•Et2O (entry 1), the donor 7 was decomposed rapidly to its hydrolysis product judged by TLC. Acceptor 8 was recovered and no disaccharide derivatives were detected. This may be attributed to the low stability of donor 7 in acid environment. Disaccharide 2 was easily separated from the hydrolysis product of



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imidate 7 by recrystallization, whereas the latter was subjected to the recycle of donor 7. The β-configuration of glycoside 2 was established from 1H and

13

C NMR spectrum with signals for the

1-β-anomeric proton at δ 4.31 ppm (d, 1H, J = 8.0 Hz, H2-1) and carbon at δ 100.78 ppm.

Table 1. Synthesis of Disaccharide Repeating Unit 2

Reagents and conditions: Toluene, -60 °C, 30 min.

entry

BF3·Et2O

donor

yield

entry

BF3·Et2O

donor

yield

1

0.4 eq.

2.0 eq.

0

3

0.15 eq.

1.5 eq.

52%

2

0.2 eq.

2.0 eq.

97%

4

0.18 eq.

1.8 eq.

96%

Elongation of the repeating unit. With the well protected disaccharide repeating unit in hand, we then proceeded to prepare the tetrasaccharide and hexasaccharide by elongation. Disaccharide donor 9 was smoothly prepared from 2 by the removal of the 4-methylphenylthiol group following trichloroacetimidate activation, with 87% yield over two steps (Scheme 3).

Scheme 3. Synthesis of Disaccharide Donor 9

Reagents and conditions: a. NIS, TFA, DCM/H2O, r.t., 1 h; b. Cl3CCN, DBU, DCM, r.t., 30 min, 87% for two steps.

Given that the disaccharide acceptor was the beginning unit in the assembly of the CS-E


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oligosaccharides, the derivatization at the C-1 position was preliminarily executed at the disaccharide stage. Both thioglycoside 2 and imidate 9 were employed as donors to couple with methanol, 3-(trimethylsilyl)propargyl alcohol and 4-methoxyphenol, respectively (Table 2). Results indicated that thioglycoside 2 was not active enough for glycosylation under the agency of NIS-TfOH. Conversely, imidate 9 was coupled with the three alcohols under the catalysis of trimethylsilyl triflate, which exclusively provided the 1,2-trans-linked glycosides 10, 11 and 12 with yields of 57%, 87% and 91%, respectively. Notably, the flexibility to functionalize the reducing end, anchored the CS-E molecule into useful biological and chemical tools, such as fluorophore for bioassays10,

28-30

and alkyne for the

synthesis of the non-natural CS-E polymerized species31-33. Here in this study, the 4-methoxyphenol was chosed as aglycone to undergo the following work.

Table 2. Preliminary Derivatization of the Reducing Terminal Protecting Group

entry

donor

1-3

2

aglycon

reaction condition

product / yeild

NIS/TfOH, 0 °C-r.t., overnight

Not work

methanol

TMSOTf/DCM, r.t., 52 h

10 / 57%

3-(Trimethylsilyl)propargyl alcohol

TMSOTf/DCM, 0 °C, overnight

11 / 87%

4-methoxyphenol

TMSOTf/DCM, -60 °C, 30 min

12 / 91%

methanol / 4-methoxyphenol / 3-(Trimethylsilyl)propargyl alcohol 4

5

6

9

The CS-E tetrasaccharide and hexasaccharide precursors were synthesized based on the above



derivatization, which characterized the scope of glycosylation of donor 9 (Scheme 4). The levulinoyl group of 12 was selectively removed by hydrazine acetate in dichloromethane, affording disaccharide acceptor 13 in 89% yield. The coupling reaction of acceptor 13 with a slight excess (1.2 equiv.) of disaccharide donor 9 under the catalysis of TMSOTf produced tetrasaccharide derivative 14 in 95% yield. Through a similar process, hexasaccharide 16 was afforded in 88% yield. All the described glycosylation reactions with imidate 9 went rapidly and efficiently, and no 1,2-cis-linked species or orthoesters were observed.

Scheme 4. Synthesis of Tetrasaccharide 14 and Hexasaccharide 16 from Disaccharide 9.

Reagents and conditions: a. p-Methoxyphenol, TMSOTf, DCM, -60 °C, 30 min, 91%; b. N2H4-H2O/Py/AcOH, r.t., 40 min, 89 %; c. TMSOTf, DCM, -60 °C, 30 min, 95%; d. N2H4-H2O/Py/AcOH, r.t., 40 min, 93%; e. TMSOTf, DCM, -60 °C, 30 min, 88%.

The herein presented results evidently indicated that disaccharide imidate 9 was a very attractive donor with a high reactivity and nearly all the corresponding glycosylations went rapidly and efficiently.


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Only a slight excess of donor was required to afford expected products in good yields, which significantly raised the economic efficiency of the CS-E oligomers’ syntheses. The improvement was attributed to two aspects: first, the elimination of deactivating effect caused by carboxylic acid moiety at C-5 of the Glc residue, and secondly, the adoption of inert azido group at C-2 of the Gal residue instead of the commonly-used acyl protecting groups which may nucleophilically attack the donor to form an unfavorable intermediate.7-8 Functional groups transformation. Having achieved intermediates 14 and 16 in hand, we then identified a series of functional group modifications to obtain the target oligomers. The reaction sequence was as follows.

Scheme 5. Application of the Postglycosylation-Oxidation Strategy on the Oligosaccharide Level

Reagents and conditions: a. DDQ, DCM/H2O, r.t., overnight, for 17; r.t., 5 h, for 19. b. TEMPO, BAIB, TBAB, CH3CN, H2O, r.t., 4 h; c. CH3I, K2CO3, DMF, r.t., 3 h, 81% for 18 (two steps), r.t., 30 min, 46% for 20 (three steps).

Tetrasaccharide 14 was transformed to 17 through the oxidative removal of p-methoxybenzyl (PMB) with 2,3-di-chloro-5,6-dicyanobenzoquinone (DDQ) in wet dichloromethane. The subsequent oxidation of the primary alcohol in 17 with (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) and (diacetoxyiodo)-benzene (BAIB), which was followed by methyl esterification, afforded C-5



carboxylic acid ester derivative 18 in 81% yield over three steps. The same sequence of reactions was repeated starting from 16 and the corresponding hexasaccharide derivative 20 was obtained, with a yield of 46% over three steps (Scheme 5). The oxidation of the primary alcohol was initially performed in wet dichloromethane as previously described for preparation of disaccharide 1.18 However, under this reaction condition, the oxidation of tetrasaccharide 17 was executed over a very long time and required a large excess of the oxidizing reagents. Otherwise, the reaction was incomplete. In the case of hexasaccharide 19, the attempt left the oxidation a list with a bar code of spots on the TLC plate. This failure was attributed to the inadequate contact between the reagents and substance in two-phase system. Therefore, acetonitrile was used instead of dichloromethane, and tetrabutylammonium bromide (TBAB) was added to make the solvent system homogeneous. As expected, the situation was greatly improved both for oligosaccharides 18 and 20 which were smoothly oxidated with good yields. To the best of our knowledge, this is the first application of the postglycosylation-oxidation strategy34 in the synthesis of CS-E oligosaccharides. The efficiency of this approach was examined. In the application of the former strategy, the glycosylations greatly benefited from the high reactivity of the non-oxidized glucosyl donor, thus making the elongation easily achievable. The total yield of the glycosylation-oxidation reactions was 77% (95% for glycosylation, 81% for oxidation) for tetrasaccharide 18 and 38% (95%, 88% for glycosylation, 46% for oxidation) for hexasaccharide 20. Therefore the postglycosylation-oxidation strategy was believed to be favorable for the synthesis of CS-E oligosaccharide. Nevertheless, the application of the reported strategy was also limited by the length of the CS-E oligomers, which was also observed in previously published synthetic approaches. Subsequently, the conversion of the azido group to the acetamide group was established at the


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oligosaccharide level (Table 3). Suitable methods were initially explored. Disaccharide 12 and tetrasaccharide 14 were subjected to four chemoselective reduction methods for the azido groups. Lindlar-catalyzed hydrogenation appeared ineffective, and reduction under a harsher condition was taken to be detrimental to 4,6-O-benzylidene acetal. With the treatment of triphenylphosphine (Ph3P) in wet tetrahydrofuran, the resulting mixture was difficult to purify. Two sulfide reagents, 1,3-propanedithiol and thioacetic acid, were then examined for azide-reduction. Under the agency of 1,3-propanedithiol in a mixture of Et3N/pyridine, the azido was reduced to free amino group and acylated with acetic anhydride to generate the corresponding acetamide 21 and 22 with yields of 86% and 59%, respectively. This two step-wise approach allowed further N-modification. The chemoselective transformation of the azides into acetamides was also fulfilled by a one-pot reduction-acetylation sequence using thioacetic acid in pyridine. The two available methods were then applied to oxidized derivatives 18 and 20. The complete conversion of these two oligosaccharides required a longer time and higher temperature, of which the results produced lower yields. Acetamide-type tetrasaccharide 23 was afforded with the employment of thioacetic acid and 1,3-propanedithiol in similar yields of 65% and 61%, respectively. Hexasaccharide 20 was directly transformed into the corresponding acetamide 24 in the presence of thioacetic acid with moderate yield of 52% (Table 3). The findings obtained above suggest that the reduction of azide tended to produce markedly lower yield when the number of azido groups increased in the molecule, which was in accordance with the results in literature7-8. Nevertheless, the reduction efficiency of hexasaccharide 20 remained acceptable. This successful conversion demonstrated that azido can serve as a good alternative for the protection of 2-amino in the syntheses of CS-E oligosaccharides, thereby providing an access to their N-derivatives.



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Table 3. Conversion of Azides to Acetamides

Reagents and conditions: a. AcSH/ Py. b. 1,3-Dithiolpropane/Et3N/Py; Ac2O, DCM, r.t., 1-3 h. entry

substance

method

reaction condition

product

yield

1

12

a

r.t., 2 h

21

89%

2

12

b

r.t., 30 min

21

86%

3

14

a

r.t., 3 h

22

76%

4

14

b

r.t., 4.5 h

22

59%

5

18

a

60 °C, 24 h

23

65%

6

18

b

r.t., 11 h

23

61%

7

20

a

60 °C, 24 h

24

52%

At this point, fully protected CS-E tetrasaccharide 23 and hexasaccharide 24 were nearly the same to their analogues reported by Tamura’s group8, and the only tiny difference of these intermediates is the distinction between benzoyl (Bz) substitutes in our compounds and methoxybenzoyl (MBz) substitutes in the reported counterparts. For this reason, we believe that the intermediates 23 and 24 can be converted to the final CS-E products based on the sulfation and de-protection procedure reported in previous literature.

CONCLUSION

In summary, starting from the known monosaccharide donor 7 and acceptor 8, we produced the CS-E tetrasaccharide and hexasaccharide precursors within 10 and 12 steps with total yield of 34% and 13%, respectively. The feature of our synthesis include the application of postoxidation-glycosylation


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strategy on CS-E oligosaccharides and a late-stage functional groups transformation. Most notably, our approach allowed the synthesis of CS-E analogues to expand research territory of this important pharmacological oligosaccharide, that is currently underway in our group, of which relevant research will be reported elsewhere in due course. The developed methodology can also be applied to other chondroitin oligomers and similar members of the glycosaminoglycan family.

EXPERIMENT SECTION

General Methods. The reactions were performed with commercial reagents and solvents, and all the chemicals were usd as received otherwise stated. Solvents were removed under reduced pressure. Traces of water in the starting materials were removed by coevaporation with toluene. Powdered molecular sieves (4 Å) used for reactions were activated by heating at 400 °C for 4 h prior to the application. All NMR spectra were recorded on a JEOL ECZ-400S (400 MHz, Japan), a Bruker AVANCE-III 400 (400 MHz, Switzerland), a WNMR-I 500 (500 MHz, China), a Bruker AVANCE-III HD 600 (600 MHz, Switzerland) spectrometers in CDCl3. High-resolution mass spectra (HRMS) were measured with a ThermoFisher Exactive Plus mass spectrometer (ThermoFisher Scientific, Bremen, Germany) equipped with a ThermoFisher Accela HPLC system (ThermoFisher Scientific, Bremen, Germany). Reactions were monitored by thin layer chromatography (TLC) (HSGF254, Branch of Qingdao Haiyang Chemical Plant, China). For column chromatography silica gel was used (HG/T2354-92, Branch of Qingdao Haiyang Chemical Plant, China). p-Methylphenyl 3,4,6-Tri-O-acetyl-2-azido-2-deoxy-1-thio-α/β-ᴅ-galactopyranoside (4a,b). 318 (49.6 g, 132.93 mmol) and p-Toluenethiol (24.8 g, 199.41 mmol) were dissolved in dry CH2Cl2 (208 mL), and BF3.Et2O (67.5 mL, 531.76 mmol) was then added dropwise at 0 °C. After being stirred for 30 min at 0°C, the system was warmed to room temperature and heated to reflux overnight. The



reaction was quenched with sat. NaHCO3 aq., and extracted with CH2Cl2 (3 ×). The organic phase was washed with brine, dried over anhydrous Na2SO4, and concentrated. Silica gel column chromatography of the residue (EtOAc/petroleum ether, 1:6) gave a mixture of 4a,b (45.2 g, 75%, a:b = 1:1) as a yellow syrup. 1H NMR (400 MHz, CDCl3, 4a, α): δ 7.41 (d, J = 8.0 Hz, 2H), 7.13 (d, J = 9.2 Hz, 2H), 5.61 (d, J = 5.6 Hz, 1H, H-1), 5.48 (d, J = 2.4 Hz, 1H, H-4), 5.18 (dd, J = 11.2, 2.8 Hz, 1H, H-3), 4.77 (t, J = 6.4 Hz, 1H, H-5), 4.30 (dd, J = 11.2, 5.6 Hz, 1H, H-2), 4.15 (d, J = 6.8 Hz, 1H, H-6a), 4.12 (d, J = 6.8 Hz, 1H, H-6b), 2.34 (s, 3H, CH3-STol), 2.15 (s, 3H, CH3-OAc), 2.06 (s, 3H, CH3-OAc), 2.01 (s, 3H, CH3-OAc); 1H NMR (400 MHz, CDCl3, 4b, β): δ 7.51 (d, J = 8.0 Hz, 2H), 7.16 (d, J = 8.8 Hz, 2H), 5.34 (d, J = 2.8 Hz, 1H, H-4), 4.86 (dd, J = 10.2, 2.8 Hz, 1H, H-3), 4.46 (d, J = 10.0 Hz, 1H, H-1), 4.09-4.07 (d, 2H, J = 6.4 Hz, H-6a,b), 3.87 (t, J = 6.4 Hz, 1H, H1-5), 3.63 (t, J = 10.0 Hz, 1H, H-2), 2.36 (s, 3H, CH3-STol), 2.09 (s, 3H, CH3-OAc), 2.04 (s, 3H, CH3-OAc), 2.03 (s, 3H, CH3-OAc); 13C NMR (100 MHz, CDCl3, α/β, 1:1.2): δ 170.5, 170.4, 170.1, 170.0, 169.8, 169.7, 139.0, 138.5, 134.1, 133.2, 130.0, 129.9, 128.7, 127.3, 87.4 (α, C-1), 86.8 (β, C-1), 74.4, 73.1, 70.2, 67.6, 67.5, 66.6, 61.8, 61.7, 59.4, 58.2, 21.3, 21.3, 20.8, 20.7, 20.7, 20.7. HRMS (ESI-TOF) m/z [M + Na]+ Calcd for C19H23N3NaO7S 460.1154, found 460.1108. p-Methylphenyl 2-azido-4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-1-thio-α/β-ᴅ-galactopyranosid e (6a,b). MeONa (2.96 g，54.78 mmol) was added into a solution of 4a,b (45.19 g, 103.37 mmol) in CH2Cl2 (104 mL) and MeOH (234 mL). The mixture was stirred for 30 min and DOWEX 50X8-200 was added. After stirred for 1 h, the mixture was filtered and concentrated to afford the deacetylated intermediate as a yellow solid. The crude product was dissolved in DMF (103 mL), then p-anisaldehyde dimethyl acetal (46.5 mL, 310.11 mmol) and p-toluene sulfonic acid monohydrate (4.72 g, 24.81 mmol) were added whereafter the system was stirred at 40 °C for 2 h at reduced pressure.


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Once complete, the reaction mixture was quenched with Et3N, diluted with EtOAc, washed with brine, dried over anhydrous Na2SO4, and concentrated to afford 5a,b as a yellow syrup. This compound was used for the next step without further purification. 1H NMR (400 MHz, CDCl3, 5a, α): δ 7.51-7.48 (m, 2H, ArH-STol), 7.40-7.36 (m, 5H, ArH-benzylidene), 7.11 (d, J = 8.0 Hz, 2H, ArH-STol), 5.67 (d, J = 5.2 Hz, 1H, H-1), 5.59 (s, 1H, CH-benzylidene), 4.31 (dd, J = 3.6, 0.8 Hz, 1H, H-4), 4.26-4.21 (m, 2H, H-2, H-5), 4.17 (dd, J = 12.4, 5.2 Hz, 1H, H-6a), 4.11 (dd, J = 12.4, 1.6 Hz, 1H, H-6b), 4.00 (td, J = 10.4, 3.6 Hz, 1H, H-3), 2.69 (d, J = 10.0 Hz, 1H, OH), 2.33 (s, 3H, CH3-STol); 13C NMR (100 MHz, CDCl3, 5a, α) δ 137.8, 137.3, 131.8, 130.0, 129.9, 129.6, 128.5, 126.4, 101.5 (CH-benzylidene), 87.8 (C-1), 75.3, 69.7, 69.3, 63.7, 61.5 (C-2), 21.2 (CH3-STol). 1H NMR (400 MHz, CDCl3, 5b, β): δ 7.62 (d, J = 8.4 Hz, 2H, ArH-STol), 7.43-7.36 (m, 5H, ArH-benzylidene), 7.11 (d, J = 8.0 Hz, 2H, ArH-STol), 5.51 (s, 1H, CH-benzylidene), 4.38 (dd, J = 12.4, 1.2 Hz, 1H, H-6a), 4.36 (d, J = 9.6 Hz, 1H, H-1), 4.16 (d, J = 3.6 Hz, 1H, H-4), 4.02 (dd, J = 12.4, 1.6 Hz, 1H, H-6b), 3.62 (td, J = 9.6, 3.6 Hz, 1H, H-3), 3.49 (d, J = 0.8 Hz, 1H, H-5), 3.48 (t, J = 9.6 Hz, 1H, H-2), 2.51 (d, 1H, J = 9.6 Hz, OH), 2.36 (s, 3H, CH3-STol); 13C NMR (100 MHz, CDCl3, 5b, β): δ 138.9, 137.5, 134.9, 129.9, 129.6, 128.4, 126.7, 126.4, 101.5 (CH-benzylidene), 85.1 (C-1), 74.6, 73.3, 69.9, 69.4, 62.1 (C-2), 21.4 (CH3-STol). HRMS (ESI-TOF) m/z [M + Na]+. Calcd for C20H21N3NaO4S 422.1150, found 422.1138. 5a,b was added to a solution of EDCI.HCl (49.5 g，258.4 mmol), DMAP (3.79 g，31.01 mmol), and levulinic acid (30.0 g，258.4 mmol) in dry CH2Cl2, and the resulting mixture was stirred for 2 h at room temperature. After that, the reaction mixture was poured into cold water, and extracted with CH2Cl2 (3×). The organic phase was washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. Silica gel column chromatography (EtOAc/petroleum ether, 1:6→1:2) gave 6a,b (44.3 g, 87% for three steps) as a yellow syrup. 1H NMR (400 MHz, CDCl3, 6b, β): δ 7.61 (d, J = 8.0 Hz, 2H,



ArH-STol), 7.43-7.36 (m, 5H, ArH-benzylidene), 7.06 (d, J = 8.4 Hz, 2H, ArH-STol), 5.47 (s, 1H, CH-benzylidene), 4.83 (dd, J = 10.4, 3.2 Hz, 1H, H-3), 4.45 (d, J = 9.6 Hz, 1H, H-1), 4.34 (dd, J = 12.8, 1.6 Hz, 1H, H-6a), 4.27 (dd, J = 3.2 Hz, 1H, H-4), 3.62 (dd, J = 12.4, 1.6 Hz, 1H, H-6b), 3.79 (t, J = 10.0 Hz, 1H, H-2), 3.51 (s, 1H, H-5), 2.72-2.69 (m, 2H, CH2-Lev), 2.60-2.55 (m, 2H, CH2-Lev), 2.33 (s, 3H, CH3-STol), 2.06 (s, 3H, CH3-Lev); 13C NMR (100 MHz, CDCl3, 6b, β): δ 206.3 (CO-Lev), 172.0 (CO-Lev), 138.7, 137.7, 134.6, 129.8, 129.2, 128.1, 126.5, 126.2, 100.9, 85.2 (C-1), 73.9, 72.6, 69.5, 69.1, 58.3 (C-2), 37.8 (CH2-Lev), 29.6 (CH3-Lev), 28.1 (CH2-Lev), 21.3 (CH3-STol). HRMS (ESI-TOF) m/z [M+Na]+ Calcd. for C25H27N3NaO6S 520.1518, found 520.1500. 2-azido-4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-1-O-trichloroacetimidoyl-α-ᴅ-galactopyranosid e (7). To a solution of 6a,b (4.87 g, 9.80 mmol) in CH2Cl2 (50 mL) and water (3.5 mL) was added N-iodosuccinimide (2.64 g, 11.75 mmol). Then trifluoroacetic acid (365 µL, 4.89 mmol) was added dropwise to the mixture at 0 °C, and the reaction was stirred for 3 h at 0 °C. After that, 5 M Na2S2O3 solution was added and the mixture was extracted with CH2Cl2 (3×), washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. Column chromatography (EtOAc/petroleum ether, 1:5 → 2:5) gave the corresponding hemiacetal (2.91 g, 76%) as a yellow syrup and the starting material 6a (271 mg) was recovered. 1H NMR (400 MHz, CDCl3, α/β, 1:0.3): δ 7.52-7.50 (m, 2H), 7.40-7.36 (m, 3H), 5.54 (s, 1H, CH-benzylidene), 5.47 (d, J = 3.2 Hz, Ha-1), 5.38 (dd, J = 10.8, 3.2 Hz, 1H, Ha-3), 4.77 (dd, J = 10.8, 3.2 Hz, 1H, Hb-3), 4.63 (d, J = 8.0 Hz, 1H, Hb-1), 4.43 (d, J = 3.2 Hz, 1H, Ha-4), 4.32-4.28 (m, 2H, Hb-4, Hb-6a), 4.22 (dd, J = 12.4, 1.2 Hz, 1H, Ha-6a), 4.07-3.98 (m, 4H, Ha-2, Ha-5, Ha-6b, Hb-6b), 3.87 (dd, J = 11.8, 8.0 Hz, 1H, Hb-2), 3.45 (s, 1H, Hb-5), 2.79-2.65 (m, 4H, CH2-Lev), 2.12 (s, 3H, CH3-Lev); 13C NMR (100 MHz, CDCl3): δ 206.5 (CO-Lev), 172.3 (CO-Lev), 137.7, 129.2, 128.33, 128.32, 126.34, 126.30, 100.9 (CH-benzylidene), 96.5 (Cb-1), 92.9 (Ca-1), 73.6, 72.7, 72.4,


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69.7, 69.1, 66.7, 62.6 (C-2), 62.1, 58.1 (C-2), 38.0 (CH2-Lev), 29.8 (CH3-Lev), 28.3 (CH2-Lev). HRMS (ESI-TOF) m/z [M + Na]+ Calcd for C18H21N3NaO7 414.1277, found 414.1256. A mixture of the hemiacetal (20.15 g, 51.53 mmol), Cl3CCN (31 mL, 309.15 mmol), and DBU (3.08 mL, 20.61 mmol) in anhyd CH2Cl2 (504 mL) was stirred at 0 °C for 4 h. Then the resulting mixture was concentrated. Column chromatography (EtOAc/PE, 1:15 + 0.5% Et3N) gave imidate 7 (23.56 g, 86%) as a white solid. 1H NMR (400 MHz, CDCl3): δ 8.77 (s, 1H, NH), 7.53-7.51 (m, 2H, ArH-benzylidene), 7.39-7.37 (m, 3H, ArH-benzylidene), 5.58 (d, J = 3.2 Hz, 1H, H-1), 5.56 (s, 1H, CH-benzylidene), 5.37 (dd, J = 10.8, 3.6 Hz, 1H, H-3), 4.55 (dd, J = 3.2 Hz, 1H, H-4), 4.32-4.28 (m, 2H, H-6a,b), 4.04 (dd, J = 12.8, 2.8 Hz, 1H, H-2), 3.98 (s, 3H, H-5), 2.80-2.66 (m, 4H, CH2-Lev), 2.12 (s, 3H, CH3-Lev); 13C NMR (100 MHz, CDCl3): δ 206.4, 172.2, 160.7 (C=NH), 137.4, 129.3, 128.3, 126.3, 100.9 (CH-benzylidene), 95.4 (C-1), 90.9 (CCl3), 72.9, 69.9, 68.8, 64.9, 57.0 (C-2), 37.9 (CH2-Lev), 29.8 (CH3-Lev), 28.2 (CH2-Lev). p-Methoxyphenyl

O-(2-Azido-4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-β-ᴅ-galactopyranosyl)

-(1→4)-2,3-di-O-benzoyl-6-O-p-methoxybenzyl-1-thio-β-ᴅ-glucopyranoside (2). Donor 7 (978.8 mg, 1.83 mmol) and acceptor 8 (625.2 mg, 1.02 mmol) were coevaporated with dry toluene (3 × 35 mL), dried in vacuo for 30 min and dissolved in anhydrous toluene (34 mL) in the presence of 4Å powdered molecular sieves. After stirring for 30 min at room temperature, a solution of BF3·Et2O in CH2Cl2 (0.76 M, 242 µL, 0.18 mmol) was added at -60 °C. The reaction was quenched 30 min later by addition of Et3N, filtered through a Celite pad and concentrated to dryness. The residue was roughly purified by column chromatography on silica gel (EtOAc/petroleum ether, 1:2)

and then was subjected to a

recrystallization (EtOAc/petroleum ether, 1:5) to afford 2 (963.1 mg, 96%) as white solid. 1H NMR (400 MHz, CDCl3): δ 7.95-7.93 (m, 2H, ArH-Bz), 7.91-7.89 (m, 2H, ArH-Bz), 7.50 (t, J = 7.4 Hz, 1H,



ArH-Bz), 7.40-7.16 (m, 14H, ArH-Bz, PMB, STol, benzylidene), 7.06 (d, J = 8.0 Hz, 1H, ArH-STol), 6.92 (d, J = 8.6 Hz, 1H, ArH-PMB), 5.69 (t, J = 9.2 Hz, 1H, H1-3), 5.35 (t, J = 9.6 Hz, 1H, H1-2), 5.21 (s, 1H, CH-benzylidene), 4.85 (d, J = 10.0 Hz, 1H，H1-1), 4.63 (d, J = 11.5 Hz, 1H, CH2-PMB), 4.54 (dd, J = 10.8, 3.6 Hz, 1H, H2-3), 4.51 (d, J = 11.6 Hz, 1H, CH2-PMB), 4.31 (d, J = 8.0 Hz, 1H, H2-1), 4.21 (t, J = 9.6 Hz, 1H, H1-4), 4.03 (d, J = 3.4 Hz, 1H, H2-4), 3.97 (dd, J = 11.2, 3.6 Hz, 1H, H1-6a), 3.91 (d, J = 10.4 Hz, 1H, H1-6b), 3.82 (s, 3H, OCH3-PMB), 3.76-3.71 (m, 2H, H2-2, H1-5), 3.56 (s, 2H, H2-6a,b), 2.90 (s, 1H, H2-5), 2.71-2.68 (m, 2H, CH2-Lev), 2.60-2.57 (m, 2H, CH2-Lev), 2.32 (s, 3H, CH3-Lev), 2.05 (s, 3H, CH3- STol); 13C NMR (100 MHz, CDCl3): δ 206.2 (CO-Lev), 172.1 (CO-Lev), 165.7 (CO-Bz), 165.3 (CO-Bz), 159.4, 138.5, 137.7, 133.7 (2C), 133.2, 132.8, 130.4, 130.0, 129.9, 129.8, 129.8, 129.7, 129.6, 129.0, 128.4, 128.4, 128.1, 128.0, 126.6, 113.9 (2C), 101.4 (PhCH), 100.8 (C2-1), 86.4 (C1-1), 79.3, 75.4, 74.6, 73.2, 72.7, 72.3, 71.0, 68.1, 68.0, 66.2, 60.6, 55.4 (OCH3-PMB), 37.9 (CH2-Lev), 29.7 (CH3-Lev), 28.2 (CH2-Lev), 21.3 (CH3-SPhMe). HRMS (ESI-TOF) m/z [M + Na]+ Calcd for C53H53N3NaO14S 1010.3146, found 1010.3127. O-(2-Azido-4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-β-ᴅ-galactopyranosyl)-(1→4)-2,3-di-O-ben zoyl-6-O-p-methoxybenzyl-1-O-trichloroacetimidoyl-α-ᴅ-glucopyranoside (9). Trifluoroacetic acid (19 µL, 0.25 mmol) was added dropwise to a stirred mixture of 2 (500 mg, 0.51 mmol) and N-iodosuccinimide (228 mg, 1.01 mmol) in CH2Cl2 (13 mL) and water (1.3 mL) at 0 °C. Then the system was stirred for 1 h at room temperature. At completion, 5 M Na2S2O3 solution was added and the mixture was extracted with CH2Cl2 (3 ×). The organic phase was washed with brine, dried over anhydrous Na2SO4 after which the solvents were evaporated to afford the corresponding hemiacetal as a white foam. HRMS (ESI-TOF) m/z [M + Na]+ Calcd for C46H47N3NaO15 904.2905, found 904.2855. The hemiacetal was dissolved in dry CH2Cl2 (10 mL). Cl3CCN (305 µL, 3.04 mmol) and DBU (30


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µL, 0.20 mmol) were added dropwise and the reaction mixture was stirred at room temperature for 30 min. After that, the volatiles were removed under reduced pressure. The obtained residue was purified by column chromatography on silica gel (EtOAc/PE, 1:5→1:2 + 0.1% Et3N) to afford imidate 9 (409 mg, 79% for two steps) as a white foam. 1H NMR (500 MHz, CDCl3): δ 8.57 (s, 1H, NH), 7.95 (m, 4H, ArH-Bz), 7.47 (t, J = 7.1 Hz, 1H, ArH-Bz), 7.39-7.15 (m, 12H, ArH-Bz, PMB, benzylidene), 6.92 (d, J = 8.3 Hz, 2H, ArH-PMB), 6.73 (d, J = 3.0 Hz, 1H, H1-1), 6.09 (t, J = 9.8 Hz, 1H, H1-3), 5.47 (dd, J = 10.1, 3.3 Hz, 1H, H1-2), 5.23 (s, 1H, CH-benzylidene), 4.66 (d, J = 11.5 Hz, 1H, CH2-PMB), 4.53 (dd, J = 10.6, 3.0 Hz, 1H, H2-3), 4.46 (d, J = 11.5 Hz, 1H, CH2-PMB), 4.38 (t, J = 9.5 Hz, 1H, H1-4), 4.26 (m, 2H, H2-4, H1-6a), 4.06 (m, 2H, H1-6b), 3.82 (s, 5H, OCH3-PMB, H2-2, H1-5), 3.58 (s, 2H, H2-6a,b), 2.93 (s, 1H, H2-5), 2.70 (m, 2H, CH2-Lev), 2.60 (m, 2H, CH2-Lev), 2.05 (s, 3H, CH3-Lev). 13C NMR (125 MHz, CDCl3): δ 206.1 (CO-Lev), 172.0 (CO-Lev), 165.6 (CO-Bz), 165.6 (CO-Bz), 160.8 (C=NH), 159.6, 137.6, 133.5, 132.8, 130.0, 129.9, 129.8, 129.0, 128.8, 128.5, 128.2, 128.0, 126.6, 114.0, 101.3 (PhCH), 100.8 (C2-1), 93.5 (C1-1), 90.9, 74.9, 73.3, 73.0, 72.9, 72.2, 71.0, 70.4, 68.1, 67.2, 66.2, 60.4, 55.4 (OCH3-PMB), 37.9 (CH2-Lev), 29.7 (CH3-Lev), 28.2 (CH2-Lev). Methyl O-(2-Azido-4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-β-ᴅ-galactopyranosyl)-(1→4)-2,3-di -O-benzoyl-6-O-p-methoxybenzyl-β-ᴅ-glucopyranoside (10). Donor 9 (197 mg, 0.19 mmol) was coevaporated with toluene (3 × 5 mL) and dried in vacuo for 30 min. To a solution of 9 in dry CH2Cl2 (4 mL) was added 4Å powdered molecular sieves. After being stirred for 30 min at room temperature, the mixture was cooled to -60 °C. Anhydrous MeOH (250 µL, 5.76 mmol) was added followed by dropwise addition of trimethylsilyl trifluoromethanesulfonate in CH2Cl2 (0.37 M, 103 µL, 0.038 mmol). After 30 min, additional of trimethylsilyl trifluoromethanesulfonate in CH2Cl2 (0.37 M, 103 µL, 0.038 mmol) was added followed by a gradual increase to room temperature and continuing stirring for 52 h.



The reaction mixture was neutralised by addition of Et3N, filtered through Celite and concentrated. The residue was purified by silica gel column chromatography (EtOAc/petroleum ether, 1:3) to afford 10 (99 mg, 57%) as white amorphous solid. 1H NMR (500 MHz, CDCl3): δ 7.95-7.92 (m, 4H, ArH-Bz), 7.47–7.19 (m, 15H, ArH-Bz, PMB, benzylidene), 6.91 (d, J = 7.0 Hz, 2H, ArH-PMB), 5.70 (t, J = 7.5 Hz, 1H, H1-3), 5.38 (t, J = 7.5 Hz, 1H, H1-2), 5.21 (s, 1H, CH-benzylidene), 4.68 (d, J = 10.0 Hz, 1H, CH2-PMB), 4.61 (d, J = 7.0 Hz, 1H, H1-1), 4.53-4.48 (m, 2H, H2-3, CH2-PMB), 4.29-4.25 (m, 2H, H2-1, H1-4), 4.03 (s, 1H, H2-4), 3.99 (d, J = 10.0 Hz, 1H, H2-6a), 3.98 (d, J = 10.0 Hz, 1H, H2-6b), 3.80-3.74 (m, 5H, OCH3-PMB, H2-2, H1-5), 3.56-3.52 (m, 5H, OCH3, H1-6a, b), 2.88 (s, 1H, H2-5), 2.69-2.59 (m, 4H, CH2CH2-Lev), 2.05 (s, 3H, CH3-Lev);

13

C NMR (125 MHz, CDCl3): δ 206.2 (CO-Lev), 172.0

(CO-Lev), 165.7 (CO-Bz), 165.4 (CO-Bz), 159.4, 137.6, 133.1, 132.7, 130.2, 129.9, 129.7, 129.6, 128.9, 128.3, 128.1, 127.9, 126.6, 125.9, 113.9, 102.0 (C1-1), 101.3 (C2-1), 100.7 (PhCH), 75.6, 75.0, 73.4, 73.2, 72.7, 72.2, 72.2, 68.1, 67.7, 66.1, 60.6 (C2-2), 57.1 (OCH3), 55.4 (OCH3 -PMB), 37.9 (CH2-Lev), 29.7 (CH3-Lev), 28.1 (CH2-Lev). HRMS (ESI-TOF) m/z [M+Na]+ Calcd for C47H49N3NaO15 918.3061, found 918.3005. 3-(Trimethylsilyl)propargyl O-(2-Azido-4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-β-ᴅ-galactopyr anosyl)-(1→4)-2,3-di-O-benzoyl-6-O-p-methoxybenzyl-β-ᴅ-glucopyranoside (11). Imidate 9 (3.32 g, 3.24 mmol) was coevaporated with dry toluene (3 × 25 mL) and dried in vacuo for 30 min. A mixture of 9, 3-(Trimethylsilyl)propargyl alcohol (3.7 mL, 24.71 mmol) and activated 4Å powdered molecular sieves in dry CH2Cl2 (55 mL) was stirred at room temperature for 30 min, and then cooled to 0 °C. Trimethylsilyl trifluoromethanesulfonate (70 µL, 0.39 mmol) was added dropwise to the reaction mixture. After being stirred overnight at 0 °C, the reaction was quenched with Et3N. Insoluble materials were removed by filtration through Celite pad and the filtrate was concentrated under reduced pressure.


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The residue was purified by silica-gel column chromatography (EtOAc/petroleum ether, 1:2) to afford 11 (2.79 g, 87%) as white amorphous solid. 1H NMR (400 MHz, CDCl3): δ 7.95-7.93 (m, 4H, ArH-Bz), 7.47–7.17 (m, 15H, ArH-Bz, PMB, benzylidene), 6.91 (d, J = 8.4 Hz, 2H, ArH-PMB), 5.73 (t, J = 9.6 Hz, 1H, H1-3), 5.40 (t, J = 9.2 Hz, 1H, H1-2), 5.21 (s, 1H, CH-benzylidene), 4.98 (d, J = 8.0 Hz, 1H, H1-1), 4.67 (d, J = 11.6 Hz, 1H, CH2-PMB), 4.52 (d, J = 10.8, 3.2 Hz, 1H, H2-3), 4.48-4.24 (m, 5H, CH2-PMB, H2-1, H1-4, CH2-alkynyl), 4.03-3.98 (m, 2H, H2-4, H2-6a), 3.88 (d, J = 10.8 Hz, 1H, H2-6b), 3.78-3.71 (m, 5H, OCH3-PMB, H2-2, H1-5), 3.55 (s, 2H, H1-6a, b), 2.87 (s, 1H, H2-5), 2.68-2.55 (m, 4H, CH2CH2-Lev), 2.02 (s, 3H, CH3-Lev), 0.13 (s, 9H, CH3-TMS); 13C NMR (125 MHz, CDCl3): δ 206.0 (CO-Lev), 171.8 (CO-Lev), 165.5 (CO-Bz), 165.2 (CO-Bz), 159.3, 137.6, 133.0, 132.7, 130.0, 129.8, 129.8, 129.7, 128.5, 128.8, 128.2, 128.0, 127.8, 126.5, 113.8, 101.2, 100.6, 100.0, 98.1, 92.3, 75.4, 74.9, 73.3, 73.1, 72.5, 72.1, 71.8, 67.9, 67.5, 66.0, 60.5, 56.5, 55.2, 37.7 (CH2-Lev), 29.5 (CH3-Lev), 28.0 (CH2-Lev), -0.3 (C-TMS). HRMS (ESI-TOF) m/z [M+Na]+ Calcd for C52H57N3NaO15 Si 1014.3457, found 1014.3472. p-Methoxyphenyl O-(2-Azido-4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-β-ᴅ-galactopyranosyl)-(1 →4)-2,3-di-O-benzoyl-6-O-p-methoxybenzyl-β-ᴅ-glucopyranoside (12). Donor 9 (390 mg, 0.38 mmol) and acceptor 15 (56.7 mg, 0.46 mmol) were coevaporated with dry toluene (3 × 5 mL), dried in vacuo for 30 min, and dissolved in dry CH2Cl2 (5 mL) in the presence of freshly activated 4Å powdered molecular sieves. After stirring for 30 min at room temperature, the system was cooled to -60 °C, and a solution of trimethylsilyl trifluoromethanesulfonate in CH2Cl2 (0.37 M, 103 µL, 0.038 mmol) was added dropwise. The reaction mixture was stirred for 30 min at -60 °C, then was quenched with Et3N, filtered through Celite pad prior to removing the solvents in vacuo. The obtained residue was purified by silica-gel column chromatography (EtOAc/petroleum ether, 1:2) to afford 12 (343 mg, 91%) as



white foam. 1H NMR (500 MHz, CDCl3): δ 7.97-7.93 (m, 4H, ArH-Bz), 7.45 (t, J = 7.0 Hz, 1H, ArH-Bz), 7.34–7.21 (m, 12H, ArH-Bz, PMB, benzylidene), 6.94 (d, J = 8.5 Hz, 2H, ArH-OMP), 6.89 (d, J = 8.0 Hz, 2H, ArH-PMB), 6.74 (d, J = 8.5 Hz, 2H, ArH-OMP), 5.77 (t, J = 9.0 Hz, 1H, H1-3), 5.63 (t, J = 9.0 Hz, 1H, H1-2), 5.22 (s, 1H, CH-benzylidene), 5.15 (d, J = 8.0 Hz, 1H, H1-1), 4.66 (d, J = 11.5 Hz, 1H, CH2-PMB), 4.57 (dd, J = 10.5, 2.5 Hz, 1H, H2-3), 4.50 (d, J = 11.5 Hz, 1H, CH2-PMB), 4.34-4.30 (m, 2H, H2-1, H1-4), 4.05 (s, 1H, H2-4), 4.00 (dd, J = 11.0, 3.0 Hz, 1H, H2-6a), 3.93 (d, J = 11.0 Hz, 1H, H2-6b), 3.88 (d, J = 9.5 Hz, 1H, H1-5), 3.78-3.74 (m, 4H, OCH3-PMB, H2-2), 3.70 (OCH3-OMP), 3.57 (brs, 2H, H1-6a,b), 2.91(s, 1H, H2-5), 2.68-2.66 (m, 2H, CH2-Lev), 2.59-2.56 (m, 2H, CH2-Lev), 2.03 (s, 3H, CH3-Lev);

13


(CO-Lev), 165.5 (CO-Bz), 165.2 (CO-Bz), 159.3, 155.6, 151.2, 137.6, 133.1, 132.8, 130.0, 129.8, 129.7, 129.6, 129.3, 128.9, 128.3, 128.1, 127.9, 126.5, 118.9, 114.0, 113.8, 101.3 (C2-1), 100.8 (C1-1), 100.6 (PhCH), 75.5 (C1-4), 75.0 (C1-5), 73.3 (C1-3), 73.1 (CH2-PMB), 72.6 (C2-3), 72.2 (C2-4), 72.1 (C1-2), 68.0 (C1-6), 67.6 (C2-6), 66.1 (C2-5), 60.6 (C2-2), 55.5 (OCH3-OMP), 55.2 (OCH3-PMB), 37.9 (CH2-Lev), 29.7 (CH3-Lev), 28.2 (CH2-Lev). HRMS (ESI-TOF) m/z [M + Na]+ Calcd for C53H53N3NaO16 1010.3324, found 1010.3294. p-Methoxyphenyl O-(2-Azido-4,6-O-benzylidene-2-deoxy-β-ᴅ-galactopyranosyl)-(1→4)-2,3-di-Obenzoyl-6-O-p-methoxybenzyl-β-ᴅ-glucopyranoside (13).

To a stirred solution of 12 (376 mg, 0.38

mmol) in pyridine (1.4 mL) was added a freshly prepared mixture of pyridine/AcOH/hydrazine acetate (6:4:0.5, 7.0 mL). After stirring for 40 min, the reaction mixture was diluted with EtOAc and washed with saturated aqueous NaHCO3 and water, dried over anhydrous Na2SO4, and concentrated to dryness. Silica gel column chromatography (CH2Cl2:Acetone, 50:1) of the residue afforded compound 13 (301 mg, 89%) as a white foam. 1H NMR (500 MHz, CDCl3): δ 7.96 (m, 4H, ArH-Bz), 7.48 (t, J = 7.3 Hz,


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1H, ArH-Bz), 7.42-7.14 (m, 12H, ArH-Bz, PMB, benzylidene), 6.94 (d, J = 8.8 Hz, 2H, ArH-OMP), 6.88 (d, J = 8.3 Hz, 2H, ArH-PMB), 6.76 (d, J = 8.9 Hz, 2H, ArH-OMP), 5.75 (t, J = 9.4 Hz, 1H, H1-3), 5.63 (t, J = 8.8 Hz, 1H, H1-2), 5.26 (s, 1H, CH-benzylidene), 5.14 (d, J = 7.8 Hz, 1H, H1-1), 4.67 (d, J = 11.6 Hz, 1H, CH2-PMB), 4.49 (d, J = 11.6 Hz, 1H, CH2-PMB), 4.29 (t, J = 9.4 Hz, 1H, H1-4), 4.19 (d, J = 8.0 Hz, 1H, H2-1), 4.02 (dd, J = 10.9, 2.9 Hz, 1H, H2-6a), 3.92 (d, J = 11.0 Hz, 1H, H2-6b), 3.89 (d, J = 2.5 Hz, 1H, H2-4), 3.85 (d, J = 9.4 Hz, 1H, H1-5), 3.80 (s, 3H, OCH3-PMB), 3.73 (s, 3H, OCH3-OMP), 3.59 (brs, 2H, H1-6a,b), 3.44 (t, J = 9.0 Hz, 1H, H2-2), 3.31 (dd, J = 10.0, 3.3 Hz, 1H, H2-3), 2.94 (s, 1H, H2-5); 13C NMR (125 MHz, CDCl3): δ 165.7 (CO-Bz), 165.4 (CO-Bz), 159.4, 155.7, 151.3, 137.4, 133.2, 132.8, 130.1, 129.9, 129.82, 129.75, 129.4, 129.2, 128.4, 128.1, 128.1, 126.6, 118.9, 114.5, 113.9, 101.4 (C2-1), 101.1 (C1-1), 100.8 (PhCH), 75.5 (C1-4), 75.2 (C1-5), 74.2 (C2-3), 73.3 (C1-3), 73.1 (CH2-PMB), 72.1 (C2-4), 71.8 (C1-2), 68.1 (C1-6), 67.6 (C2-6), 66.4 (C2-5), 64.2 (C2-2), 55.6 (OCH3-OMP), 55.3 (OCH3-PMB). HRMS (ESI-TOF) m/z [M + Na]+ Calcd for C48H47N3NaO14 912.2956, found 912.2917. p-Methoxyphenyl O-(2-Azido-4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-β-ᴅ-galactopyranosyl)-(1 →4)-O-(2,3-di-O-benzoyl-6-O-p-methoxybenzyl-β-ᴅ-glucopyranosyl)-(1→3)-O-(2-Azido-4,6-O-benzy lidene-2-deoxy-β-ᴅ-galactopyranosyl)-(1→4)-2,3-di-O-benzoyl-6-O-p-methoxybenzyl-β-ᴅ-glucopyran oside (14). Donor 9 (208.9 mg，0.20 mmol) and acceptor 13 (150.0 mg, 0.17 mmol) were coevaporated with dry toluene (3 × 5 mL), dried in vacuo for 30 min, and dissolved in dry CH2Cl2 (5 mL) in the presence of 4Å powdered molecular sieves. The mixture was stirred at room temperature for 30 min and then cooled to -60 °C. A solution fo trimethylsilyl trifluoromethanesulfonate in CH2Cl2 (0.19 M, 106 µL, 0.020 mmol) was added dropwise. The reaction mixture was neutralised 30 min later with Et3N before being allowed to warm to room temperature, and filtered with Celite pad. The filtrate was



concentrated in vacuo, and the obtained residue was purified by silica-gel column chromatography (petroleum ether/EtOAc, 1:3→2:3) to afford 14 (283.2 mg, 95%) as white foam. 1H NMR (500 MHz, CDCl3): δ 7.93-7.90 (m, 8H, ArH-Bz), 7.48 (m, 2H, ArH-Bz), 7.46-7.20 (m, 24H, ArH-Bz, PMB, benzylidene), 6.91 (d, J = 8.8 Hz, 2H, ArH-OMP), 6.89 (d, J = 8.0 Hz, 2H, ArH-PMB), 6.86 (d, J = 8.0 Hz, 2H, ArH-PMB), 6.73 (d, J = 8.5 Hz, 2H, ArH-OMP), 5.71-5.66 (m, 2H, H1，3-3), 5.59 (t, J = 9.5 Hz, 1H, H1-2), 5.37 (t, J = 9.0 Hz, 1H, H3-2), 5.22 (s, 1H, CH-benzylidene), 5.17 (s, 1H, CH-benzylidene), 5.09 (d, J = 8.0 Hz, 1H, H1-1), 4.95 (d, J = 7.0 Hz, 1H, H3-1), 4.64 (d, J = 12 Hz, 1H, CH2-PMB), 4.57-4.54 (m, 2H, H4-3, CH2-PMB), 4.44 (dd, J = 12 Hz, 2 Hz, 2H, CH2-PMB), 4.28-4.17 (m, 4H, H1， 3-4,

H2，4-1), 4.08 (s, 1H, H4-4), 4.02 (s, 1H, H2-4), 3.90-3.72 (m, 16H, H4-2,H1-5, H3-5, H3-6a,b, H4-6ab,

OCH3-PMB, OCH3-OMP) ,3.60-3.49 (m, 4H, H2-6ab, H2-2, H1-6a), 3.44 (d, J = 12.0 Hz, 1H, H1-6b), 3.21 (d, J = 10.0 Hz, 1H, H2-3), 2.85 (s, 1H, H2-5), 2.80 (s, 1H, H4-5), 2.70-2.69 (m, 2H, CH2-Lev), 2.60-2.58 (m, 2H, CH2-Lev), 2.06 (s, 3H, CH3-Lev); 13C NMR (125 MHz, CDCl3): δ 206.1 (CO-Lev), 172.0 (CO-Lev), 165.5 (2C, CO-Bz), 165.3 (CO-Bz), 165.1 (CO-Bz), 159.4, 159.3, 155.6, 151.3, 137.8, 137.6, 133.1, 133.0, 132.9, 132.7, 130.2, 130.0, 129.9 (2C), 129.8 (8C), 129.7 (2C), 129.7, 129.6, 129.6 (4C), 129.5, 129.4, 128.9, 128.7, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 126.5 (2C), 126.5 (2C), 118.9 (2C), 114.5 (2C), 113.9 (2C), 113.9 (2C), 101.8 (C4-1), 101.7 (C1-1), 101.6 (C3-1), 100.7 (C2-1), 100.7 (PhCH), 100.5 (PhCH), 79.3, 76.2, 75.5, 75.1, 74.6, 74.5, 73.7, 73.4 (CH2-PMB), 73.3, 73.2 (CH2-PMB), 72.6, 72.2, 72.2, 72.1, 68.6, 68.0, 67.5, 66.4, 66.1, 62.0, 60.7, 55.6 (OCH3-OMP), 55.4 (OCH3-PMB), 55.3 (OCH3-PMB), 37.8 (CH2-Lev), 29.7 (CH3-Lev), 28.1 (CH2-Lev). HRMS (ESI-TOF) m/z [M + Na]+ Calcd for C94H92N6NaO28 1775.5857, found 1775.5819. p-Methoxyphenyl O-(2-Azido-4,6-O-benzylidene-2-deoxy-β-ᴅ-galactopyranosyl)-(1→4)-O-(2,3-diO-benzoyl-6-O-p-methoxybenzyl-β-ᴅ-glucopyranosyl)-(1→3)-O-(2-Azido-4,6-O-benzylidene-2-deoxy


Page 24 of 41

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-β-ᴅ-galactopyranosyl)-(1→4)-2,3-di-O-benzoyl-6-O-p-methoxybenzyl-β-ᴅ-glucopyranoside (15). 14 (600 mg, 0.34 mmol) was dissolved in pyridine (1.2 mL), and a freshly prepared mixture of pyridine/AcOH/hydrazine acetate (6:4:0.5, 6 mL) was added. After stirring for 40 min, the resulting mixture was diluted with EtOAc, washed with saturated aqueous NaHCO3 and water, dried over anhydrous Na2SO4, and concentrated to dryness. Silica gel column chromatography (CH2Cl2:Acetone, 25:1) afforded compound 15 (529 mg, 93%) as a white foam. 1H NMR (500 MHz, CDCl3): δ 8.00-7.86 (m, 8H, ArH-Bz), 7.46 (t, J = 6.5 Hz, 2H, ArH-Bz), 7.36-7.22 (m, 25H, ArH-Bz, PMB, benzylidene, overlapped with solvent peak), 6.92-6.83 (m, 4H, ArH-OMP), 6.83 (d, J = 8.0 Hz, 2H, ArH-PMB), 6.74 (d, J = 8.0 Hz, 2H, ArH-PMB), 6.73 (d, J = 8.5 Hz, 2H, ArH-OMP), 5.71-5.66 (m, 2H, H1，3-3), 5.59 (t, J = 8.0 Hz, 1H, H1-2), 5.39 (t, J = 7.5 Hz, 1H, H3-2), 5.26 (s, 1H, CH-benzylidene), 5.17 (s, 1H, CH-benzylidene), 5.09 (d, J = 8.0 Hz, 1H, H1-1), 4.94 (d, J = 7.0 Hz, 1H, H3-1), 4.63 (d, J = 11.5 Hz, 1H, CH2-PMB), 4.58 (d, J = 11.5 Hz, 1H, CH2-PMB), 4.45-4.42 (m, 2H, CH2-PMB), 4.26 (t, J = 9.0 Hz, 1H, H1-4), 4.17-4.08 (m, 5H, H3-4, H2，4-1, H2，4-4), 3.94-3.72 (m, 17H, H4-3, H4-2, H1-5, H3-5, H3-6a,b, H4-6ab, OCH3-PMB, OCH3-OMP) ,3.59-3.44 (m, 6H, H2-6ab, H2-2, H1-6a), 3.33 (dd, J = 9.5 Hz, , 1H, H1-6b), 3.21 (d, J = 10.0 Hz, 1H, H2-3), 2.86 (s, 1H, H2-5), 2.82 (s, 1H, H4-5); 13C NMR (125 MHz, CDCl3): δ 165.6 (CO-Bz), 165.5 (CO-Bz), 165.4 (CO-Bz), 165.1 (CO-Bz), 159.4, 159.3, 155.6, 151.3, 137.9 (ArC-benzylidene), 137.4, 133.2, 133.1, 132.9, 132.8, 130.2, 130.0, 129.9, 129.9, 129.8, 129.7, 129.6, 129.5, 129.4, 129.3, 128.7, 128.4, 128.3, 128.2, 128.1, 127.9, 126.5, 126.5, 118.9 (2C), 114.5 (2C), 113.9 (2C), 101.9 (C4-1), 101.8 (C2-1), 101.6 (C3-1), 101.1 (C1-1), 100.7 (PhCH), 100.5 (PhCH), 79.3, 76.1, 75.5, 75.1, 74.6, 74.6, 74.1, 73.7, 73.3 (CH2-PMB), 73.3, 73.2 (CH2-PMB), 72.1, 71.9, 68.5, 68.1, 67.5, 66.5, 66.4, 64.4, 62.0, 60.5, 55.6 (OCH3-OMP), 55.5 (OCH3-PMB), 55.4 (OCH3-PMB). HRMS (ESI-TOF) m/z [M + Na]+ Calcd for C89H86N6NaO26 1677.5489, found



1677.5470. p-Methoxyphenyl O-(2-Azido-4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-β-ᴅ-galactopyranosyl)-(1 →4)-O-(2,3-di-O-benzyl-6-O-p-methoxybenzyl-β-ᴅ-glucopyranosyl)-(1→3)-O-(2-Azido-4,6-O-benzyli dene-2-deoxy-β-ᴅ-galatpyrnsyl)-(1→4)-O-(2,3-di-O-benzoyl-6-O-p-methoxybenzyl-β-ᴅ-glucopyranos yl)-(1→3)-O-(2-Azido-4,6-O-benzylidene-2-deoxy-β-ᴅ-galactopyranosyl)-(1→4)-2,3-di-O-benzoyl-6O-p-methoxybenzyl-β-ᴅ-glucopyranoside (16). Donor 9 (628 mg, 0.61 mmol) and acceptor 15 (845 mg, 0.51 mmol) were coevaporated with dry toluene (3 × 10 mL), and dried in vacuo for 30 min. The mixture was then dissolved in dry CH2Cl2 (10 mL), and freshly activated 4Å molecular sieves were added. After stirring for 30 min at room temperature, the reaction mixture was cooled to -60 °C, and a solution of trimethylsilyl trifluoromethanesulfonate in CH2Cl2 (0.19 M, 316 µL, 0.061 mmol) was added dropwise. The mixture was stirred for 30 min at room temperature, then was neutralised with Et3N, filtered and concentrated. Silica-gel column chromatography (CH2Cl2/Acetone, 60:1→20:1) afforded 16 (1.13 g, 88%) as white foam. 1H NMR (500 MHz, CDCl3): δ 7.94-7.86 (m, 12H, ArH-Bz), 7.45-7.17 (m, 40H, ArH-Bz, PMB, benzylidene, overlapped with solvent peak), 6.91-6.86 (m, 4H, ArH-OMP, PMB), 6.82 (m, 4H, ArH-PMB), 6.73 (d, J = 8.5 Hz, 2H, ArH-OMP), 5.71-5.56 (m, 4H, 3H1/3/5 -3, H1/3/5-2), 5.38 (t, J = 8.0 Hz, 1H, H1/3/5-2), 5.34 (t, J = 8.0 Hz, 1H, H1/3/5-2), 5.22 (s, 1H, CH-benzylidene), 5.17 (s, 1H, CH-benzylidene), 5.15 (s, 1H, CH-benzylidene), 5.09 (d, J = 8.0 Hz, 1H, H1/3/5-1), 4.95 (d, J = 7.0 Hz, 1H, H1/3/5-1), 4.89 (d, J = 7.0 Hz, 1H, H1/3/5-1), 4.62 (d, J = 11.5 Hz, 1H, CH2-PMB), 4.56-4.50 (m, 3H, CH2-PMB), 4.43-4.41 (m, 2H, CH2-PMB, H6-3), 4.36 (d, J = 11.5 Hz, 1H, CH2-PMB), 4.27-4.05 (m, 8H, 3H1/3/5-4, 3H2/4/6-1, 2H2 ,4 -4), 4.02 (s, 1H, H6-4), 3.89-3.73 (m, 19H, H6-2, 3H1/3/5-5, 6H-6a,b, OCH3-PMB), 3.70 (s, 3H, OCH3-OMP), 3.60-3.51 (m, 6H, 4H -6a,b, H2/4-2), 3.41 (d, J = 12.0 Hz, 1H, H-6a), 3.37 (d, J = 11.5 Hz, 1H, H-6b), 3.23 (dd, J = 10.5 Hz, 2.5 Hz, 1H,


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H2/4-3), 3.18 (dd, J = 10.0 Hz, 2.0 Hz, 1H, H2/4-3), 2.82 (s, 1H, H2/4/6-5), 2.79 (s, 1H, H2/4/6-5), 2.73 (s, 1H, H2/4/6-5), 2.68-2.67 (m, 2H, CH2-Lev), 2.59-2.57 (m, 2H, CH2-Lev), 2.04 (s, 3H, CH3-Lev);

13

C

NMR (100 MHz, CDCl3): δ 206.2 (CO-Lev), 172.0 (CO-Lev), 165.5 (CO-Bz), 165.5 (CO-Bz), 165.4 (CO-Bz), 165.1 (CO-Bz), 165.1 (CO-Bz), 159.5, 159.4, 159.3, 155.6, 151.3, 137.9, 137.6, 133.2, 133.03, 133.00, 132.9, 132.8, 132.7, 130.2, 130.1, 130.0, 129.9, 129.8, 129.77, 129.75, 129.72, 129.7, 129.6, 129.6, 129.6, 129.5, 129.4, 129.0, 128.7, 128.6, 128.4, 128.3, 128.2, 128.2, 128.1, 128.0, 127.9, 127.9, 126.5, 126.5, 126.4, 118.9 (2C), 114.5 (2C), 113.9 (2C), 113.9 (4C), 101.9 (C-1), 101.7 (C-1), 101.7 (2C, C-1), 101.6 (C-1), 100.7 (C-1), 100.7 (PhCH), 100.4 (2C, PhCH), 79.4, 79.3, 76.23, 76.19, 75.5, 75.1, 74.53, 74.51, 74.46 (2C), 73.8, 73.7 (CH2-PMB), 73.39 (CH2-PMB), 73.35 (CH2-PMB), 73.2 (2C), 72.6, 72.2, 72.2, 72.14, 72.11, 68.6 (2C), 68.0 (3C), 67.5, 66.5 (2C), 66.2, 62.2, 62.0, 60.8, 55.7 (OCH3-OMP), 55.5 (OCH3-PMB), 55.44 (OCH3-PMB), 55.36 (OCH3-PMB), 37.8 (CH2-Lev), 29.7 (CH3-Lev), 28.1 (CH2-Lev). HRMS (ESI-TOF) m/z [M + Na]+ Calcd for C135H131N9NaO40 2540.8391, found 2540.8352. p-Methoxyphenyl O-(2-Azido-4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-β-ᴅ-galactopyranosyl)-(1 →4)-O-(2,3-di-O-benzyl-β-ᴅ-glucopyranosyl)-(1→3)-O-(2-Azido-4,6-O-benzylidene-2-deoxy-β-ᴅ-gal actopyranosyl)-(1→4)-2,3-di-O-benzoyl-β-ᴅ-glucopyranoside (17). To a solution of 14 (73 mg, 0.04 mmol) in CH2Cl2 (2 mL) coverd with aluminum foil were added water (106 µL) and DDQ (23 mg, 0.1 mmol). The reaction mixture was stirred for 5 h at room temperature, quenched with sat. NaHCO3 aq., and extracted with CH2Cl2 (3 ×). The organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated. Silica gel column chromatography (CH2Cl2/MeOH, 10:1) afforded 17 (64 mg) quantitatively as a white foam. 1H NMR (500 MHz, CDCl3): δ 7.94-7.92 (m, 8H, ArH-Bz), 7.48 (m, 2H, ArH-Bz), 7.41-7.20 (m, 24H, ArH-Bz, benzylidene, overlapped with solvent peak), 6.88 (d, J =



8.5 Hz, 2H, ArH-OMP), 6.75 (d, J = 8.5 Hz, 2H, ArH-OMP), 5.75 (t, J = 9.5 Hz, 1H, H1-3), 5.68 (t, J = 8.5 Hz, 1H, H3-3), 5.56 (t, J = 8.5 Hz, 1H, H1-2), 5.36 (t, J = 7.5 Hz, 1H, H3-2), 5.27 (s, 1H, CH-benzylidene), 5.23 (s, 1H, CH-benzylidene), 5.18 (d, J = 8.0 Hz, 1H, H1-1), 5.10 (d, J = 6.5 Hz, 1H, H3-1), 4.63 (dd, J = 10.5, 3.0 Hz, 1H, H4-3), 4.38 (d, J = 8.0 Hz, 1H, H2-1), 4.32-4.28 (m, 3H, H1，3-4, H4-1), 4.12 (s, 1H, H4-4), 4.05-3.97 (m, 5H, H2-4, H1，3-6a,b), 3.80 (t, J = 9.0 Hz, 1H, H1-5), 3.72-3.67 (m, 6H, H3-5, OCH3-OMP, H2-6a,b), 3.61 (br-s, 2H, H4-6a,b), 3.56-3.49 (m, 3H, H2-3, H2，4-2), 3.06 (s, 1H, H2-5), 2.93 (s, 1H, H4-5), 2.71-2.69 (m, 2H, CH2-Lev), 2.59-2.57 (m, 2H, CH2-Lev), 2.06 (s, 3H, CH3-Lev);

13

C NMR (125 MHz, CDCl3): δ 206.2 (CO-Lev), 172.0 (CO-Lev), 165.6 (CO-Bz), 165.4

(2C, CO-Bz), 165.2 (CO-Bz), 155.8, 151.1, 137.8, 137.6, 133.3, 133.2, 133.0, 132.9, 129.9, 129.9, 129.7, 129.7, 129.4, 129.4, 129.0, 129.0, 128.5, 128.4, 128.3, 128.2, 128.1, 126.6, 118.7 (2C), 114.7 (2C), 102.2, 101.9, 101.1, 100.8, 100.7, 100.7, 78.7, 75.8, 75.5, 75.3, 75.1, 74.6, 73.7, 73.2, 72.8, 72.3, 72.1, 68.2, 68.1, 66.6, 66.3, 62.3, 61.2, 60.8, 60.7, 55.7 (OCH3-OMP), 53.6, 37.9 (CH2-Lev), 29.7 (CH3-Lev), 28.2 (CH2-Lev). HRMS (ESI-TOF) m/z [M + Na]+ Calcd for C78H76N6NaO26 1535.4707, found 1535.4673. Methy [p-Methoxyphenyl O-(2-Azido-4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-β-ᴅ-galacto pyranosyl)-(1→4)-O-(methy 2,3-di-O-benzoyl-β-ᴅ-glucopyranosyluronate)-(1→3)-O-(2-Azido-4,6 -O-benzylidene-2-deoxy-β-ᴅ-galactopyranosyl)-(1→4)-(2,3-di-O-benzoyl-β-ᴅ-glucopyranoside)] Uronate (18). To a solution of glucoside 16 (377 mg, 0.25 mmol) in a mixture of acetonitrile/buffer (Na2CO3/ NaHCO3, PH 9.5) (1/1, 5 mL), (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO, 46.7 mg, 0.30 mmol), (diacetoxyiodo)-benzene (BAIB, 602.1 mg, 1.87 mmol) and tetrabutylammonium bromide (TBAB, 11.7 mg, 0.075 mmol) were added. The reaction mixture was stirred at room temperature for 4 h. Then the reaction was quenched by addition of sat. Na2S2O3 aq. followed by separation of the layers.


Page 28 of 41

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The aqueous phase was extracted with EtOAc (3 ×), and the combined organic phase was washed with brine, dried over Na2SO4, and concentrated to afford a yellow syrup. The crude acid, K2CO3 (103.2 mg, 0.75 mmol) and CH3I (124 µL, 1.99 mmol) were suspended in DMF (4 mL). After stirring for 30 min, the reaction mixture was queched with water, diluted with EtOAc (3 ×), washed with brine, dried over unhydrous Na2SO4 and concentrated. Silica gel column chromatography (Acetone/petroleum ether/CH2Cl2, 4:1:4) afforded 18 (318 mg, 81% for two steps) as a white foam. 1H NMR (600 MHz, CDCl3): δ 7.97-7.93 (m, 5H, ArH-Bz), 7.90 (d, J = 7.2 Hz, 2H, ArH-Bz), 7.52 - 7.24 (m, 23H, ArH-Bz, benzylidene), 6.91 (d, J = 9.0 Hz, 2H, ArH-OMP), 6.77 (d, J = 9.6 Hz, 2H, ArH-OMP), 5.77 (t, J = 9.0 Hz, 1H, H1-3), 5.65-5.60 (m, 2H, H3-3, H4-3), 5.34 (t, J = 6.6 Hz, 1H, H3-2), 5.38 (s, 1H, CH-benzylidene), 5.21 (d, J = 6.6 Hz, 1H, H1/3-1), 5.21 (s, 1H, CH-benzylidene), 5.12 (d, J = 6.0 Hz, 1H, H1/3-1), 4.55 (dd, J = 9.6, 8.4 Hz, 1H, H1-2), 4.51-4.48 (m, 2H, H1,3-4), 4.36 (d, J = 7.8 Hz, 1H, H4-1), 4.31-4.29 (m, 2H, H1,3-5), 4.25 (d, J = 3.0 Hz, 1H, H4-4), 4.25 (d, J = 8.4 Hz, 1H, H2-1), 3.99 (d, J = 3.0 Hz, 1H, H2-4), 3.82 (s, 3H, OCH3-COOMe), 3.75 (s, 3H, OCH3-COOMe), 3.74 (s, 3H, OCH3-COOMe), 3.72-3.69 (m, 1H, H4-2), 3.66-3.61 (m, 2H, H2-2, H-6a), 3.43-3.40 (m, 3H, H2-3, 2H-6a,b), 3.34 (d, J = 9.0 Hz, 1H, H-6b), 3.11 (s, 1H, H4-5), 2.72-2.69 (m, 3H, CH2-Lev, H2-5), 2.61-2.58 (m, 2H, CH2-Lev), 2.07 (s, 3H, CH3-Lev); 13C NMR (150 MHz, CDCl3): δ 206.2 (CO-Lev), 172.1 (CO-Lev), 168.7 (CO-COOMe), 168.3 (CO-COOMe), 165.4 (CO-Bz), 165.3 (CO-Bz), 165.2 (CO-Bz), 164.9 (CO-Bz), 155.9, 151.0, 138.3, 137.7, 133.4, 133.2, 133.0, 130.1, 129.9, 129.6, 129.6, 129.2, 129.1, 128.9, 128.5, 128.4, 128.2, 128.1, 128.1, 126.7, 126.6, 118.8, 114.7, 102.7, 102.5, 101.0, 101.0, 100.7, 100.6, 78.9, 78.0, 77.8, 74.5, 74.1, 73.5, 73.39, 72.37, 72.2 (2C), 71.9, 71.8, 68.0, 67.9, 66.7, 66.2, 62.1, 60.6, 55.8 (CH3-OMP), 53.1 (CH3-COOMe), 53.0 (CH3-COOMe), 37.9 (CH2-Lev), 29.8 (CH3-Lev), 28.2 (CH2-Lev). HRMS (ESI-TOF) m/z [M + Na]+. Calcd for C80H76N6NaO28



Page 30 of 41

1591.4605, found 1591.4579. p-Methoxyphenyl O-(2-Azido-4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-β-ᴅ-galactopyranosyl)-(1 →4)-O-(2,3-di-O-benzyl-β-ᴅ-glucopyranosyl)-(1→3)-O-(2-Azido-4,6-O-benzylidene-2-deoxy-β-ᴅ-gal actopyranosyl)-(1→4)-O-(2,3-di-O-benzoyl-β-ᴅ-glucopyranosyl)-(1→3)-O-(2-Azido-4,6-O-benzylide ne-2-deoxy-β-ᴅ-galactopyranosyl)-(1→4)-2,3-di-O-benzoyl-β-ᴅ-glucopyranoside (19). To a solution of 16 (1.27 g, 0.51 mmol) in CH2Cl2 (13 mL) coverd with aluminum foil were added water (700 µL) and DDQ (413.4 mg，1.82 mmol). The reaction mixture was stirred for 5 h at room temperature, quenched with sat. NaHCO3 aq., and extracted with CH2Cl2 (3 ×). The organic layer was washed with brine, dried over unhydrous Na2SO4 and concentrated. Column chromatography (CH2Cl2/MeOH, 10:1) afforded 19 (1.15 g) quantitatively as a white foam. 1H NMR (500 MHz, CDCl3): δ 7.93-7.88 (m, 12H, ArH-Bz), 7.48-7.21 (m, 33H, ArH-Bz, benzylidene), 6.87 (d, J = 8.0 Hz, 2H, ArH-OMP), 6.75 (d, J = 8.5 Hz, 2H, ArH-OMP), 5.74 (t, J = 9.5 Hz, 1H, H1/3/5-3), 5.68-5.62 (m, 2H, H1/3/5-3), 5.56 (t, J = 8.5 Hz, 1H, H1/3/5-2), 5.36-5.32 (m, 2H, H1/3/5-2), 5.25 (s, 1H, CH-benzylidene), 5.23 (s, 2H, CH-benzylidene), 5.16 (d, J = 7.5 Hz, 1H, H1/3/5-1), 5.08-5.04 (m, 2H, H1/3/5-1), 4.63 (dd, J = 10.5, 2.5 Hz, 1H, H6-3), 4.38 (d, J = 8.0 Hz, 1H, H2/4/6-1), 4.30-4.23 (m, 5H, 3H1/3/5-5 or 4, 2H2/4/6-1), 4.12-3.90 (m, 8H, 3H2/4/6-4, 2H2/4/6-2, 2H1/3/5-4 or 5, H1/3/5-6a,b), 3.79 (t, J = 9.0 Hz, 1H, H1/3/5-4), 3.74-3.43 (m, 16H, H2/4/6-2, 2H2/4-3, OCH3-OMP, 6H2/4/6-6a,b, 4H1/3/5-6a,b), 3.05 (s, 1H, H2/4/6-5), 2.94 (s, 2H, H2/4/6-5), 2.72-2.67 (m, 2H, CH2-Lev), 2.59-2.57 (m, 2H, CH2-Lev), 2.05 (s, 3H, CH3-Lev) ;

13

C NMR (125

MHz, CDCl3): δ 206.3 (CO-Lev), 172.0 (CO-Lev), 165.6 (CO-Bz), 165.5 (CO-Bz), 165.4 (2C, CO-Bz), 165.3 (CO-Bz), 165.2 (CO-Bz), 155.8, 151.1, 137.7, 137.6, 133.3, 133.2 (2C), 133.0, 132.9 (2C), 129.8, 129.7, 129.3, 129.0, 128.9, 128.4, 128.3, 128.2, 128.2, 128.1, 128.0, 126.5, 118.7 (2C), 114.7 (2C), 102.1 (2C), 101.9, 101.2, 101.0, 100.7 (4C), 78.6, 78.5, 75.7, 75.5, 75.4, 75.2, 75.0 (2C), 74.53, 74.48,


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73.7, 73.5, 73.2, 72.7, 72.2 (2C), 72.1, 68.1 (2C), 68.0, 66.4 (2C), 66.2, 62.2 (2C), 61.0, 60.9, 60.7, 55.7 (OCH3-OMP), 37.9 (CH2-Lev), 29.7 (CH3-Lev), 28.1 (CH2-Lev). HRMS (ESI-TOF) m/z [M + Na]+ Calcd for C111H107N9NaO37 2180.6666, found 2180.6694. Methy [p-Methoxyphenyl O-(2-Azido-4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-β-ᴅ-galactopyra nosyl)-(1→4)-O-(methy 2,3-di-O-benzoyl-β-ᴅ-glucopyranosyluronate)-(1→3)-O-(2-Azido-4,6-O-ben zylidene-2-deoxy-β-ᴅ-galactopyranosyl)-(1→4)-(2,3-di-O-benzoyl-β-ᴅ-glucopyranoside)-(1→3)-O-(2Azido-4,6-O-benzylidene-2-deoxy-β-ᴅ-galactopyranosyl)-(1→4)-(2,3-di-O-benzoyl-β-ᴅ-glucopyra-no side)] Uronate (20). To a solution of glucoside 19 (1.15 g, 0.53 mmol) in a mixture of acetonitrile/buffer (Na2CO3/ NaHCO3, PH 9.5) (1/1, 20 mL), TEMPO (100 mg, 0.64 mmol), BAIB (1.28 g, 3.98 mmol) and TBAB (77 mg, 0.24 mmol) were added. The reaction mixture was stirred at room temperature for 4 h. Then the reaction was quenched by addition of sat. Na2S2O3 aq. followed by separation of the layers. The aqueous layer was extracted with EtOAc (3 ×). The combined organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated to afford a yellow syrup. The crude acid, K2CO3 (629.4 mg, 4.55 mmol) and CH3I (756 µL, 12.14 mmol) were suspended in DMF (10 mL). After 30 min at room temperature, the resulting mixture was queched by addition of water, diluted with EtOAc (3 ×), washed with brine, dried over Na2SO4 and concentrated. Silica gel column chromatography (EtOAc/petroleum ether/CH2Cl2, 1:1:1) afforded 20 (1.13 g, 46% for two steps) as a white foam. 1H NMR (600 MHz, CDCl3): δ 7.97-7.83 (m, 11H, ArH-Bz), 7.51-7.24 (m, 34H, ArH-Bz, benzylidene), 6.91 (d, J = 9.0 Hz, 2H, ArH-OMP), 6.76 (d, J = 9.0 Hz, 2H, ArH-OMP), 5.76 (t, J = 9.0 Hz, 1H, H1-3), 5.65-5.60 (m, 3H, H3,5-3, H6-3), 5.39-5.35 (m, 4H, H3,5-2, 2H-CH-benzylidene), 5.22 (d, J = 6.6 Hz, 1H, H1/3-1), 5.21 (s, 1H, CH-benzylidene), 5.12 (d, J = 6.0 Hz, 1H, H1/3-1), 5.09 (d, J = 6.0 Hz, 1H, H5-1), 4.56 (t, J = 9.0 Hz, 1H, H1-2), 4.52-4.48 (m, 3H,



H1,3,5-4), 4.36 (d, J = 7.8 Hz, 1H, H6-1), 4.32 (d, J = 10.2 Hz, 1H, H1/3-5), 4.28 (d, J = 9.0 Hz, 1H, H1/3-5), 4.24 (d, J = 9.6 Hz, 1H, H5-5), 4.21 (d, J = 2.4 Hz, 1H, H6-4), 4.18 (d, J = 3.0 Hz, 1H, H4-4), 4.11 (d, J = 8.4 Hz, 1H, H2/4-1), 4.09 (d, J = 8.4 Hz, 1H, H2/4-1), 3.98 (d, J = 3.0 Hz, 1H, H2-4), 3.81 (s, 3H, OCH3-COOMe), 3.73 (s, 3H, OCH3-COOMe), 3.72 (s, 6H, OCH3-COOMe), 3.69 (dd, J = 10.8, 6.4 Hz, 1H, H6-2), 3.64-3.56 (m, 3H, H2,4-2, H-6a), 3.56 (d, J = 12.0 Hz, 1H, H-6b), 3.42-3.39 (m, 3H, H2-3, 2H-6a,b), 3.35-3.34 (m, 2H, H-6a,b), 3.30 (dd, J = 10.2, 3.0 Hz, 1H, H4-3), 3.09 (s, 1H, H6-5), 2.74 (s, 1H, H4-5), 2.71-2.68 (m, 3H, CH2-Lev, H2-5), 2.60-2.57 (m, 2H, CH2-Lev), 2.06 (s, 3H, CH3-Lev); 13

C NMR (150 MHz, CDCl3): δ 206.2 (CO-Lev), 172.0 (CO-Lev), 168.7 (CO-COOMe), 168.6

(CO-COOMe), 168.2 (CO-COOMe), 165.4 (CO-Bz), 165.3 (CO-Bz), 165.2 (CO-Bz), 165.1 (CO-Bz), 164.9 (CO-Bz), 155.9, 151.0, 138.2, 138.2, 137.7, 133.4, 133.3, 133.2, 133.1, 133.0, 130.0, 129.89, 129.87, 129.6, 129.5, 129.2, 129.0, 128.8, 128.7, 128.5, 128.4, 128.4, 128.3, 128.2, 128.1, 128.04, 127.95, 126.6, 126.5, 118.8, 114.6, 102.7, 102.7, 102.4, 101.1, 100.9, 100.8, 100.7, 100.5, 100.4, 78.8, 78.7, 78.0, 77.9, 77.7, 74.4, 74.1, 74.0, 73.6, 73.5, 73.4, 73.3, 72.3, 72.3, 72.2, 71.9, 71.82, 71.75, 68.0, 67.93, 67.87, 66.6, 66.5, 66.1, 62.2, 62.1, 60.6, 55.7 (CH3-OMP), 53.0 (CH3-COOMe), 52.9 (CH3-COOMe), 52.9 (CH3-COOMe), 37.9 (CH2-Lev), 29.8 (CH3-Lev), 28.1 (CH2-Lev). HRMS (ESI-TOF) m/z [M + Na]+ Calcd for C114H107N9NaO40 2264.6513, found 2264.6526. p-Methoxyphenyl O-(2-Acetamido-4,6-O-benzylidene-2-deoxy-β-ᴅ-galactopyranosyl)-(1→4)-2,3 -di-O-benzoyl-6-O-p-methoxybenzyl-β-ᴅ-glucopyranoside (21). Method a: To azide 12 (50 mg, 0.051 mmol) in pyridine (1 mL) was added 2.0 mL of AcSH and the mixture was stirred at room temperature for 2 h. After that, the volatiles were removed and the obtained residue was subjected to silica gel column chromatography (EtOAc/petroleum ether, 1:1→CH2Cl2/MeOH, 20:1) to give compoud 21 (50.8 mg, 89%) as a white solid. Method b: Water (1.3 mL), triethylamine (125 µL, 0.9 mmol), and


Page 32 of 41

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1,3-propanedithiol (123 µL, 1.23 mmol) were added to a solution of disaccharide 12 (55 mg, 0.06 mmol) in pyridine (5.0 mL), and the resulting mixture was stirred for 30 min at rt. After that, the reaction mixture was concentrated and dried in vacuo. The crude residue was dissolved in MeOH (3 mL), Ac2O (23 µL, 0.24 mmol) was added, and the resulting suspension was stirred under argon for 3 h at room temperature. The reaction mixture was then concentrated and dried in vacuo. Silica gel column chromatography (EtOAc/petroleum ether, 3:5→CH2Cl2/MeOH, 20:1) afforded compound 21 (48 mg, 86%) as a white solid. 1H NMR (500 MHz, CDCl3): δ 7.97-7.93 (m, 4H, ArH-Bz), 7.49 (t, J = 7.5 Hz, 1H), 7.41–7.25 (m, 12H, ArH-Bz, PMB, benzylidene), 6.94-6.89 (m, 4H, ArH-PMB, OMP), 6.74 (d, J = 9.0 Hz, 2H, ArH-OMP), 5.79 (t, J = 9.5 Hz, 1H, H1-3), 5.55 (t, J = 9.0 Hz, 1H, H1-2), 5.36 (d, J = 7.5 Hz, 1H, NH), 5.25 (s, 1H, CH-benzylidene), 5.16 (dd, J = 11.0, 3.0 Hz, 1H, H2-3), 5.11 (d, J = 8.0 Hz, 1H, H1-1), 5.01 (d, J = 8.5 Hz, 1H, H2-1), 4.65 (d, J = 12.0 Hz, 1H, CH2-PMB), 4.55 (d, J = 11.5 Hz, 1H, CH2-PMB), 4.25 (t, J = 9.0 Hz, 1H, H1-4), 4.00 (d, 1H, J = 2.5 Hz, H2-4), 3.87-3.70 (m, 11H, OCH3-PMB, OCH3-OMP, H2-2, H1-5, H2-6a,b, H1-6a,), 3.51 (d, 1H, J = 12.0 Hz, H1-6b), 2.83 (s, 1H, H2-5), 2.74-2.44 (m, 4H, CH2CH2-Lev), 2.05 (s, 3H, CH3-Lev), 1.82 (s, 3H, NHAc); 13C NMR (100 MHz, CDCl3): δ 206.8 (CO-Lev), 172.2 (CO-Lev), 170.8 (CO-NHAc), 165.5 (CO-Bz), 165.3 (CO-Bz), 159.5, 155.7, 151.4, 137.8, 133.2, 133.1, 130.4, 130.0, 129.9, 129.8, 129.6, 129.5, 128.9, 128.5, 128.0, 126.6, 119.0, 114.6, 114.0, 100.8, 100.7, 100.3, 77.4, 75.8, 75.2, 74.1, 73.30, 72.95, 72.5, 70.4, 68.4, 68.0, 66.3, 55.7 (OCH3-PMB), 55.4 (OCH3-PMB), 52.4 (OCH3-OMP), 38.0 (CH2-Lev), 29.8 (CH3-Lev), 28.3 (CH2-Lev), 23.5 (CH3-NHAc). HRMS (ESI-TOF) m/z [M + Na]+ Calcd for C55H57NNaO17 1026.3524, found 1026.3502. p-Methoxyphenyl O-(2-Acetamido-4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-β-ᴅ-galactopyrano syl)-(1→4)-O-(2,3-di-O-benzoyl-6-O-p-methoxybenzyl-β-ᴅ-glucopyranosyl)-(1→3)-O-(2-Acetamido-



Page 34 of 41

4,6-O-benzylidene-2-deoxy-β-ᴅ-galactopyranosyl)-(1→4)-2,3-di-O-benzoyl-6-O-p-methoxybenzyl-β-ᴅ -glucopyranoside (22). Method a: To azide 14 (50 mg, 0.029 mmol) in pyridine (0.7 mL) was added 1.4 mL of AcSH and the mixture was stirred at room temperature for 3 h. The reaction mixture was concentrated,

and

the

residue

was

subjected

to

silica

gel

column

chromatography

(CH2Cl2→CH2Cl2/Acetone, 20:1→CH2Cl2/MeOH, 20:1) to give compoud 22 (39 mg, 76%) as a white solid. Method b: Water (0.8 mL), triethylamine (113 µL, 0.81 mmol), and 1,3-propanedithiol (109 µL, 1.08 mmol) were added to a solution of tetrasaccharide 14 (47.5 mg, 0.027 mmol) in pyridine (3.0 mL), and the resulting mixture was stirred for 4.5 h at room temperature. After that, the reaction mixture was concentrated and dried in vacuo. The crude residue was dissolved in MeOH (2 mL) and CH2Cl2 (1 mL), Ac2O (90 µL, 0.94 mmol) was added, and the resulting suspension was stirred under argon for 1 h at room temperature. The reaction mixture was then concentrated and dried in vacuo. The residue was purified by silica gel column chromatography (CH2Cl2→CH2Cl2/Acetone, 10:1→CH2Cl2/MeOH, 40:1) to afford compound 22 (28.7 mg, 59%) as a white solid. 1H NMR (500 MHz, CDCl3): δ 7.93-7.89 (m, 8H, ArH-Bz), 7.49-7.18 (m, 30H, ArH-Bz, PMB, benzylidene, overlapped with solvent peak), 6.90 (d, J = 9.0 Hz, 2H, ArH-PMB), 6.83-6.82 (m, 4H, ArH-OMP, PMB), 6.72 (d, J = 9.0 Hz, 2H, ArH-OMP), 5.72-5.66 (m, 2H, H1,3-3), 5.50-5.47 (m, 2H, H1,3-2), 5.29-5.26 (m, 2H, CH-benzylidene, NH), 5.20 (d, J = 9.0 Hz, 1H, H4-1), 5.17 (s, 1H, CH-benzylidene), 5.12 (dd, J = 11.0, 2.5 Hz, 1H, NH), 5.06 (d, J = 7.5 Hz, 1H, H2-1), 4.95 (d, J = 7.5 Hz, 1H, H1-1), 4.89 (d, J = 7.5 Hz, 1H, H3-1), 4.53-4.51 (m, 4H, CH2-PMB, H4-3), 4.40 (d, 1H, J = 11.0 Hz, CH2-PMB), 4.14 (t, J = 9.0 Hz, 1H, H3-4), 4.09 (s, 1H, H4-4), 4.17 (t, J = 9.5 Hz, 1H, H1-4), 3.98 (d, J = 1.5 Hz, H2-4), 3.79-3.62 (m, 21H, H2,4-2, H2,4-5, H1,3-5, 6H-H6a,b, 6H-OCH3-PMB, OCH3-OMP), 3.51 (d, J = 12.0 Hz, 1H, H-6a), 3.36 (d, J = 12.0 Hz, 1H, H-6b), 3.20-3.15 (m, 1H, H2-3), 2.78 (s, 1H, H4-5), 2.75 (s, 1H, H2-5), 2.70-2.44 (m, 4H, CH2-Lev),


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2.05 (s, 3H, CH3-Lev), 1.74 (s, 3H, CH3-NHAc), 1.35 (s, 3H, CH3-NHAc);

13

C NMR (125 MHz,

CDCl3): δ 206.8, 172.2, 171.3, 170.8, 165.43, 165.36, 165.22, 165.16, 159.5, 159.3, 155.7, 151.4, 138.1, 137.8, 133.3, 133.22, 133.15, 133.0, 130.5, 130.2, 129.9, 129.9, 129.8, 129.7, 129.6, 129.4, 129.0, 128.7, 128.5, 128.4, 128.1, 128.0, 126.6, 126.5, 119.0, 114.6, 114.0, 113.8, 101.1, 100.7, 100.7, 100.4, 100.0, 98.8, 76.1, 75.7, 75.4, 75.3, 75.0, 74.24, 74.15, 74.1, 73.4, 73.0, 72.9, 72.7, 72.4, 70.3, 68.5, 68.5, 68.0, 66.6, 66.3, 55.7, 55.4, 53.6, 52.4, 38.0, 29.5, 28.2, 23.5, 23.1. HRMS (ESI-TOF) m/z [M+Na]+ Calcd. for C98H100N2NaO30 1807.6259, found 1807.6231. Methy [p-Methoxyphenyl O-(2-Acetamido-4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-β-ᴅ-galacto pyranosyl)-(1→4)-O-(methy 2,3-di-O-benzoyl-β-ᴅ-glucopyranosyluronate)-(1→3)-O-(2-Acetamido -4,6-O-benzylidene-2-deoxy-β-ᴅ-galactopyranosyl)-(1→4)-(2,3-di-O-benzoyl-β-ᴅ-glucopyranoside)] Uronate (23). Method a: To azide 18 (100 mg, 0.064 mmol) in pyridine (0.8 mL) was added 3.2 mL of AcSH while stirring. After 8 h at 60°C, additional 1 mL of pyridine and 1.5 mL of AcSH were added, and stirring was continued for 16 h. The reaction mixture was concentrated, and the residue was subjected to silica gel column chromatography (CH2Cl2→CH2Cl2/Acetone, 20:1→CH2Cl2/MeOH, 30:1) to give compoud 23 (66.0 mg, 65%) as a white solid. Method b: Water (0.8 mL), triethylamine (247 µL, 1.77 mmol), and 1,3-propanedithiol (238 µL, 2.37 mmol) were added to a solution of tetrasaccharide 18 (93 mg, 0.059 mmol) in pyridine (3.3 mL), and the resulting mixture was stirred for 11 h at room temperature. After that, the reaction mixture was concentrated and dried in vacuo. The crude residue was dissolved in MeOH (2 mL) and CH2Cl2 (2 mL), Ac2O (200 µL, 0.24 mmol) was added, and the resulting suspension was stirred under argon for 1 h at room temperature. The reaction mixture was then concentrated and dried in vacuo. The residue was purified by silica gel column chromatography (CH2Cl2→CH2Cl2/Acetone, 20:1→CH2Cl2/MeOH, 30:1) to afford compound 23 (58



mg, 61%) as a white solid. 1H NMR (600 MHz, CDCl3): δ 8.05 (d, J = 7.2 Hz, 2H, ArH-Bz), 7.98-7.93 (m, 6H, ArH-Bz), 7.59-7.25 (m, 22H, ArH-Bz, benzylidene), 6.90 (d, J = 9.0 Hz, 2H, ArH-OMP), 6.75 (d, J = 9.6 Hz, 2H, ArH-OMP), 5.76 (t, J = 9.0 Hz, 1H, H1-3), 5.60 (dd, J = 7.8, 4.2 Hz, 1H, H3-3), 5.57 (s, 1H, CH3-benzylidene), 5.53 (dd, J = 8.4, 7.2 Hz, 1H, H1-2), 5.50 (d, J = 6.6 Hz, 1H, NH), 5.25 (s, 1H, CH1-benzylidene), 5.23 (t, J = 4.2 Hz, 1H, H3-2), 5.19 (d, J = 9.0 Hz, 1H, H1-1), 5.18 (d, J = 6.6 Hz, 1H, H4-1), 5.04 (d, J = 4.2 Hz, 1H, H3-1), 4.75 (dd, J = 9.6, 7.8 Hz, 1H, H3-4), 4.70 (dd, J = 10.8, 3.6 Hz, 1H, H4-3), 4.56-4.53 (m, 2H, H2-3, H1-4), 4.42 (d, J = 3.6 Hz, 1H, H4-4), 4.39 (d, J = 9.0 Hz, 1H, NH), 4.34 (d, J = 8.4 Hz, 1H, H4-1), 4.30 (d, J = 10.2 Hz, 1H, H1-5), 4.23 (d, J = 9.0 Hz, 1H, H3-5), 4.02-3.98 (m, 1H, H4-2), 4.31 (d, J = 3.6 Hz, 1H, H2-4), 3.80-3.72 (m, 11H, 2H2/4-6a,b, OCH3-COOMe, OCH3-OMP), 3.79 (d, J = 11.4 Hz, 1H, H2/4-6a), 4.49 (d, J = 11.4 Hz, 1H, H2/4-6b), 3.31-3.27 (m, 1H, H4-2), 3.03 (s, 1H, H4-5), 2.71-2.41 (m, 6H, H2-2, H2-5, CH2-Lev), 2.04 (s, 3H, CH3-Lev), 1.80 (s, 3H, CH3-NHAc), 1.79 (s, 3H, CH3-NHAc);

13


(CO-Lev), 171.7 (CO-COOMe), 170.2 (CO-COOMe), 169.3 (CO-NHAc), 167.9 (CO-NHAc), 165.31 (CO-Bz), 165.25 (CO-Bz), 164.93 (CO-Bz), 164.87 (CO-Bz), 155.8, 151.0, 138.7, 137.6, 133.7, 133.6, 133.4, 133.2, 130.1, 130.0, 129.9, 129.7, 129.6, 129.43, 129.36, 129.3, 129.1, 129.0, 128.9, 128.6, 128.49, 128.47, 128.11, 128.08, 126.6, 126.5, 118.8, 114.6, 101.6, 100.8, 100.7, 99.8, 99.7, 99.0, 77.0, 75.9, 75.0, 74.7, 74.6, 73.1, 72.6, 72.5, 72.0, 71.1, 68.8, 68.3, 66.7, 66.2, 55.7 (OCH3-OMP), 55.1, 53.1 (CH3-COOMe), 52.8 (CH3-COOMe), 49.9, 37.9 (CH2-Lev), 29.8 (CH3-Lev), 28.3 (CH2-Lev), 23.6 (CH3-NHAc), 23.5 (CH3-NHAc). HRMS (ESI-TOF) m/z [M+Na]+ Calcd. for C84H84N2NaO30 1623.5007, found 1623.4974. Methy [p-Methoxyphenyl O-(2-Acetamido-4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-β-ᴅ-galacto pyranosyl)-(1→4)-O-(methy 2,3-di-O-benzoyl-β-ᴅ-glucopyranosyluronate)-(1→3)-O-(2-Acetamido-


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Page 37 of 41 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


4,6-O-benzylidene-2-deoxy-β-ᴅ-galactopyranosyl)-(1→4)-(2,3-di-O-benzoyl-β-ᴅ-glucopyranoside)-(1 →3)-O-(2-Acetamido-4,6-O-benzylidene-2-deoxy-β-ᴅ-galactopyranosyl)-(1→4)-(2,3-di-O-benzoyl-βᴅ-glucopyranoside)] Uronate (24). To azide 20 (114.9 mg, 0.051 mmol) in pyridine (1 mL) was added 2 mL of AcSH while stirring. After 7 h at 60°C, additional 0.5 mL of pyridine and 2 mL of AcSH were added, and stirring was continued for 17 h. The reaction mixture was concentrated, and the residue was subjected to silica gel column chromatography (CH2Cl2→CH2Cl2/MeOH, 50:1) to give compoud 24 (61.6 mg, 52%) as a white solid. 1H NMR (600 MHz, CDCl3): δ 8.06-7.92 (m, 12H, ArH-Bz), 7.60-7.25 (m, 33H, ArH-Bz, benzylidene, overlapped with solvent peak), 6.89 (d, J = 9.0 Hz, 2H, ArH-OMP), 6.75 (d, J = 9.6 Hz, 2H, ArH-OMP), 5.74 (t, J = 9.0 Hz, 1H, H1/3/5-3), 5.63 (s, 1H, CH-benzylidene), 5.62-5.58 (m, 2H, H1/3/5-3, 2), 5.52 (dd, J = 8.4, 7.2 Hz, 1H, H1/3/5-2), 5.44 (s, 1H, CH-benzylidene), 5.40 (d, 1H, J = 6.6 Hz, NH), 5.25 (s, 1H, CH-benzylidene), 5.23 (t, J = 5.9 Hz, 1H, H1/3/5-3), 5.18 (d, J = 7.2 Hz, 1H, H1/3/5-1), 5.17 (d, J = 8.4 Hz, 1H, H1/3/5-1), 5.09 (t, 1H, J = 3.3 Hz, H1/3/5-2), 5.00 (d, J = 3.6 Hz, 1H, H1/3/5-1), 4.99 (d, J = 5.4 Hz, 1H, H2/4/6-1),4.81 (d, J = 7.8, 7.8 Hz, 1H, H1/3/5-4), 4.78 (br-d, J = 7.8 Hz, 1H, NH), 4.70 (d, J = 8.4 Hz, 1H, H2/4/6-1), 4.66 (dd, J = 11.4, 3.6 Hz, 1H, H6-3), 4.63 (t, J = 8.4 Hz, 1H, H1/3/5-4), 4.52 (t, J = 9.0 Hz, 1H, H1/3/5-4), 4.43 (d, J = 3.6 Hz, 1H, H6-4), 4.37 (br-d, J = 7.2 Hz, 1H, H2/4-3), 4.32-4.31 (m, 2H, H1/3/5-5, H2/4-4), 4.22 -4.19 (m, 3H, H2/4/6-1, 2H1/3/5-5), 4.04-4.02 (m, 2H, H2/4/6-2, H2/4-3), 3.88 (d, J = 3.6 Hz, 1H, H2/4-4), 3.85 (d, 2H, J = 12.0 Hz, H2/4/6-6a,b), 3.78 (d, J = 11.4 Hz, 1H, H2/4/6-6a), 3.73-3.64 (m, 13H, OCH3-OMP, COOMe, H2/4/6-6b), 3.60-3.56 (m, 1H, H2/4/6-2), 3.51(d, J = 11.4 Hz, 2H, H2/4/6-6a,b), 3.26-3.22 (m, 1H, H2/4/6-2), 2.99 (s, 1H, H2/4/6-5), 2.80 (s, 1H, H2/4/6-5), 2.70-2.39 (m, 5H, H2/4/6-5, CH2-Lev), 2.03 (s, 3H, CH3-Lev), 1.75 (s, 6H, CH3-NHAc), 1.65 (s, 3H, CH3-NHAc);

13


(CO-Lev), 171.7, 170.9, 170.0, 169.6, 168.8, 167.9, 165.3 (CO-Bz), 165.2 (CO-Bz), 165.0 (3C,



CO-Bz), 164.9 (CO-Bz), 155.8, 151.0, 138.64, 138.58, 137.6, 133.7 (2C), 133.5, 133.44, 133.35, 133.2, 130.1, 130.0, 129.9, 129.8, 129.7, 129.6, 129.4, 129.30, 129.25, 129.1, 129.0, 128.9, 128.8, 128.70, 128.65, 128.6, 128.5, 128.5, 128.2, 128.1, 128.1, 126.7, 126.6, 126.5, 118.8 (2C), 114.6 (2C), 101.8, 100.8, 100.6, 100.3, 100.0, 99.9, 99.4, 99.4, 98.9, 76.4, 76.3, 75.9, 75.2, 75.1, 74.9, 74.7, 74.5, 74.2, 73.3, 73.1, 72.9, 72.5, 72.4, 72.2, 72.0 (2C), 71.3, 68.8, 68.7, 68.3, 66.7, 66.6, 66.2, 55.8 (OCH3-OMP), 54.9, 53.3 (CH3-COOMe), 53.1, 52.9 (CH3-COOMe), 52.8 (CH3-COOMe), 49.4, 37.8 (CH2-Lev), 29.8 (CH3-Lev), 28.3 (CH2-Lev), 23.5 (CH3-NHAc), 23.5 (CH3-NHAc), 23.4 (CH3-NHAc). HRMS (ESI-TOF) m/z [M + Na]+ Calcd for C120H119N3NaO43 2312.7115, found 2312.7107.

ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. 1

HNMR and 13CNMR spectra of compounds 2, 4-7, 9-24 and the target CS-E tetrasaccharide and

hexasaccharide; DEPT spectra of compounds 12, 14, 16, 18-20, 23-24, the target CS-E tetrasaccharide and hexasaccharide; HSQC spectra of compounds 12, 14, 16-20, 23-24 and the target CS-E tetrasaccharide (PDF)

AUTHOR INFORMATION

Corresponding Author *

E-mail: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS

This work was supported by the CAMS Innovation Fund for Medical Sciences (Item Number:


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2017-I2M-3-011 and 2016-I2M-3-009).

REFERENCES

(1) Djerbal, L.; Lortat-Jacob, H.; Kwok, J. Glycoconj J. 2017, 34, 363-376. (2) (a) Bergefall, K.; Trybala, E.; Johansson, M.; Uyama, T.; Naito, S.; Yamada, S.; Kitagawa, H.; Sugahara, K.; Bergström, T. J Biol Chem. 2005, 280, 32193-32199. (b) Kato, D.; Era, S.; Watanabe, I.; Arihara, M.; Sugiura, N.; Kimata, K.; Suzuki, Y.; Morita, K.; Hidari, KI.; Suzuki, T. Antiviral Res. 2010, 88, 236-243. (3) Mizumoto, S.; Fongmoon, D.; Sugahara, K. Glycoconj J. 2013, 30, 619-632. (4) Gama, CI.; Tully, SE.; Sotogaku, N.; Clark, PM.; Rawat, M.; Vaidehi, N.; Goddard, WA.; Nishi, A.; Hsieh-Wilson, LC. Nat Chem Biol. 2006, 2, 467-473. (5) Sotogaku, N.; Tully, SE.; Gama, CI.; Higashi, H.; Tanaka, M.; Hsieh-Wilson, LC.; Nishi, A. J Neurochem. 2007, 103, 749-760. (6) Tamura, J.; Neumann, K.W.; Kurono, S.; Ogawa, T. Carbohydr. Res.1998, 305, 43–63. (7) Tamura, J.; Tokuyoshi, M. Biosci. Biotechnol. Biochem. 2004, 12, 2436–2443. (8) Tamura, J.; Nakada, Y.; Taniguchi, K.; Yamane, M. Carbohydr. Res. 2008, 343 , 39–47. (9) Tully, S. E.; Ross, M.; Gama, C. I.; Tsai, S. M.; Xuewei, L.; Hsieh-Wilson, L. C. J. Am. Chem. Soc. 2004, 126, 7736-7737. (10) Vibert, A.; Lopin-Bon, C.; Jacquinet, J.-C. Chem. Eur. J. 2009, 15, 9561–9578. (11) Jacquinet, J.-C.; Lopin-Bon, C.; Vibert, A. Chem. Eur. J. 2009, 15, 9579–9595. (12) Vibert, A.; Lopin-Bon, C.; Jacquinet, J.-C. Eur. J. Org. Chem. 2011, 22, 4183–4204. (13) Lopin, C.; Jacquinet, J.-C. Angew. Chem. Int. Ed. 2006, 45, 2574-2578. (14) Macchione, G.; Maza, S.; Kayser, M. M.; de Paz, J. L.; Nieto, P. M., Eur. J. Org. Chem. 2014, 18,



3868-3884. (15) Solera, C.; Macchione, G.; Maza, S.; Kayser, M. M.; Corzana, F.; de Paz, J. L.; Nieto, P. M., Chem. Eur. J. 2016, 22, 2356-2369. (16) Miyachi, K.; Wakao, M.; Suda, Y. Bioorg Med Chem Lett. 2015, 25, 1552-1555. (17) Wakao, M.; Obata, R.; Miyachi, K.; Kaitsubata, Y.; Kondo, T.; Sakami, C.; Suda, Y. Bioorg Med Chem Lett. 2015, 25, 1407-1411. (18) Shuang, Y.; Apeng, W.; Guangyan, Z.; Xiangjie, D.; Zhehui, Z.; Pingsheng, L. Tetrahedron 2016, 72, 5659-5670. (19) Jacquinet, J.-C. Carbohydrate Research 1990, 199, 153-181. (20) Tamura, J.; Neumann, K. W.; Ogawa, T. Liebigs Ann. 1996, 8, 1239-1257. (21) Cirillo, L.; Bedini, E.; Molinaro, A.; Parrilli, M. Tetrahedron Lett. 2010, 51, 1117-1120. (22) Lassaletta, J.; Schmidt, RR. Tetrahedron Lett. 1995, 36, 4209-4212. (23) Lassaletta, Jose´ M.; Carlsson, Karin; Garegg, Per J.; Schmidt, RR. J. Org. Chem. 1996, 61, 6873-6880. (24) Vankar, Y. D.; Vankar, P. S.; Behrendt, M.; Schmidt, RR. Tetrahedron 1991, 47, 9981-9992. (25) Wulff, G.; Röhle, G. Angew. Chem. Int. Ed. 1974, 13, 157-170. (26) Banoub, J.; Boullanger, P.; Lafont, D. Chem. Rev. 1992, 92,1167-1195. (27) Schmidt, R. R. Angew. Chem. Int. Ed. 1986, 25, 212-235. (28) Tully, SE.; Rawat, M.; Hsieh-Wilson, LC. J. Am. Chem. Soc. 2006, 128, 7740-7741. (29) Aït-Mohand, K.; Mirault, A.; Jacquinet, JC.; Lopin-Bon, C. Org. Biomol. Chem., 2016, 14, 7962-7971. (30) Tamura, J.; Tsutsumishita-Nakai, N.; Nakao, Y.; Kawano, M.; Kato, S.; Takeda, N.; Nadanaka, S.;


Page 40 of 41

Page 41 of 41 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


Kitagawa, H. Bioorg. Med. Chem. Lett. 2012, 22, 1371-1374. (31) Domínguez-Rodríguez, P.; Reina, JJ.; Gil-Caballero, S.; Nieto, PM.; de Paz, JL.; Rojo, J. Chem. Eur. J. 2017, 23, 11338-11345. (32) Rawat, M.; Gama, C.; Matson, JB.; Hsieh-Wilson, LC. J. Am. Chem. Soc. 2008, 130, 2959-2961. (33) Liu, P.; Chen, L.; Toh, JKC.; Ang, YL.; Jee, JE.; Lim, J.; Lee, SS.; Lee, SG. Chem. Sci. 2015, 6, 450-456. (34) Huang, L.; Huang, X. Chem. Eur. J. 2007, 13, 529-540.


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