Chemical Synthesis of the Repeating Unit of Type II Group B

Subscriber access provided by Kaohsiung Medical University

Article

Chemical Synthesis of the Repeating Unit of Type II Group B Streptococcus Capsular Polysaccharide Liming Shao, Han Zhang, Yaoyao Li, Guofeng Gu, Feng Cai, Zhongwu Guo, and Jian Gao J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b00396 • Publication Date (Web): 30 Apr 2018 Downloaded from http://pubs.acs.org on May 4, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30 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

The Journal of Organic Chemistry

Chemical Synthesis of the Repeating Unit of Type II Group B Streptococcus Capsular Polysaccharide Liming Shao,† Han Zhang,† Yaoyao Li,† Guofeng Gu,† Feng Cai,† Zhongwu Guo,*,†,‡ and Jian Gao*,† †

National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Jinan, Shandong 250100, China

‡

Department of Chemistry, University of Florida, 214 Leigh Hall, Gainesville, Florida 32611, United States

Abstract. The first chemical synthesis of the repeating unit of serotype II group B Streptococcus capsular polysaccharide, a branched heptasaccharide α-Neup5Ac-(2→3)-β-D-Galp-(1→4)-β-DGlcNpAc-(1→3)-{[β-D-Galp-(1→6)]-β-D-Galp}-(1→4)-β-D-Glcp-(1→3)-β-D-Glcp-(1→, was achieved by convergent [4 + 2 + 1] glycosylation, after probing different synthetic strategies and overcoming a series of difficulties. The title compound was designed to carry a free amino group at its downstream end to enable further regioselective elaboration. This work also revealed that the

-Neu5Ac-(2→3)--D-Gal-(1→4)--D-GlcNAc motif, which is common in natural glycans, had low reactivity as glycosyl donors so it was rather difficult to directly couple this trisaccharide with sterically hindered acceptors. The motif was efficiently constructed via on-site glycan elongation using properly protected GlcN and -Neu5Ac-(2→3)--D-Gal as consecutive glycosyl donors.

Introduction Group B Streptococcus (GBS) is one of the primary causes of bacterial infections among neonates and pregnant women, resulting in many severe diseases such as sepsis, meningitis, abortion and so on.1-3 GBS can be classified into at least ten different serotypes according to their structurally distinctive capsular polysaccharides (CPSs).4,5 Type II GBS is associated with about 15% of the invasive infections in adults and infants;6,7 therefore, it represents an important human pathogen. The structure of type II GBS CPS (1, Figure 1) was characterized by Jennings et al. in 1983.8 It consists of a repeating heptasaccharide having a pentasaccharide backbone, →2-β-D-Galp-(1→4)β-D-GlcNpAc-(1→3)-β-D-Galp-(1→4)-β-D-Glcp-(1→3)-β-D-Glcp-(1→, and two decorating 1 ACS Paragon Plus Environment

The Journal of Organic Chemistry 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

monosaccharide branches, an α-Neup5Ac linked to the GalV 3-O-position and a β-D-Galp linked to the GalIII 6-O-position.8 Direct linkage of an α-NeuNAc moiety to the main polysaccharide chain is a unique structural feature in type II and type VIII GBS CPSs.

Figure 1. Structures of type II GBS CPS 1 and synthetic target 2 CPSs have been shown to be excellent haptens for developing carbohydrate-based antibacterial vaccines.9-12 However, natural CPSs are structurally heterogeneous, so homogeneous CPSs needed for various studies are difficult to access from biological sources. As a result, chemical synthesis to obtain structurally well-defined repeating units of CPSs and related derivatives has become an attractive topic, because these synthetic oligosaccharides would enable not only detailed structural, functional and other biological studies of CPSs but also the development of novel bacterial detection methods and fully and semi-synthetic CPS-based glycoconjugate vaccines.13,14 The latter strategy has been extensively explored and demonstrated.12-14 Presently, there has been no reported synthesis of the repeating unit of type II GBS CPS yet. This unit has two branches and can be presented in multiple forms. Heptasaccharide 2 (Figure 1) was selected as our first synthetic target and model compound for probing the chemical synthesis and immunological properties since the -Neu5Ac-(2→3)--D-Gal-(1→4)--D-GlcNAc trisaccharide at the non-reducing end is a rather common motif in the structure of GBS CPSs, including that of serotypes Ia, IV, V, VII and IX.4,5 It has been also discovered that to confer immune protection it is not always a prerequisite for the conjugate vaccines to contain the whole repeating sequence of bacterial CPSs.15 The synthetic target was designed to have an aminoethyl group at the downstream end to facilitate its subsequent regioselective elaboration, such as conjugation with carrier proteins for the preparation of synthetic glycoconjugate vaccines. 2 ACS Paragon Plus Environment

Page 2 of 30

Page 3 of 30 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


Results and Discussion We envisioned two viable synthetic strategies for 2 via protected 3 (Scheme 1), both of which were supposed to have the first bond cleavage around the branched sugar unit. One strategy was to disconnect the glycosidic linkage between GlcNAcIV and GalIII in 3a (Scheme 1A), resulting in trisaccharide 4 and tetrasaccharide 5. This might lead to a highly convergent [4 + 3] glycosylation scheme. In turn, trisaccharide 4 could be derived from 616 and 717 by Huang and Ye’s preactivationbased glycosylation,16,18 and tetrasaccharide 5 could be convergently constructed from disaccharides 8 and 9 that would be assembled from monosaccharides 10-13. Alternatively, disconnection of the glycosidic bond between the side chain Gal and main chain GalIII in 3b (Scheme 1B) would result in monosaccharide donor 15 and the key linear hexasaccharide 14, which could be assembled from trisaccharides 4 and 16. In both designs, acyl groups would be used to protect the 2-O-position of glycosyl donors including 8, 10-12 and 15, since the capability of acyl groups to participate in the glycosylation process was expected to promote stereoselective 1,2-trans-glycosylation. Scheme 1. Retrosynthetic Analysis of 3

3 ACS Paragon Plus Environment


First, we attempted the [4 + 3] synthetic strategy (Scheme 1A). The synthesis was commenced with preparing monosaccharides 10-12 according to reported procedures.16,19 In the meantime, the terminal Glc residue 13 was prepared from 1720 through a series of established transformations including tin-mediated regioselective 3-O-allylation, removal of the benzylidene group under acidic conditions, 2,4,6-O-benzylation under basic conditions, and PdCl2-catalyzed deallylation (Scheme 2). Subsequently, 13 was glycosylated with thioglycoside 12 in the presence of N-iodosuccinimide (NIS) and triflic acid (TfOH) to afford disaccharide 19 in a good yield (85%) and stereoselectivity. The newly formed β-glycosidic linkage was confirmed by its 1H NMR data, which showed a large coupling constant (7.8 Hz) between H-1' and H-2'. Finally, the benzylidene ring in 19 was opened regioselectively upon reaction with NaBH3CN and HCl to provide 9, one of the two disaccharides employed to construct tetrasaccharide 5. Scheme 2. Synthesis of Disaccharide 9

On the other hand, disaccharide 8 was obtained upon glycosylation of 11 with 10 under the Schmidt condition, namely, utilizing trimethylsilyl triflate (TMSOTf) as the promoter (Scheme 3). Once disaccharides 8 and 9 were in hand, we performed their glycosylation reaction under the promotion of NIS and TfOH, which gave the desired tetrasaccharide 20 in a good yield (83%) and stereoselectivity. The β-configuration of the new glycosidic linkages in 20 was confirmed by its 1

H NMR spectrum, which exhibited large coupling constants for both anomeric protons (JH1'',H2'' =

7.8 Hz, JH1''',H2''' = 7.8 Hz). Eventually, the Fmoc group in 20 was selectively removed upon Et3N treatment to afford tetrasaccharide 5 smoothly.


Page 4 of 30

Page 5 of 30 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


Scheme 3. Synthesis of Tetrasaccharide 5

For the final-stage assembly of 3a by the [3 + 4] strategy (Scheme 4), we attempted first a onepot glycosylation approach. Preactivation of thioglycoside 6 with stoichiometric p-toluenesulfenyl triflate (TolSOTf) generated in situ from the reaction of p-toluenesulfenyl chloride (TolSCl) with silver triflate (AgOTf) at -78 ℃ was followed by the addition of 7 at the same temperature. The reaction was allowed to warm up to room temperature and stirred for another 20 min to finish the first glycosylation reaction (→4a).16 Thereafter, 5 was added to the reaction mixture, which was followed by adding AgOTf and TolSCl to perform the next glycosylation. To our disappointment, however, only a trace amount of desired heptasaccharide 3a was observed by MS with the recovery of glycosyl acceptor 5, whereas glycosyl donor 4a was completely consumed to give the hydrolytic product. To improve the reaction, other promoters such as NIS/TfOH and NIS/AgOTf systems were probed (Scheme 4), but both conditions gave low to negligible yields of the desired product. Next, trisaccharide 4a was converted into the more reactive glycosyl phosphate donor 4b by reaction with dibutyl phosphate under the promotion of NIS and TfOH. Its coupling reaction with 5 in the presence of TMSOTf was also attempted but failed. At this point, we suspected that the increased steric hindrance in 5, due to the presence of a side chain at the GalIII 6-O-position, might have rendered the 3-OH group of GalIII unreactive.



Scheme 4. Attempted Assembly of 3a by the [3 + 4] Strategy

To overcome the problem, next, we attempted plan B shown in Scheme 1, namely, introducing the side chain on the GalIII 6-O-position at the final synthetic stage. To prepare the required linear hexasaccharide 14, monosaccharide 21, obtained upon silylation of 11, was glycosylated with 9 in the presence of NIS and TfOH, which was followed by removal of the Fmoc group with Et3N to afford 16 in a 66% yield from 11 (Scheme 5). To our disappointment, however, the reactions of 16 with glycosyl donors 4a and 4b in the presence of various promoters including NIS/TfOH, NIS/AgOTf, TolSCl/AgOTf and TMSOTf failed to provide an acceptable yield of the desired hexasaccharide 23, once again with consumption of donors to give the hydrolytic product. The highest yield (15%) of 23 was observed with NIS/TfOH as the promoter. These results indicated that probably the failed [3 + 4] glycosylation in Scheme 4 was not mainly due to the presence of the side chain at the GalIII 6-O-position of 5. Instead, it was more likely because of the relatively low reactivity of sialotrisaccharide 4 as a donor, although 4a was used to successfully glycosylate a tetrasaccharide acceptor containing a more reactive primary hydroxyl group.16


Page 6 of 30

Page 7 of 30 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


Scheme 5. Attempted Synthesis of 23 as a Precursor of 14 by a [3 + 3] Strategy

Consequently, we had to revise our synthetic design for the linear hexasaccharide. Our new plan was to perform on-site assembly of the sialotrisaccharide (Scheme 6), namely, to attach a less bulky monosaccharide to 16 first followed by elongation of the carbohydrate chain using a disaccharide such as 6. Delightfully, the reaction between 16 and 2419 in the presence of NIS and TfOH went smoothly to afford the desired tetrasaccharide 25. It was followed by Et3N-mediated removal of the Fmoc group to give 26. The stereochemistry of the new glycosidic bond was confirmed by 1H NMR spectrometry (JH1''',2''' = 8.4 Hz). This result further indicated that the failed glycosylation reactions in Schemes 4 and 5 were probably because of the low reactivity of trisaccharide 4.21 It was worth noting that the glycosylation reaction of 16 with a 4-O-Fmoc derivative of diol 7 gave a pentasaccharide byproduct, which could be avoided by using donor 24. Subsequently, 26 was glycosylated with sialodisaccharide 6 under the promotion of NIS and TfOH to provide 27 (JH1'''',2'''' = 8.4 Hz) smoothly, which was eventually desilylated with 3HF•Et3N to furnish 14. At this stage, the galactose side chain was introduced to 14 via glycosylation with 15 to finally produce the fully protected heptasaccharide 3b.



Scheme 6. Optimized Synthesis of the Target Molecule 2

Global deprotection of 3b that contained different types of protecting groups, including acyl, amido, azido, benzylidene, benzyl, carboxylate, and carbamate groups, was realized by a five-step protocol (Scheme 6). First, 3b was treated with LiI in anhydrous pyridine at refluxing temperature to selectively deprotect the sialic acid methyl ester.22 Next, the carbamate and acyl groups in the resultant intermediate were simultaneously removed using hydrazine in EtOH at refluxing temperature.17,23 It was followed by selective N-acetylation of the exposed amino groups in two steps including acetylation using acetic anhydride and pyridine and selective removal of O-acetyl groups using NaOMe at room temperature. The N-acetylated intermediate was purified with a Sephadex LH-20 column and then subjected to Pd(OH)2-catalyzed hydrogenolysis in a mixture of MeOH and H2O (4:1) to remove the remaining benzyl and benzylidene groups and reduce the 8 ACS Paragon Plus Environment

Page 8 of 30

Page 9 of 30 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


azido group. The target heptasaccharide 2 was eventually obtained in a 48% overall yield after purification on a Sephadex G-25 column with H2O as the eluent and lyophilization. The structure of 2 was rigorously confirmed by 1D and 2D NMR and HR MS. It is also worth noting that careful monitoring of the reaction progress in every step by TLC and MS during the deprotection process was important because the completion of each reaction could facilitate convenient purification of the final product 2.

Conclusion We have achieved the first chemical synthesis of the repeating unit of type II GBS CPS. After probing different synthetic strategies, it was found that the target molecule could be efficiently synthesized via [4 + 2 + 1] glycosylation and the tetrasaccharide was constructed through linear assembly. All glycosylation reactions were optimized to produce excellent yields and stereoselectivity. Disaccharide 6 containing the α-NeuNAc epitope was assembled at the very beginning of the synthesis and utilized as a building block in subsequent reactions to avoid the necessity to construct the difficult α-sialyl bond at a later stage. Furthermore, we have also found that the sialotrisaccharide moiety, α-Neu5Ac-(2→3)-β-D-Gal-(1→4)-β-D-GlcNAc, which is quite common in natural glycans including cancer-associated carbohydrate antigens, had low reactivity as a glycosyl donor, thus it can only be used to glycosylate reactive glycosyl acceptors as shown previouly.15 On the other hand, disaccharide 6 was demonstrated to be a very robust glycosyl donor, while similar donors were utilized by different groups for the synthesis of other glycans containing the same trisaccharide unit.19,24-35 Consequently, for the construction of this difficult trisaccharide in the context of complex glycan syntheses, a relatively reliable strategy would be on-site assembly, i.e., to introduce GlcNAc to the main carbohydrate chain first and then attach the sialodisaccharide. Alternatively, Pozsgay and Jennings et al36-39 developed an efficient chemo-enzymatic strategy for the synthesis of type Ia and III GBS oligosaccharides. All of these works together with the method established in this paper have provided useful solutions for the preparation of glycans containing the challenging α-Neu5Ac-(2→3)-β-D-Gal-(1→4)-β-D-GlcNAc motif. It is also worth mentioning that the free amino group in the structure of 2 would enable its further regioselective elaboration, such as conjugation with other molecules, to obtain compounds useful for many biological studies including the development of novel anti-GBS vaccines. 9 ACS Paragon Plus Environment


Experimental Section General Experimental Methods: Chemical and materials purchased from commercial sources were used as received without further purification unless otherwise noted. Molecular sieve 4Å (MS 4Å) was flame-dried under high vacuum and cooled under a N2 atmosphere immediately before use. Analytical TLC was carried out on Silica Gel 60Å F254 plates with detection by a UV detector and/or by charring with 15% H2SO4 in EtOH (w/v). Mass spectrometry (MS) was performed on a high resolution ESI-TOF MS machine. NMR spectra were recorded on a 600 MHz machine with chemical shifts reported in ppm (δ) downfield from internal tetramethylsilane (TMS) reference. Signals were described as s (singlet), d (doublet), t (triplet), or m (multiplet), and the coupling constants were reported in Hz. 2-Azidoethyl 3-O-Allyl-4,6-benzylidene-β-D-glucopyranoside (18). A mixture of 17 (1.8 g, 4.811 mmol) and Bu2SnO (2.4 g, 9.623 mmol) in toluene (70 mL) was refluxed under a N2 atmosphere for 8 h. After the reaction was cooled to room temperature, toluene was removed under vacuum, and the residue was dissolved in 20 mL of anhydrous DMF and mixed with CsF (2.2 g, 14.434 mmol) and AllylBr (0.6 mL, 7.217 mmol). The mixture was stirred at room temperature for 24 h, at the end of which time TLC indicated a complete reaction. The solution was diluted with EtOAc (200 ml) and washed with saturated aq. NaCl solution. The organic layer was dried over Na2SO4 and then concentrated under vacuum. The residue was purified by silica gel column chromatography with EtOAc and hexanes (1:3) as the eluents to give 18 (1.2 g, 65%) as colorless syrup. 1H NMR (600 MHz, CDCl3) δ: 7.48 (d, J = 7.2 Hz, 2H, Ph), 7.40 – 7.33 (m, 3H, Ph), 5.99 – 5.91 (m, 1H, -CH2CH=CH2), 5.55 (s, 1H, Ph-CH-), 5.31 (d, J = 17.4 Hz, 1H, -CH=CH2), 5.19 (d, J = 10.2 Hz, 1H, -CH2CH=CH2), 4.46 (d, J = 7.8 Hz, 1H, H-1Glc), 4.43 (dd, J = 12.6, 6.0 Hz, 1H, -OCH2CH=CH2), 4.34 (dd, J = 10.8, 4.8 Hz, 1H, H-6aGlc), 4.28 (dd, J = 12.6, 6.0 Hz, 1H, OCH2CH=CH2), 4.09 – 4.04 (m, 1H, -OCH2CH2-), 3.82 – 3.76 (m, 2H, H-6bGlc, -OCH2CH2-), 3.65 (t, J = 9.0 Hz, 1H, H-4Glc), 3.60 (t, J = 9.0 Hz, 1H, H-3Glc), 3.57 – 3.51 (m, 2H, H-2Glc, CH2CH2N3), 3.45 (td, J = 9.0, 4.8 Hz, 1H, H-5Glc), 3.42 – 3.37 (dt, J = 13.2, 4.2 Hz, 1H, CH2CH2N3), 2.60 (s, 1H, -OH). 13C NMR (150 MHz, CDCl3) δ: 137.1, 134.8, 129.0, 128.3, 126.0, 117.5, 103.4 (C-1), 101.2 (Ph-CH-), 81.2, 79.8, 74.1, 73.6, 69.0, 68.6, 66.5, 50.7. HR ESI-TOF MS (m/z): calcd for C18H24N3O6 [M + H]+, 378.1665; found 378.1669.


Page 10 of 30

Page 11 of 30 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


2-Azidoethyl 2,3,6-tri-O-Benzyl-β-D-glucopyranoside (13). A mixture of 18 (1.0 g, 2.653 mmol) in 10% TFA in CH2Cl2 (20 mL) and H2O (2 mL) was stirred at room teperature for 2 h, at which time TLC indicated a complete reaction. The mixture was co-evaporated with toluene under vacuum to provide a residue. To a solution of this residue in 20 mL of anhydrous DMF was added NaH (530 mg, 13.257 mmol) and BnBr (1.3 mL, 10.606 mmol) at 0 ℃ under a N2 atmosphere. One hour later, CH3OH was added slowly to quench the reaction before H2O was added. The aqueous phase was extracted with EtOAc (3 × 100 mL), and the organic layer was combined, dried over Na2SO4 and concentrated under vacuum. The residue was purified by a flash silica gel column chromatography with EtOAc and hexanes (1:11) as the eluents to give colorless syrup. The solution of this residue in CH3OH-CH2Cl2 (V/V, 1:1, 30 mL) was mixed with PdCl2 (50 mg) and stirred under a N2 atmosphere for 2 h, at which time TLC indicated a complete reaction. The mixture was filtered, and the filtrate was then concentrated under vacuum. The residue was purified by silica gel chromatography with EtOAc and hexanes (1:5) as the eluents to afford 13 (964 mg, 70%) as colorless syrup. 1H NMR (600 MHz, CDCl3) δ: 7.40 – 7.23 (m, 15H, Ph), 4.99 (d, J = 11.4 Hz, 1H, Bn), 4.85 (d, J = 11.4 Hz, 1H, Bn), 4.69 (d, J = 11.4 Hz, 1H, Bn), 4.62 (d, J = 12.6 Hz, 1H, Bn), 4.57 – 4.54 (m, 2H, Bn), 4.42 (d, J = 7.8 Hz, 1H, H-1Glc), 4.10 – 4.06 (m, 1H, OCH2CH2-), 3.76 – 3.67 (m, 4H, H-3Glc, -OCH2CH2-), 3.54 – 3.50 (m, 2H, -CH2CH2N3), 3.49 – 3.46 (m, 1H), 3.45 – 3.41 (m, 1H, -CH2CH2N3), 3.31 (dd, J = 9.0, 7.8 Hz, 1H, H-2Glc), 2.47 (s, 1H, -OH).

13

C NMR (150 MHz, CDCl3) δ: 138.4, 138.2, 138.1, 128.6, 128.4, 128.3, 128.1, 128.0,

127.9, 127.8, 127.7, 127.6, 103.2 (C-1), 81.3, 76.6, 74.8, 74.6, 74.5, 73.5, 68.9, 68.1, 51.0. HR ESI-TOF MS (m/z): calcd for C29H33N3O6Na [M + Na]+, 542.2267; found, 542.2272. 2-Azidoethyl

(2-O-Benzoyl-3-O-benzyl-4,6-benzylidene-β-D-glucopyranosyl)-(1→3)-2,3,6-

tri-O-benzyl-β-D-glucopyranoside (19). To a stirred mixture of 13 (700 mg, 1.349 mmol), 12 (919 mg, 1.618 mmol) and freshly activated MS 4Å in anhydrous CH2Cl2 (10 ml) were added NIS (546 mg, 2.427 mmol) and TfOH (14 μL, 0.162 mmol) under a N2 atmosphere at 0 ℃. After the mixture was stirred for another 30 min, it was neutralized with Et3N, diluted with CH2Cl2 (50 ml) and filtered. The filtrate was washed with saturated aq. Na2S2O3 and brine, dried over Na2SO4, and concentrated under vacuum. The residue was purified by silica gel column chromatography with EtOAc and toluene (1:30) as the eluents to give 19 (1.1 g, 85%) as a white solid. 1H NMR (600 MHz, CDCl3) δ: 7.92 – 7.89 (m, 2H, Ph), 7.57 – 7.51 (m, 3H, Ph), 7.45 – 7.25 (m, 20H, Ph), 7.20 11 ACS Paragon Plus Environment


– 7.10 (m, 5H, Ph), 5.58 (s, 1H, Ph-CH-), 5.35 (t, J = 7.8 Hz, 1H, H-2Glc-A), 5.29 (d, J = 7.8 Hz, 1H, H-1Glc-A), 5.00 (d, J = 10.2 Hz, 1H, Bn), 4.84 (d, J = 12.0 Hz, 1H, Bn), 4.77 – 4.71 (m, 2H, Bn), 4.57 (d, J = 12.0 Hz, 1H, Bn), 4.52 (d, J = 12.0 Hz, 1H, Bn), 4.49 (d, J = 10.2 Hz, 1H, Bn), 4.34 (d, J = 10.8 Hz, 1H, Bn), 4.33 – 4.30 (m, 2H, H-1Glc-B), 4.03 (t, J = 8.4 Hz, 1H, H-3Glc-B), 3.96 (m, 1H, -OCH2CH2-), 3.86 – 3.82 (m, 2H, H-3Glc-A), 3.71 – 3.58 (m, 4H, -OCH2CH2-), 3.49 (dd, J = 9.6, 8.4 Hz, 1H, H-4Glc-B), 3.44 – 3.38 (m, 3H, -CH2CH2N3, H-5Glc-B). 3.35 – 3.31 (m, 1H, CH2CH2N3), 3.25 (dd, J = 9.0, 7.8 Hz, 1H, H-2Glc-B). 13C NMR (150 MHz, CDCl3) δ: 165.2, 138.4, 138.3, 138.0, 137.7, 137.2, 133.2, 129.8, 129.6, 129.0, 128.5, 128.38, 128.37, 128.35, 128.3, 128.29, 128.23, 128.21, 128.1, 127.9, 127.7, 127.69, 127.67, 127.64, 127.3, 126.0, 103.2 (C-1GlcB

), 101.2 (Ph-CH-), 101.0 (C-1Glc-A), 82.6, 82.1, 80.6, 77.7, 75.6, 75.0, 74.6, 74.3, 74.0, 73.8, 73.5,

68.8, 68.7, 68.0, 66.1, 50.9. HR ESI-TOF MS (m/z): calcd for C56H57N3O12Na [M + Na]+, 986.3840; found, 986.3845. 2-Azidoethyl (2-O-Benzoyl-3,6-di-O-benzyl-β-D-glucopyranosyl)-(1→3)-2,3,6-tri-O-benzylβ-D-glucopyranoside (9). After the mixture of 19 (1.0 g, 1.038 mmol), NaBH3CN (978 mg, 15.570 mmol), and freshly activated MS 4Å in anhydrous THF (20 mL) was stirred under N2 protection at room temperature for 2 h, it was cooled to 0 ℃. Then, HCl in dry ether (1 M) was added dropwise until the pH reached 2. The reaction mixture was stirred at 0 ℃ for 1 h and at room temperature for another 2 h, and then quenched with Et3N. The mixture was diluted with CH2Cl2 (100 ml) and filtered. The filtrate was washed with saturated aq. NaHCO3 and brine, dried over Na2SO4, and concentrated under vacuum. The residue was purified by silica gel column chromatography with EtOAc and hexanes (1:7) as the eluents to give 9 (881 mg, 88%) as a white solid. 1H NMR (600 MHz, CDCl3) δ: 7.93 (d, J = 8.4 Hz, 2H, Ph), 7.55 (t, J = 7.2 Hz, 1H, Ph), 7.41 – 4.16(m, 27H, Ph), 5.30 (t, J = 8.4 Hz, 1H, H-2Glc-A), 5.22 (d, J = 7.8 Hz, 1H, H-1Glc-A), 5.01 (d, J = 10.2 Hz, 1H, Bn), 4.80 (d, J = 10.8 Hz, 1H, Bn), 4.76 – 4.71(m, 2H, Bn), 4.56 – 4.46 (m, 5H, Bn), 4.39 (d, J = 11.4 Hz, 1H, Bn), 4.30 (d, J = 7.8 Hz, 1H, H-1Glc-B), 4.01 (t, J = 9.0 Hz, 1H, H-3Glc-B), 3.98 – 3.94 (m, 1H, -OCH2CH2-), 3.84 (t, J = 9.0 Hz, 1H, H-4Glc-A), 3.71 – 3.57 (m, 6H, H-6aGlc-A, H-6aGlc-B, H-6bGlc-A, H-3Glc-A, -OCH2CH2-, H-6bGlc-B), 3.49 – 3.38 (m, 4H, H-5Glc-A, H4Glc-B, -CH2CH2N3, H-5Glc-B), 3.35 – 3.31 (m, 1H, -CH2CH2N3), 3.24 (t, J = 7.8 Hz, 1H, H-2Glc-B), 3.01 (s, 1H, -OH). 13C NMR (150 MHz, CDCl3) δ: 165.3, 138.5, 138.4, 138.0, 137.9, 137.6, 133.1, 129.8, 129.7, 128.48, 128.47, 128.43, 128.41, 128.36, 128.35, 128.1, 128.0, 127.8, 127.7, 127.69, 12 ACS Paragon Plus Environment

Page 12 of 30

Page 13 of 30 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


127.65, 127.5, 127.4, 103.2 (C-1Glc-B), 100.4 (C-1Glc-A), 82.7, 81.8, 80.4, 75.6, 74.9, 74.6, 74.5, 74.4, 73.9, 73.8, 73.7, 73.4, 73.3, 71.0, 69.0, 68.0, 51.0. HR ESI-TOF MS (m/z): calcd for C56H59N3O12Na [M + Na]+, 988.3996; found, 988.4014. 2-Azidoethyl

(2,3,4,6-tetra-O-Benzoyl-β-D-galactopyranosyl)-(1→6)-[2-O-benzoyl-4-O-

benzyl-3-O-(9-fluorenylmethoxycarbonyl)-β-D-galactopyranosyl]-(1→4)-(2-O-benzoyl-3,6di-O-benzyl-β-D-glucopyranosyl)-(1→3)-2,3,6-tri-O-benzyl-β-D-glucopyranoside (20): To a stirred mixture of 10 (316 mg, 0.427 mmol), 11 (250 mg, 0.356 mmol), and freshly activated MS 4Å in anhydrous CH2Cl2 (7 ml) was added TMSOTf (7.7 μL, 0.043 mmol) under N2 protection at 0 ℃. After the reaction mixture was stirred for another 30 min, it was neutralized with Et 3N, filtered, and concentrated. The residue was purified by silica gel column chromatography with EtOAc and hexanes (1:5) as the eluents to give 8 (406 mg, 89%) as a white solid. HR ESI-TOF MS (m/z): calcd for C76H68NO17S [M + NH4]+, 1298.4208; found 1298.4223. To a stirred mixture of 8 (398 mg, 0.311 mmol), 9 (250 mg, 0.259 mmol), and freshly activated MS 4Å in anhydrous CH2Cl2 (7 mL) were added NIS (104 mg, 0.466 mmol) and TfOH (2.7 μL, 0.031mmol) under a N2 atmosphere at 0 ℃. After the reaction mixture was stirred for another 30 min, it was neutralized with Et3N, diluted with CH2Cl2 (40 ml) and filtered. The filtrate was washed with saturated aq. Na2S2O3 and brine, dried over Na2SO4, and concentrated under vacuum. The residue was purified by silica gel column chromatography with EtOAc and hexanes (1:4) as the eluents to give 20 (456 mg, 83%) as colorless syrup. 1H NMR (600 MHz, CDCl3) δ: 8.04 – 8.01 (m, 2H, Ph), 8.00 – 7.94 (m, 6H, Ph), 7.91 – 7.88 (m, 2H, Ph), 7.73 – 7.66 (m, 4H, Ph), 7.57 – 7.24 (m, 35H, Ph), 7.22 – 7.04 (m, 17H), 6.99 (m, 2H, Ph), 6.04 (d, J = 3.0 Hz, 1H, H-4Gal-B), 5.74 (dd, J = 10.2, 7.8 Hz, 1H, H-2Gal-B), 5.70 - 5.64 (m, 2H, H-3Gal-B, H-2Gal-C), 5.32 (dd, J = 9.6, 8.4 Hz, 1H, H-2Glc-A), 5.13 (d, J = 7.8 Hz, 1H, H-1Glc-A), 5.09 (d, J = 11.4 Hz, 1H, Bn), 4.92 (d, J = 11.4 Hz, 1H, Bn), 4.81 (d, J = 11.4 Hz, 1H, Bn), 4.79 – 4.73 (m, 3H, Bn, H-1Gal-B, H-3Gal-C), 4.68 (d, J = 10.8 Hz, 1H, Bn), 4.57 – 4.55 (m, 2H, Bn, H-1Gal-C), 4.51 (d, J = 10.8 Hz, 1H, Bn), 4.48 (s, 2H, Bn), 4.45 – 4.38 (m, 3H, H-6aGal-B, Bn), 4.30 – 4.24 (m, 3H, -OCH2CH-, H-6bGal-B, H-1Glc-B), 4.21 – 4.15 (m, 3H, OCH2CH-, H-5Gal-B, Bn), 4.07 (t, J = 7.8 Hz, 1H, -OCH2CH-), 4.03 (t, J = 9.6 Hz, 1H, H-4Glc-A), 3.98 - 3.91 (m, 4H, H-3Glc-B, H-4Gal-C, -OCH2CH2-), 3.67 – 3.64 (m, 1H), 3.62 – 3.55 (m, 2H, H3Glc-A, -OCH2CH2-), 3.53 – 3.46 (m, 4H, H-6aGlc-A, H-6bGlc-A), 3.44 – 3.35 (m, 4H, H-4Glc-B, CH2CH2N3), 3.32 – 3.28 (m, 1H, -CH2CH2N3), 3.24 (t, J = 8.4 Hz, 1H, H-2Glc-B), 3.15 – 3.12 (m, 13 ACS Paragon Plus Environment


1H, H-5Glc-A).

13

C NMR (150 MHz, CDCl3) δ: 165.9, 165.5, 165.4, 165.3, 164.9, 154.4, 143.3,

142.9, 141.2, 141.1, 138.5, 138.4, 138.3, 138.2, 138.1, 138.0, 133.4, 133.36, 133.33, 133.2, 133.1, 133.0, 129.9, 129.8, 129.74, 129.72, 129.6, 129.33, 129.31, 129.2, 129.1, 128.8, 128.58, 128.55, 128.49, 128.45, 128.34, 128.33, 128.17, 128.15, 128.0, 127.9, 127.82, 127.80, 127.75, 127.72, 127.6, 127.5, 127.4, 127.3, 127.2, 127.1, 127.0, 125.2, 125.0, 119.95, 119.94, 103.1 (C-1Glc-B), 100.9 (C-1Gal-B), 100.5 (C-1Gal-C), 100.3 (C-1Glc-A), 82.8, 80.8, 80.5, 77.3, 77.2, 75.8, 75.3, 75.2, 74.9, 74.6, 74.4, 73.7, 73.6, 73.4, 73.1, 73.0, 71.4, 71.0, 70.6, 70.1, 69.2, 68.2, 67.9, 67.7, 66.5, 61.6, 50.9, 46.4. HR ESI-TOF MS (m/z): calcd for C125H115N3O29Na [M + Na]+, 2144.7514; found, 2144.7591. 2-Azidoethyl

(2,3,4,6-tetra-O-Benzoyl-β-D-galactopyranosyl)-(1→6)-(2-O-benzoyl-4-O-

benzyl-β-D-galactopyranosyl)-(1→4)-(2-O-benzoyl-3,6-di-O-benzyl-β-D-glucopyranosyl)(1→3)-2,3,6-tri-O-benzyl-β-D-glucopyranoside (5): The solution of 20 (400 mg, 0.189 mmol) in CH2Cl2 (6 mL) mixed with Et3N (1.5 mL) was stirred at room temperature for 2 h. Then, it was concentrated under vacuum to give a residue, which was purified by silica gel column chromatography with EtOAc and hexanes (1:3) as the eluents to give 5 (301 mg, 84%) as colorless syrup. 1H NMR (600 MHz, CDCl3) δ: 8.01 – 7.93 (m, 8H, Ph), 7.91 – 7.88 (m, 2H, Ph), 7.76 – 7.72 (m, 2H, Ph), 7.58 – 7.55 (m, 2H, Ph), 7.53 – 7.48 (m, 3H, Ph), 7.45 – 7.26 (m, 24H, Ph), 7.25 – 7.01 (m, 19H, Ph), 6.02 (d, J = 3.6 Hz, 1H, H-4Gal-B), 5.77 (dd, J = 10.2, 7.8 Hz, 1H, H-2Gal-B), 5.66 (dd, J = 10.2, 3.0 Hz, 1H, H-3Gal-B), 5.32 – 5.29 (dd, J = 9.6, 7.8 Hz, 1H, H-2Glc-A), 5.19 (dd, J = 10.2, 7.8 Hz, 1H, H-2Gal-C), 5.12 (d, J = 7.8 Hz, 1H, H-1Glc-A), 5.07 (d, J = 11.4 Hz, 1H, Bn), 4.92 (m, 2H, Bn), 4.77 – 4.72 (m, 3H, Bn, H-1Gal-B), 4.58 (d, J = 10.8 Hz, 1H, Bn), 4.52 – 4.43 (m, 6H, H-1Gal-C, Bn, H-6aGal-B), 4.39 (d, J = 11.4 Hz, 1H, Bn), 4.29 – 4.22 (m, 4H, H-6bGal-B, H-1GlcB

, Bn, H-5Gal-B), 4.06 – 4.01 (m, 2H), 3.97 (t, J = 8.4 Hz, 1H, H-3Glc-B), 3.94 – 3.91 (m, 1H, -

OCH2CH2-), 3.78 (d, J = 3.6 Hz, 1H), 3.65 (dd, J = 10.8, 1.8 Hz, 1H), 3.61 – 3.54 (m, 4H, H-3Glc, -OCH2CH2-), 3.52 – 3.49 (m, 2H), 3.43 – 3.35 (m, 3H, -CH2CH2N3), 3.33 – 3.26 (m, 3H, -

A

CH2CH2N3, H-3Gal-C), 3.22 (dd, J = 9.0, 7.8 Hz, 1H, H-2Glc-B), 3.17 – 3.14 (m, 1H, H-5Glc-A), 2.12 (d, J = 9.0 Hz, 1H, -OH).

C NMR (150 MHz, CDCl3) δ: 166.2, 166.0, 165.4, 165.3, 165.26,

13

165.22, 138.5, 138.36, 138.35, 138.30, 138.1, 138.0, 133.5, 133.4, 133.3, 133.2, 133.0, 129.9, 129.8, 129.75, 129.71, 129.69, 129.67, 129.61, 129.2, 129.1, 129.0, 128.8, 128.7, 128.59, 128.58, 128.5, 128.4, 128.35, 128.33, 128.30, 128.2, 128.08, 128.05, 128.01, 128.00, 127.9, 127.7, 127.6, 14 ACS Paragon Plus Environment

Page 14 of 30

Page 15 of 30 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


127.5, 127.4, 127.4, 127.3, 127.2, 103.1 (C-1Glc-B), 101.1 (C-1Gal-B), 100.3 (C-1Gal-C), 100.2 (C1Glc-A), 82.7, 80.7, 80.4, 77.2, 75.8, 75.7, 75.3, 75.0, 74.9, 74.8, 74.6, 74.4, 74.2, 73.7, 73.4, 73.1, 72.8, 72.4, 71.4, 71.1, 70.0, 69.2, 68.1, 67.9, 66.4, 61.6, 50.9. HR ESI-TOF MS (m/z): calcd for C110H109N4O27 [M + NH4]+, 1917.7279; found, 1917.7280. 2-Azidoethyl (Methyl 5-acetamido-7,8,9-tri-O-acetyl-3,5-dideoxy-D-glycero-α-D- galactonon-2-ulopyranosylonate)-(2→3)-(2-O-benzoyl-4,6-benzylidene-β-D-galactopyranosyl)(1→4)-(6-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1→3)-{[(2,3,4,6-tetra-Obenzoyl-β-D-galactopyranosyl)-(1→6)]-(2-O-benzoyl-4-O-benzyl-β-D-galactopyranosyl)}(1→4)-(2-O-benzoyl-3,6-di-O-benzyl-β-D-glucopyranosyl)-(1→3)-2,4,6-tri-O-benzyl-β-Dglucopyranoside (3a): To a stirred mixture of 4a (21.6 mg, 0.016 mmol), 5 (26.0 mg, 0.014 mmol), and freshly activated MS 4Å in anhydrous CH2Cl2 (2 mL) were added NIS (5.4 mg, 0.025 mmol) and TfOH (0.1 μL, 1.6 μmol) under a N2 atmosphere at 0 ℃. After the reaction mixture was stirred for another 30 min, it was neutralized with Et3N, diluted with CH2Cl2 (20 mL), and filtered. The filtrate was washed with saturated aq. Na2S2O3 and brine, dried over Na2SO4, and concentrated under vacuum. The residue was purified by silica gel column chromatography with EtOAc and toluene (1:5) as the eluents to give 3a (3.5 mg, 8%) as a white solid. 1H NMR (600 MHz, CDCl3) δ: 8.18 (d, J = 7.8 Hz, 2H, Ph), 8.00 – 7.97 (d, 3H, Ph), 7.83 (d, J = 8.4 Hz, 2H, Ph), 7.79 (d, J = 8.4 Hz, 2H, Ph), 7.74 (d, J = 7.2 Hz, 1H, Ph), 7.59 – 7.45 (m, 11H, Ph), 7.41 – 7.26 (m, 17H, Ph), 7.26 – 7.20 (m, 18H, Ph), 7.19 – 7.12 (m, 5H, Ph), 7.09 – 6.95 (m, 11H, Ph), 6.93 – 6.83 (m, 5H, Ph), 6.81 – 6.77 (m, 2H, Ph), 6.10 (d, J = 3.0 Hz, 1H, H-4Gal-B), 5.83 (dd, J = 10.2, 3.0 Hz, 1H, H-3Gal-B), 5.68 – 5.64 (m, 1H, H-2Gal-B), 5.60 – 5.56 (m, 1H, H-8NeuAc), 5.55 – 5.51 (m, 2H, H-2Gal-A, H-7NeuAc), 5.35 – 5.29 (m, 3H, H-2Glc-A, Ph-CH-, H-2Gal-C), 5.15 (d, J = 8.4 Hz, 1H, H-1GlcN), 5.12 – 5.09 (m, 3H, Bn, H-1Glc-A), 5.06 – 5.02 (m, 2H, H-1Gal-B, Bn), 4.91 (d, J = 11.4 Hz, 1H, Bn), 4.81 (d, J = 7.8 Hz, 1H, H-1Gal-A), 4.78 (d, J = 10.8 Hz, 1H, Bn), 4.58 – 4.52 (m, 3H, Bn, H-3Gal-A), 4.47 – 4.43 (m, 4H, H-9NeuAc, H-6NeuAc, Bn), 4.39 – 4.34 (m, 2H, Bn), 4.22 (d, J = 7.8 Hz, 1H, H-1Glc-B), 4.18 (d, J = 7.8 Hz, 1H, H-1Gal-C), 4.16 – 4.11 (m, 4H, H-4Gal-A, Bn, H-6aGalB

, H-2GlcN), 4.08 – 4.00 (m, 4H, Bn), 3.99 – 3.88 (m, 6H, Bn, H-5Gal-B, H-9NeuAc, H-3Glc-B, -

OCH2CH2-), 3.84 - 3.77 (m, 2H), 3.76 - 3.70 (m, 2H, H-4NeuAc), 3.65 – 3.60 (m, 3H), 3.58 – 3.53 (m, 3H, H-5NeuAc, -OCH2CH2-), 3.51 – 3.48 (s, 2H), 3.44 – 3.34 (m, 11H, H-3Gal-C, H-3Glc-A, CH2CH2N3, -COOCH3), 3.32 – 3.22 (m, 5H, -CH2CH2N3, H-2Glc-B), 2.98 – 2.91 (m, 2H, H15 ACS Paragon Plus Environment


3eqNeuAc), 2.45 (s, 3H, -OAc), 2.16 (s, 3H, -OAc), 1.98 (s, 3H, -OAc), 1.93 (s, 3H, -OAc), 1.71 (t, J = 12.6 Hz, 1H, H-3axNeuAc). 13C NMR (150 MHz, CDCl3) δ: 172.2, 171.1, 170.4, 170.0, 168.5, 167.6, 167.5, 165.8, 165.5, 165.3, 165.2, 164.87, 164.85, 164.3, 153.3, 138.62, 138.60, 138.4, 138.3, 138.1, 138.0, 137.9, 137.4, 133.6, 133.3, 133.2, 133.18, 133.14, 133.0, 132.8, 132.6, 131.4, 131.1, 129.99, 129.97, 129.9, 129.7, 129.6, 129.5, 129.4, 129.0, 128.7, 128.5, 128.44, 128.41, 128.37, 128.31, 128.29, 128.28, 128.23, 128.20, 128.15, 128.12, 127.88, 127.86, 127.7, 127.6, 127.56, 127.50, 127.4, 127.3, 127.2, 127.0, 126.4, 103.1 (C-1Glc-B), 101.6 (C-1Gal-C), 100.6 (PhCH-), 100.43, 100.41, 100.40, 100.3, 99.5 (C-1GlcN), 96.8, 82.8, 82.5, 81.6, 80.8, 80.3, 76.6, 75.89, 75.86, 75.3, 75.1, 75.0, 74.9, 74.8, 74.7, 74.6, 74.3, 73.6, 73.3, 73.2, 72.9, 72.7, 72.4, 72.2, 71.6, 71.4, 71.2, 70.8, 70.3, 70.1, 69.3, 69.2, 68.9, 68.6, 68.3, 67.9, 67.7, 67.6, 66.4, 64.1, 58.8, 56.0, 55.9, 53.0, 50.9, 37.2, 24.7, 21.4, 20.9, 20.8. HR ESI-TOF MS (m/z): calcd for C170H165N5O51Na2 [M + 2Na]2+, 1569.0134; found, 1569.0249. 2-Azidoethyl

[2-O-Benzoyl-4-O-benzyl-6-O-tert-butyldimethylsilyl-3-O-(9-

fluorenylmethoxycarbonyl)-β-D-galactopyranosyl]-(1→4)-(2-O-benzoyl-3,6-di-O-benzyl-βD-glucopyranosyl)-(1→3)-2,4,6-tri-O-benzyl-β-D-glucopyranoside (22). To a solution of 11 (650 mg, 0.926 mmol) in anhydrous DMF (10 mL) were added imidazole (126 mg, 1.852 mmol) and TBSCl (154 mg, 1.018 mmol) at 0 ℃. The mixture was stirred at room temperature for 2 h and then diluted with EtOAc (100 mL). The organic phase, after being washed with saturated aq. NaCl solution, was dried over Na2SO4 and concentrated under vacuum. The residue was purified by silica gel column chromatography with EtOAc and hexanes (1:30) as the eluents to give 21 (612 mg, 81%) as a white solid. HR ESI-TOF MS (m/z): calcd for C48H56NO8SSi [M + NH4]+, 834.3496; found, 834.3495. To a stirred mixture of 21 (608 mg, 0.746 mmol), 9 (600 mg, 0.622 mmol), and freshly activated MS 4Å in anhydrous CH2Cl2 (10 mL) were added NIS (252 mg, 1.119 mmol) and TfOH (6.6 μL, 0.075 mmol) under a N2 atmosphere at 0 ℃. After the reaction mixture was stirred for another 30 min, it was neutralized with Et3N, diluted with CH2Cl2 (50 mL), and filtered. The filtrate was washed with saturated aq. Na2S2O3 and brine, dried over Na2SO4, and concentrated under vacuum. The residue was purified by silica gel column chromatography with EtOAc and hexanes (1:7) as the eluents to give 22 (896 mg, 87%) as a white solid. Compound 22: 1H NMR (600 MHz, CDCl3) δ: 7.99 – 7.90 (m, 4H, Ph), 7.69 – 7.65 (m, 2H, Ph), 7.56 – 7.51 (m, 2H, Ph), 7.45 (d, J = 7.2 Hz, 1H, Ph), 7.42 – 7.39 (m, 4H, Ph), 7.37 – 7.20 (m, 25H, Ph), 7.17 – 7.04 (m, 16 ACS Paragon Plus Environment

Page 16 of 30

Page 17 of 30 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


8H, Ph), 7.01 – 6.96 (m, 2H, Ph), 5.69 (dd, J = 10.2, 7.8 Hz, 1H, H-2Gal), 5.29 – 5.26 (m, 1H, H2Glc-A), 5.08 (d, J = 7.8 Hz, 1H，H-1Glc-A), 5.05 (d, J = 11.4 Hz, 1H, Bn), 4.94 (d, J = 10.8 Hz, 1H, Bn), 4.89 (dd, J = 10.8, 3.0 Hz, 1H, H-3Gal), 4.77 – 4.71 (m, 3H, Bn, H-1Gal), 4.58 – 4.46 (m, 6H, Bn), 4.37 (d, J = 10.8 Hz, 1H, Bn), 4.30 (dd, J = 10.8, 7.2 Hz, 1H, -OCH2CH-), 4.26 – 4.21 (m, 3H, Bn, -OCH2CH-, H-1Glc-B), 4.16 (t, J = 9.6 Hz, 1H, H-4Glc-A), 4.09 – 4.05 (m, 2H, -OCH2CH-, H-4Gal), 3.97 – 3.91 (m, 2H, H-3Glc-B, -OCH2CH2-), 3.67 – 3.54 (m, 5H, H-3Glc-A, -OCH2CH2-, H6aGlc-A), 3.53 – 3.48 (m, 3H, H-6bGlc-A), 3.41 – 3.35 (m, 4H, H-4Glc-B, -CH2CH2N3, H-5Gal), 3.32 – 3.27 (m, 1H, -CH2CH2N3), 3.21 (t, 1H, J = 8.4 Hz, H-2Glc-B), 3.09 (d, J = 9.6 Hz, 1H, H-5Glc-A), 0.87 (s, 9H, -tBu), -0.01 (s, 3H, -SiCH3), -0.03 (s, 3H, -SiCH3). 13C NMR (150 MHz, CDCl3) δ: 165.3, 164.9, 154.6, 143.3, 142.8, 141.2, 141.1, 138.43, 138.40, 138.37, 138.30, 138.2, 138.1, 133.3, 133.0, 130.0, 129.7, 129.6, 129.4, 128.5, 128.48, 128.43, 128.39, 128.36, 128.33, 128.2, 128.1, 128.0, 127.9, 127.8, 127.76, 127.75, 127.73, 127.70, 127.6, 127.5, 127.4, 127.3, 127.07, 127.06, 127.03, 125.2, 125.0, 119.93, 119.92, 103.1 (C-1Glc-B), 100.3 (C-1Gal), 100.2 (C-1Glc-A), 82.8, 80.6, 80.2, 77.9, 76.5, 75.8, 75.2, 75.0, 74.9, 74.8, 74.6, 74.5, 74.3, 73.8, 73.7, 73.4, 73.2, 70.9, 70.0, 69.1, 67.9, 67.6, 59.9, 50.9, 46.5, 25.9, 18.1, -5.43, -5.45. HR ESI-TOF MS (m/z): calcd for C97H103N3O20SiK [M + K]+, 1696.6541; found, 1696.6557. 2-Azidoethyl

(2-O-Benzoyl-4-O-benzyl-6-O-tert-butyldimethylsilyl-β-D-galactopyranosyl)-

(1→4)-(2-O-benzoyl-3,6-di-O-benzyl-β-D-glucopyranosyl)-(1→3)-2,4,6-tri-O-benzyl-β-Dglucopyranoside (16). The solution of 22 (800 mg, 0.483 mmol) in CH2Cl2 (10 mL) mixed with Et3N (2.5 mL) was stirred at room temperature for 2 h. Then, it was concentrated under vacuum to give a residue, which was purified by silica gel column chromatography with EtOAc and hexanes (1:6) as the eluent to give 16 (644 mg, 93%) as a white solid. 1H NMR (600 MHz, CDCl3) δ: 7.98 (d, J = 8.4 Hz, 2H, Ph), 7.92 (d, J = 8.0 Hz, 2H, Ph), 7.58 (t, J = 7.2 Hz, 1H, Ph), 7.53 (t, J = 7.2 Hz, 1H, Ph), 7.43 (t, J = 7.8 Hz, 2H, Ph), 7.40 – 7.21 (m, 24H, Ph), 7.17 – 7.13 (m, 5H, Ph), 7.09 (t, J = 7.2 Hz, 1H, Ph), 7.07 – 7.03 (m, 2H, Ph), 5.32 – 5.25 (m, 2H, H-2Glc-A, H-2Gal), 5.11 (d, J = 7.8 Hz, 1H, H-1Glc-A), 5.08 (d, J = 11.4 Hz, 1H, Bn), 4.96 (d, J = 11.4 Hz, 1H, Bn), 4.84 (d, J = 12.0 Hz, 1H, Bn), 4.76 – 4.70 (m, 2H, Bn), 4.67 (d, J = 7.8 Hz, 1H, H-1Gal), 4.61 – 4.56 (m, 2H, Bn), 4.51 – 4.48 (m, 3H, Bn), 4.38 (d, J = 11.4 Hz, 1H, Bn), 4.30 (d, J = 12.6 Hz, 1H, Bn), 4.25 (d, J = 7.8 Hz, 1H, H-1Glc-B), 4.17 (t, J = 9.6 Hz, 1H, H-4Glc-A), 4.00 – 3.92 (m, 3H, H-3Glc-B, -OCH2CH2-), 3.69 – 3.66 (m, 2H), 3.64 – 3.55 (m, 6H, H-3Glc-A, H-3Gal, -OCH2CH2-), 3.53 (dd, J 17 ACS Paragon Plus Environment


= 10.8, 4.8 Hz, 1H), 3.43 – 3.37 (m, 3H, H-4Glc-B, -CH2CH2N3), 3.35 – 3.28 (m, 2H, -CH2CH2N3), 3.22 (t, J = 9.0 Hz, 1H, H-2Glc-B), 3.14 (d, J = 9.6 Hz, 1H, H-5Glc-A), 2.29 - 2.26 (m, 1H, -OH), 0.90 (s, 9H, -tBu), 0.02 (s, 3H, -SiCH3), -0.00 (s, 3H, -SiCH3). 13C NMR (150 MHz, CDCl3) δ: 166.3, 165.3, 138.5, 138.4, 138.3, 138.3, 138.1, 133.3, 133.0, 129.9, 129.7, 129.6, 128.5, 128.4, 128.39, 128.36, 128.35, 128.1, 127.9, 127.86, 127.80, 127.7, 127.66, 127.60, 127.5, 127.45, 127.43, 127.3, 127.1, 103.1 (C-1Glc-B), 100.2, 100.1, 82.8, 80.6, 80.2, 76.6, 76.2, 75.8, 75.3, 74.9, 74.89, 74.86, 74.62, 74.61, 74.5, 74.4, 73.8, 73.4, 73.3, 72.9, 69.1, 67.9, 67.8, 60.0, 50.9, 25.8, 18.0, -5.4, -5.5. HR ESI-TOF MS (m/z): calcd for C82H93N3O18SiNa [M + Na]+, 1458.6121; found, 1458.6155. 2-Azidoethyl (Methyl 5-acetamido-7,8,9-tri-O-acetyl-3,5-dideoxy-D-glycero-α-D- galactonon-2-ulopyranosylonate)-(2→3)-(2-O-benzoyl-4,6-benzylidene-β-D-galactopyranosyl)(1→4)-(6-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-Obenzyl-6-O-tert-butyldimethylsilyl-β-D-galactopyranosyl)-(1→4)-(2-O-benzoyl-3,6-di-Obenzyl-β-D-glucopyranosyl)-(1→3)-2,4,6-tri-O-benzyl-β-D-glucopyranoside (23): To a stirred mixture of 4a (33.0 mg, 0.025 mmol), 16 (30.0 mg, 0.021 mmol), and freshly activated MS 4Å in anhydrous CH2Cl2 (2 mL) were added NIS (8.5 mg, 0.038 mmol) and TfOH (0.2 μL, 2.5 μmol) under a N2 atmosphere at 0 ℃. After the reaction mixture was stirred for another 30 min, it was neutralized with Et3N, diluted with CH2Cl2 (10 mL), and filtered. The filtrate, after being washed with saturated aq. Na2S2O3 and NaCl, was dried over Na2SO4 and concentrated under vacuum. The residue was purified by silica gel column chromatography with EtOAc and hexanes (1:2) as the eluents to give 23 (8.2 mg, 15%) as a white solid. 1H NMR (600 MHz, CDCl3) δ: 8.18 - 8.12 (m, 2H, Ph), 7.89 – 7.83 (m, 2H, Ph), 7.71 (d, J = 7.8 Hz, 1H, Ph), 7.63 – 7.60 (m, 1H, Ph), 7.56 – 7.43 (m, 9H, Ph), 7.41 – 7.04 (m, 38H, Ph), 7.00 – 6.94 (m, 3H, Ph), 6.92 – 6.85 (m, 3H, Ph), 5.58 5.50 (m, 3H, H-7NeuAc, H-8NeuAc, H-2Gal-A), 5.35 – 5.30 (m, 2H, H-2Gal-C, Ph-CH-), 5.26 (d, J = 8.4 Hz, 1H, H-1GlcN), 5.15 (dd, J = 9.6, 8.4 Hz, 1H, H-2Glc-A), 5.12 (d, J = 11.4 Hz, 1H, Bn), 4.99 (d, J = 10.8 Hz, 1H, Bn), 4.93 (d, J = 8.4 Hz, 1H, H-1Glc-A), 4.84 – 4.79 (m, 2H, Bn, H-1Gal-A), 4.66 (d, J = 10.8 Hz, 1H, Bn), 4.52 (dd, J = 10.2, 3.6 Hz, 1H, H-3Gal-A), 4.49 – 4.34 (m, 10H, Bn, H6NeuAc, H-9NeuAc, H-1Gal-C), 4.29 (d, J = 10.8 Hz, 1H, Bn), 4.20 – 4.07 (m, 8H, H-1Glc-B, H-2GlcN, H4Gal-A, Bn), 4.04 – 3.95 (m, 3H, H-9NeuAc), 3.91 – 3.85 (m, 2H, -OCH2CH2-, H-3Glc-B), 3.75 – 3.70 (m, 2H, H-4NeuAc), 3.68 – 3.61 (m, 4H, H-3Gal-C), 3.60 – 3.45 (m, 7H, -OCH2CH2-, H-5NeuAc), 3.43 – 3.21 (m, 12H, H-3Glc-A, -COOCH3, -CH2CH2N3), 3.14 (t, J = 8.4 Hz, 1H, H-2Glc-B), 2.91 (dd, J = 18 ACS Paragon Plus Environment

Page 18 of 30

Page 19 of 30 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


12.0, 3.0 Hz, 1H, H-3eqNeuAc), 2.79 (d, J = 9.6 Hz, 1H), 2.44 (s, 3H, -OAc), 2.16 (s, 3H, -OAc), 1.96 (s, 3H, -OAc), 1.91 (s, 3H, -OAc), 1.72 (t, J = 12.6 Hz,1H, H-3axNeuAc), 0.75 (s, 9H, -tBu), 0.13 (s, 3H, -SiCH3), -0.15 (s, 3H, -SiCH3). 13C NMR (150 MHz, CDCl3) δ: 172.1, 171.0, 170.4, 170.0, 168.5, 167.6, 167.4, 165.2, 164.9, 164.4, 153.3, 139.3, 138.5, 138.4, 138.3, 138.23, 138.18, 138.0, 137.5, 133.5, 133.3, 133.2, 132.8, 132.7, 131.5, 131.2, 130.0, 129.91, 129.90, 129.6, 129.5, 129.3, 129.0, 128.6, 128.41, 128.39, 128.38, 128.30, 128.28, 128.13, 128.09, 128.06, 128.0, 127.92, 127.89, 127.80, 127.76, 127.72, 127.69, 127.6, 127.47, 127.45, 127.3, 127.2, 127.0, 126.8, 126.4, 123.2, 122.6, 103.1 (C-1Glc-B), 101.5 (C-1Gal-A), 100.6 (Ph-CH-), 100.14 (C-1Gal-C), 100.09 (C-1Glc-A), 99.5 (C-1GlcN), 96.8, 82.7, 82.0, 80.7, 80.6, 80.1, 76.1, 75.74, 75.72, 75.03, 74.97, 74.8, 74.67, 74.66, 74.6, 74.5, 73.9, 73.7, 73.4, 73.2, 73.1, 72.4, 72.2, 72.1, 71.5, 70.2, 69.21, 69.15, 69.0, 68.3, 67.9, 67.8, 67.3, 66.4, 64.0, 60.7, 58.8, 56.1, 52.93, 50.88, 37.2, 30.9, 25.9, 24.7, 21.3, 20.80, 20.77, 18.0, -5.51, -5.53. HR ESI-TOF MS (m/z): calcd for C142H153N5O42SiNa2 [M + 2Na]2+, 1336.9778; found, 1336.9852. 2-Azidoethyl [3,6-di-O-Benzyl-2-deoxy-2-phthalimido-4-O-(9-fluorenylmethoxycarbonyl)-βD-glucopyranosyl]-(1→3)-(2-O-benzoyl-4-O-benzyl-6-O-tert-butyldimethylsilyl-β-Dgalactopyranosyl)-(1→4)-(2-O-benzoyl-3,6-di-O-benzyl-β-D-glucopyranosyl)-(1→3)-2,4,6tri-O-benzyl-β-D-glucopyranoside (25). To a stirred mixture of 24 (421 mg, 0.515 mmol), 16 (370 mg, 0.258 mmol), and freshly activated MS 4Å in anhydrous CH2Cl2 (8 mL) were added NIS (174 mg, 0.773 mmol) and TfOH (4.6 μL, 0.052 mmol) under a N2 atmosphere at 0 ℃. After the reaction mixture was stirred for another 30 min, it was neutralized with Et3N, diluted with CH2Cl2 (40 mL), and filtered. The filtrate, after being washed with saturated aq. Na2S2O3 and NaCl, was dried over Na2SO4 and concentrated under vacuum. The residue was purified by silica gel column chromatography with EtOAc and hexanes (1:6) as the eluents to give 25 (428 mg, 78%) as a white solid. 1H NMR (600 MHz, CDCl3) δ: 7.87 (d, J = 7.8 Hz, 2H, Ph), 7.75 – 7.66 (m, 3H, Ph), 7.63 – 7.06 (m, 45H, Ph), 7.04 – 6.89 (m, 6H, Ph), 6.86 – 6.81 (m, 2H, Ph), 6.80 – 6.74 (m, 3H, Ph), 6.69 (d, J = 6.6 Hz, 1H, Ph), 5.32 – 5.29 (m, 1H, H-2Gal-C), 5.21 (d, J = 7.8 Hz, 1H, H-1GlcN), 5.18 (t, J = 9.0 Hz, 1H, H-2Glc-A), 5.08 (d, J = 11.4 Hz, 1H, Bn), 5.00 (d, J = 10.8 Hz, 1H, Bn), 4.97 – 4.92 (m, 2H, H-1Glc-A), 4.84 (d, J = 10.8 Hz, 1H, Bn), 4.68 (d, J = 10.8 Hz, 1H, Bn), 4.56 (d, J = 11.4 Hz, 1H, Bn), 5.34 – 4.49 (m, 3H, Bn), 4.48 – 4.37 (m, 7H, Bn, H-1Gal-C), 4.34 – 4.18 (m, 6H, OCH2CH-, Bn, H-2GlcN, H-1Glc-B), 4.13 – 4.06 (m, 3H, Bn, -OCH2CH-), 4.02 (t, J = 9.6 Hz, 1H, 19 ACS Paragon Plus Environment


Page 20 of 30

H-4Glc-A), 3.93 - 3.85 (m, 3H, -OCH2CH2-, H-3Glc-B), 3.74 – 3.62 (m, 4H, H-3Gal-C), 3.57 – 3.52 (m, 2H, -OCH2CH2-), 3.50 – 3.40 (m, 3H, H-3Glc-A), 3.39 – 3.26 (m, 6H, -CH2CH2N3, H-6aGlc-A), 3.22 (d, J = 10.8 Hz, 1H, H-6bGlc-A), 3.16 (t, J = 7.8 Hz, 1H, H-2Glc-B), 2.79 (d, J = 9.6 Hz, 1H, H-5Glc), 0.84 (s, 9H, -tBu), -0.06 (s, 3H, -SiCH3). 13C NMR (150 MHz, CDCl3) δ: 167.8, 166.7, 165.2,

A

164.3, 154.3, 143.3, 143.0, 141.3, 141.2, 139.2, 138.4, 138.36, 138.2, 138.1, 137.7, 137.5, 133.3, 133.2, 132.9, 132.7, 131.3, 130.8, 130.0, 129.6, 129.5, 129.3, 128.4, 128.39, 128.32, 128.2, 128.1, 128.0, 127.9, 127.89, 127.86, 127.79, 127.74, 127.70, 127.6, 127.57, 127.55, 127.50, 127.4, 127.3, 127.2, 127.17, 127.12, 126.9, 125.1, 124.9, 123.2, 122.7, 120.0, 103.1 (C-1Glc-B), 100.2 (C-1Gal-C), 100.1 (C-1Glc-A), 99.4 (C-1GlcN), 82.7, 80.6, 80.3, 80.1, 77.3, 76.2, 75.89, 75.87, 75.7, 75.1, 74.8, 74.7, 74.6, 74.5, 74.4, 74.1, 73.7, 73.6, 73.4, 73.1, 72.6, 72.2, 70.2, 69.9, 69.2, 67.9, 67.2, 60.4, 55.6, 50.9, 46.7, 25.9, 18.1, -5.45, -5.47. HR ESI-TOF MS (m/z): calcd for C125H128N4O26SiNa [M + Na]+, 2151.8484; found, 2151.8517. 2-Azidoethyl

(3,6-di-O-Benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1→3)-(2-O-

benzoyl-4-O-benzyl-6-O-tert-butyldimethylsilyl-β-D-galactopyranosyl)-(1→4)-(2-O-benzoyl3,6-di-O-benzyl-β-D-glucopyranosyl)-(1→3)-2,4,6-tri-O-benzyl-β-D-glucopyranoside

(26).

The solution of 25 (400 mg, 0.188 mmol) in CH2Cl2 (6 mL) mixed with Et3N (1.5 mL) was stirred at room temperature for 2 h. Then, it was concentrated under vacuum to give a residue, which was purified by silica gel column chromatography with EtOAc and hexanes (1:4) as the eluents to give 26 (319 mg, 89%) as a white solid. 1H NMR (600 MHz, CDCl3) δ: 7.88 (d, J = 7.8 Hz, 2H, Ph), 7.72 (d, J = 7.2 Hz, 1H, Ph), 7.59 – 6.87 (m, 50H, Ph), 6.72 (d, J = 7.2 Hz, 1H, Ph), 5.33 (t, J = 9.0 Hz, 1H, H-2Gal-C), 5.25 (d, J = 8.4 Hz, 1H, H-1GlcN), 5.18 (t, J = 9.0 Hz, 1H, H-2Glc-A), 5.08 (d, J = 11.4 Hz, 1H, Bn), 5.00 (d, J = 10.8 Hz, 1H, Bn), 4.96 (d, J = 7.8 Hz, 1H, H-1Glc-A), 4.84 (d, J = 10.8 Hz, 1H, Bn), 4.70 – 4.61 (m, 3H, Bn), 4.59 – 4.53 (m, 2H, Bn), 4.48 – 4.38 (m, 7H, Bn, H1Gal-C), 4.32 (d, J = 10.8 Hz, 1H, Bn), 4.25 – 4.14 (m, 4H, H-1Glc-B, H-2GlcN, Bn), 4.06 – 4.01 (m, 2H, H-4Glc-A), 3.92 – 3.85 (m, 3H, -OCH2CH2-, H-3Glc-B), 3.82 – 3.75 (m, 2H), 3.72 – 3.63 (m, 3H, H-3Gal-C), 3.57 – 3.52 (m, 2H, -OCH2CH2-), 3.49 (dd, J = 10.8, 4.2 Hz, 1H), 3.45 – 3.41 (m, 2H, H-3Glc-A), 3.39 – 3.36 (m, 1H, -CH2CH2N3), 3.35 – 3.23 (m, 6H, H-6aGlc-A, -CH2CH2N3, H-6bGlcA

), 3.16 (t, J = 8.4 Hz, 1H, H-2Glc-B), 2.99 (s, 1H, -OH), 2.81 (d, J = 9.6 Hz, 1H, H-5Glc-A), 0.85 (s,

9H, -tBu), -0.05 (s, 3H, -SiCH3), -0.06 (s, 3H, -SiCH3). 13C NMR (150 MHz, CDCl3) δ: 167.9, 167.1, 165.2, 164.3, 139.3, 138.5, 138.4, 138.2, 138.1, 138.0, 137.3, 133.3, 133.2, 132.9, 132.7, 20 ACS Paragon Plus Environment

Page 21 of 30 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


131.4, 130.9, 130.0, 129.6, 129.5, 129.4, 128.6, 128.4, 128.37, 128.35, 128.31, 128.2, 128.07, 128.06, 128.05, 128.0, 127.9, 127.8, 127.72, 127.71, 127.6, 127.58, 127.56, 127.50, 127.3, 127.2, 127.1, 126.9, 123.2, 122.6, 103.1 (C-1Glc-B), 100.3 (C-1Gal-C), 100.1 (C-1Glc-A), 99.7 (C-1GlcN), 82.8, 80.7, 80.6, 80.1, 78.2, 75.9, 75.7, 75.1, 74.8, 74.74, 74.70, 74.6, 74.55, 74.4, 74.2, 74.0, 73.7, 73.4, 73.1, 73.0, 72.2, 71.3, 69.2, 67.9, 67.4, 60.2, 55.5, 50.9, 25.9, 18.0, -5.46, -5.48. HR ESI-TOF MS (m/z): calcd for C110H118N4O24SiK [M + K]+, 1945.7542; found, 1945.7495. 2-Azidoethyl (Methyl 5-acetamido-7,8,9-tri-O-acetyl-3,5-dideoxy-D-glycero-α-D- galactonon-2-ulopyranosylonate)-(2→3)-(2-O-benzoyl-4,6-benzylidene-β-D-galactopyranosyl)(1→4)-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1→3)-(2-O-benzoyl4-O-benzyl-6-O-tert-butyldimethylsilyl-β-D-galactopyranosyl)-(1→4)-(2-O-benzoyl-3,6-diO-benzyl-β-D-glucopyranosyl)-(1→3)-2,4,6-tri-O-benzyl-β-D-glucopyranoside (27). To a stirred mixture of 6 (177 mg, 0.189 mmol), 26 (300 mg, 0.157 mmol), and freshly activated MS 4Å in anhydrous CH2Cl2 (5 mL) were added NIS (64 mg, 0.283 mmol) and TfOH (1.7 μL, 0.019 mmol) under a N2 atmosphere at 0 ℃. After the reaction mixture was stirred for another 30 min, it was neutralized with Et3N, diluted with CH2Cl2 (50 mL), and filtered. The filtrate, after being washed with saturated aq. Na2S2O3 and NaCl, was dried over Na2SO4 and concentrated under vacuum. The residue was purified by silica gel column chromatography with EtOAc and toluene (1:5) as the eluents to give 27 (325 mg, 76%) as a white solid. 1H NMR (600 MHz, CDCl3) δ: 8.15 (d, J = 7.8 Hz, 2H, Ph), 7.86 (d, J = 7.8 Hz, 2H, Ph), 7.66 – 7.58 (m, 2H, Ph), 7.54 – 7.46 (m, 5H, Ph), 7.44 – 7.02 (m, 42H, Ph), 7.01 – 6.95 (m, 3H, Ph), 6.92 – 6.87 (t, J = 7.2 Hz, 2H, Ph), 6.83 (d, J = 7.2 Hz, 2H, Ph), 6.68 – 6.60 (m, 3H, Ph), 5.59 – 5.51 (m, 3H, H-7NeuAc, H-8NeuAc, H-2Gal), 5.33 (s, 1H, Ph-CH-), 5.27 (t, J = 9.6 Hz, 1H, H-2Gal-C), 5.17 – 5.09 (m, 3H, H-2Glc-A, Bn, H-

A

1GlcN), 5.00 – 4.96 (m, 3H, Bn, H-1Gal-A), 4.93 (d, J = 7.8 Hz, 1H, H-1Glc-A), 4.82 (d, J = 10.8 Hz, 1H, Bn), 4.66 (d, J = 11.4 Hz, 1H, Bn), 4.54 (d, J = 11.4 Hz, 1H, Bn), 4.51 – 4.35 (m, 12H, H-3Gal, Bn, H-9NeuAc, H-6NeuAc, H-1Gal-C), 4.31 – 4.26 (m, 2H, Bn), 4.21 – 4.13 (m, 4H, H-1Glc-B), 4.10

A

(d, J = 12.0 Hz, 1H, Bn), 4.07 (d, J = 2.4 Hz, 1H, H-4Gal-A), 4.04 – 3.97 (m, 4H, H-9NeuAc, H-4Glc), 3.91 – 3.84 (m, 2H, -OCH2CH2-, H-3Glc-B), 3.80 – 3.73 (m, 2H, H-4NeuAc), 3.69 (d, J = 10.2 Hz,

A

1H), 3.64 – 3.60 (m, 2H, H-3Gal-C), 3.56 – 3.44 (m, 9H, H-5NeuAc, -OCH2CH2-, -COOCH3), 3.42 – 3.32 (m, 5H, H-3Glc-A, -CH2CH2N3, H-4Glc-B), 3.30 – 3.24 (m, 3H, -CH2CH2N3, H-6aGlc-A), 3.19 (d, J = 10.8 Hz, 1H, H-6bGlc-A), 3.14 (t, J = 8.4 Hz, 1H, H-2Glc-B), 2.90 (d, J = 10.2 Hz, 1H, H-3eqNeuAc), 21 ACS Paragon Plus Environment


2.76 (d, J = 9.6 Hz, 1H, H-5Glc-A), 2.45 (s, 3H, -OAc), 2.18 (s, 3H, -OAc), 1.97 (s, 3H, -OAc), 1.83 (s, 3H, -OAc), 1.78 (t, J = 12.6 Hz, 1H, H-3axNeuAc), 0.78 (s, 9H, -tBu), -0.11 (s, 3H, -SiCH3), 0.12 (s, 3H, -SiCH3). 13C NMR (150 MHz, CDCl3) δ: 172.0, 170.8, 170.2, 170.0, 168.4, 167.6, 167.0, 165.2, 164.9, 164.3, 153.4, 139.3, 138.7, 138.4, 138.3, 138.2, 138.17, 138.0, 137.9, 133.3, 133.0, 132.89, 132.85, 132.7, 131.4, 130.9, 130.0, 129.9, 129.8, 129.6, 129.5, 129.3, 128.9, 128.6, 128.4, 128.38, 128.34, 128.31, 128.2, 128.09, 128.05, 128.99, 127.92, 127.8, 127.72, 127.70, 127.6, 127.57, 127.55, 127.51, 127.47, 127.42, 127.3, 127.0, 126.8, 126.6, 126.4, 123.0, 122.6, 103.0 (C-1Glc-B), 100.9 (Ph-CH-), 100.4 (C-1Gal-A), 100.2 (C-1Gal-C), 100.1(C-1Glc-A), 99.5 (C-1GlcN

), 97.1, 82.7, 80.6, 80.4, 80.1, 77.8, 77.1, 76.0, 75.8, 75.7, 75.1, 75.03, 75.01, 74.9, 74.8, 74.7,

74.6, 74.5, 74.4, 73.7, 73.37, 73.35, 73.0, 72.9, 72.8, 72.1, 71.5, 71.2, 69.2, 68.8, 68.6, 67.9, 67.8, 67.3, 66.0, 63.7, 60.6, 58.9, 56.0, 52.9, 50.9, 37.0, 25.9, 24.7, 21.3, 20.8, 20.7, 18.1, -5.48, -5.49. HR ESI-TOF MS (m/z): calcd for C149H159N5O42SiNa2 [M + 2Na]2+, 1382.0012, found 1382.0098. 2-Azidoethyl (Methyl 5-acetamido-7,8,9-tri-O-acetyl-3,5-dideoxy-D-glycero-α-D- galactonon-2-ulopyranosylonate)-(2→3)-(2-O-benzoyl-4,6-benzylidene-β-D-galactopyranosyl)(1→4)-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1→3)-(2-O-benzoyl4-O-benzyl-β-D-galactopyranosyl)-(1→4)-(2-O-benzoyl-3,6-di-O-benzyl-β-Dglucopyranosyl)-(1→3)-2,4,6-tri-O-benzyl-β-D-glucopyranoside (14). A solution of 27 (280 mg, 0.103 mmol) in CH2Cl2 and CH3CN (V/V, 2:1, 3 mL) and triethylamine trihydrofluoride (1.5 ml) was stirred at room temperature overnight under N2 atmosphere. The reaction was quenched with dropwise addition of saturated aq. NaHCO3 solution. The aq. phase was extracted with CH2Cl2 (3 × 50 mL), and the organic layer was dried over Na2SO4, and concentrated under vacuum. The residue was purified by silica gel column chromatography with EtOAc and toluene (1:4) as the eluents to give 14 (223 mg, 83%) as a white solid. 1H NMR (600 MHz, CDCl3) δ: 8.19 (d, J = 7.8 Hz, 2H, Ph), 7.84 (d, J = 7.8 Hz, 2H, Ph), 7.65 (d, J = 6.6 Hz, 1H, Ph), 7.61 – 7.58 (m, 1H, Ph), 7.55 – 7.45 (m, 5H, Ph), 7.43 – 7.36 (m, 4H, Ph), 7.36 – 7.08 (m, 38H, Ph), 7.06 – 6.59 (m, 5H, Ph), 6.87 – 6.82 (m, 2H, Ph), 6.68 – 6.61 (m, 3H, Ph), 6.58 – 6.54 (d, J = 7.2 Hz, 1H, Ph), 5.61 – 5.53 (m, 3H, H-7NeuAc, H-8NeuAc, H-2Gal-A), 5.32 (s, 1H, Ph-CH-), 5.24 - 5.20 (m, 1H, H2Gal-C), 5.13 (t, J = 9.0 Hz, 1H, H-2Glc-A), 5.05 (d, J = 8.4 Hz, 1H, H-1GlcN), 5.01 – 4.92 (m, 5H, Bn, H-1Gal-A, H-1Glc-A), 4.79 (d, J = 10.8 Hz, 1H, Bn), 4.67 (d, J = 11.4 Hz, 1H, Bn), 4.52 (dd, J = 10.2, 3.6 Hz, 1H, H-3Gal-A), 4.49 – 4.29 (m, 13H, Bn, H-9NeuAc, H-6NeuAc), 4.25 (d, J = 8.4 Hz, 1H, 22 ACS Paragon Plus Environment

Page 22 of 30

Page 23 of 30 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


H-1Gal-C), 4.22 – 4.13 (m, 3H, H-1Glc-B, H-2GlcN), 4.07 – 3.97 (m, 5H, Bn, H-9NeuAc), 3.92 – 3.87 (m, 2H, H-4Glc-A, -OCH2CH2-), 3.85 (t, J = 8.4 Hz, 1H, H-3Glc-B), 3.77 – 3.71 (m, 3H, H-4NeuAc), 3.66 – 3.61 (m, 2H), 3.58 – 3.44 (m, 9H, H-5NeuAc, -OCH2CH2-, H-3Gal-C, -COOCH3), 3.40 – 3.32 (m, 5H, H-3Glc-A, -CH2CH2N3), 3.29 – 3.25 (m, 1H, -CH2CH2N3), 3.22 (dd, J = 10.8, 2.4 Hz, 1H, H-6aGlc-A), 3.17 – 3.11 (m, 3H, H-2Glc-B, H-6bGlc-A), 3.05 (td, J = 10.8, 3.0 Hz, 1H), 2.92 (dd, J = 12.0, 3.0 Hz, 1H, H-3eqNeuAc), 2.78 (d, J = 9.6 Hz, 1H, H-5Glc-A), 2.44 (s, 3H, -OAc), 2.18 (s, 3H, -OAc), 1.98 (s, 3H, -OAc), 1.85 (s, 3H, -OAc), 1.76 (t, J = 12.6 Hz, 1H, H-3axNeuAc), 1.49 (d, J = 9.0 Hz, 1H, -OH). 13C NMR (150 MHz, CDCl3) δ: 172.0, 170.9, 170.3, 170.0, 168.5, 167.5, 167.1, 165.2, 164.9, 164.3, 153.4, 138.7, 138.6, 138.4, 138.25, 138.22, 138.1, 137.8, 133.4, 133.1, 132.93, 132.90, 132.7, 131.4, 130.8, 130.1, 129.9, 129.6, 129.5, 129.1, 129.0, 128.6, 128.4, 128.36, 128.33, 128.2, 128.1, 128.0, 127.89, 127.87, 127.76, 127.74, 127.71, 127.6, 127.5, 127.4, 127.3, 127.2, 126.6, 126.4, 123.0, 122.6, 103.0 (C-1Glc-B), 100.9 (Ph-CH-), 100.8 (C-1Gal-A), 100.5 (C-1Gal-C), 100.1 (C-1Glc-A), 99.5 (C-1GlcN), 96.9, 82.7, 80.6, 80.3, 78.8, 77.5, 76.4, 75.8, 75.7, 75.18, 75.16, 74.9, 74.88, 74.82, 74.7, 74.6, 74.5, 74.47, 74.40, 74.3, 73.6, 73.4, 73.1, 73.0, 72.8, 72.7, 71.6, 71.5, 71.2, 69.2, 68.9, 68.7, 67.9, 67.8, 67.0, 66.0, 63.8, 61.8, 58.9, 56.1, 52.9, 50.9, 37.1, 24.7, 21.4, 20.8, 20.7. HR ESI-TOF MS (m/z): calcd for C143H145N5O42Na2 [M + 2Na]2+, 1324.9580; found, 1324.9543. 2-Azidoethyl (Methyl 5-acetamido-7,8,9-tri-O-acetyl-3,5-dideoxy-D-glycero-α-D- galactonon-2-ulopyranosylonate)-(2→3)-(2-O-benzoyl-4,6-benzylidene-β-D-galactopyranosyl)(1→4)-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1→3)-{[(2-O-acetyl3,4,6-tri-O-benzyl-β-D-galactopyranosyl)-(1→6)]-(2-O-benzoyl-4-O-benzyl-β-Dgalactopyranosyl)}-(1→4)-(2-O-benzoyl-3,6-di-O-benzyl-β-D-glucopyranosyl)-(1→3)-2,4,6tri-O-benzyl-β-D-glucopyranoside (3b). To a stirred mixture of 14 (170 mg, 0.066 mmol), 15 (47 mg, 0.079 mmol), and freshly activated MS 4Å in anhydrous CH2Cl2 (3 mL) were added NIS (27 mg, 0.118 mmol) and TfOH (0.7 μL, 7.9 μmol) under a N2 atmosphere at 0 ℃. After the mixture was stirred for another 30 min, it was neutralized with Et3N, diluted with CH2Cl2 (30 mL), and filtered. The filtrate, after being washed with saturated aq. Na2S2O3 and NaCl, was dried over Na2SO4 and concentrated under vacuum. The residue was purified by silica gel column chromatography with EtOAc and toluene (1:5) as the eluents to give 3b (169 mg, 84%) as a white solid. 1H NMR (600 MHz, CDCl3) δ: 8.18 (d, J = 7.8 Hz, 2H, Ph), 7.83 (d, J = 7.8 Hz, 2H, Ph), 23 ACS Paragon Plus Environment


7.66 – 7.60 (m, 2H, Ph), 7.55 – 7.46 (m, 5H, Ph), 7.46 – 7.00 (m, 55H, Ph), 6.99 – 6.93 (m, 4H, Ph), 6.90 – 6.83 (m, 3H, Ph), 6.78 – 6.74 (m, 2H, Ph), 6.66 – 6.61 (m, 3H, Ph), 6.56 (d, J = 7.2 Hz, 1H, Ph), 5.61 – 5.54 (m, 3H, H-7NeuAc, H-8NeuAc, H-2Gal-A), 5.33 (s, 1H, Ph-CH-), 5.25 – 5.20 (m, 2H, H-2Gal-B, H-2Gal-C), 5.11 (t, J = 9.0 Hz, 1H, H-2Glc-A), 5.03 (d, J = 8.4 Hz, 1H, H-1GlcN), 5.01 – 4.95 (m, 4H, H-1Glc-A, H-1Gal-A, Bn), 4.90 (d, J = 11.4 Hz, 1H, Bn), 4.84 (d, J = 10.8 Hz, 1H, Bn), 4.79 (d, J = 12.0 Hz, 1H, Bn), 4.75 (d, J = 12.0 Hz, 1H, Bn), 4.70 – 4.65 (m, 2H, Bn), 4.54 – 4.39 (m, 10H, H-3Gal-A, Bn, H-9NeuAc, H-6NeuAc, H-1Gal-B), 4.35 – 4.20 (m, 9H, Bn, H-1Gal-C), 4.19 – 4.12 (m, 3H, H-1Glc-B, Bn, H-2GlcN), 4.10 – 4.03 (m, 4H, H-4Gal-A, Bn), 4.02 – 3.95 (m, 2H, H-9NeuAc), 3.90 – 3.83 (m, 3H, -OCH2CH2-, H-3Glc-B), 3.79 – 3.73 (m, 3H, H-4NeuAc), 3.70 – 3.66 (m, 2H), 3.62 (d, J = 10.8 Hz, 1H), 3.57 – 3.49 (m, 5H, H-5NeuAc, -OCH2CH2-), 3.45 – 3.22 (m, 14H, H3Gal-C, -COOCH3, H-3Glc-A, -CH2CH2N3), 3.18 – 3.15 (m, 2H, H-2Glc-B), 3.05 (dd, J = 7.8, 4.8 Hz, 1H), 2.91 (d, J = 9.6 Hz, 1H, H-3eqNeuAc), 2.82 – 2.77 (m, 2H), 2.45 (s, 3H, -OAc), 2.18 (s, 3H, OAc), 1.98 (s, 3H, -OAc), 1.88 (s, 3H, -OAc), 1.82 – 1.74 (m, 2H, H-3axNeuAc, -OAc). 13C NMR (150 MHz, CDCl3) δ: 172.0, 170.9, 170.3, 170.0, 168.4, 167.5, 167.1, 165.0, 164.9, 164.3, 153.4, 139.0, 138.9, 138.6, 138.5, 138.4, 138.3, 138.2, 138.1, 138.0, 137.9, 137.8, 137.7, 133.5, 133.1, 132.9, 132.8, 132.7, 131.4, 130.8, 130.0, 129.9, 129.8, 129.7, 129.5, 129.1, 128.9, 128.7, 128.6, 128.59, 128.55, 128.4, 128.3, 128.2, 128.1, 128.05, 128.02, 127.9, 127.8, 127.7, 127.6, 127.55, 127.53, 127.4, 127.3, 127.2, 127.1, 127.0, 127.0, 126.6, 126.5, 123.0, 122.6, 103.1 (C-1Glc-B), 100.9 (Ph-CH-), 100.8, 100.5, 100.4 (C-1Gal-B), 100.1 (C-1Gal-C), 99.4 (C-1GlcN), 97.0, 82.8, 81.0, 80.7, 79.9, 78.7, 77.5, 76.6, 75.8, 75.7, 75.69, 75.62, 75.2, 75.95, 74.90, 74.6, 74.5, 74.44, 74.40, 74.38, 74.2, 73.5, 73.32, 73.30, 73.14, 73.10, 73.0, 72.8, 72.7, 72.3, 72.1, 71.9, 71.5, 71.4, 71.1, 69.1, 68.8, 68.7, 67.9, 67.8, 67.7, 67.1, 66.0, 63.8, 58.8, 56.1, 52.9, 50.9, 37.0, 24.7, 21.4, 21.0, 20.8, 20.7. HR ESI-TOF MS (m/z): calcd for C172H175N5O48NaK [M + Na + K]2+, 1570.0471; found, 1570.0594. 2-Aminoethyl

(5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-non-2-ulopyranosylonic

acid)-(2→3)-β-D-galactopyranosyl-(1→4)-(2-acetamido-2-deoxy-β-D-glucopyranosyl)(1→3)-{[β-D-galactopyranosyl-(1→6)]-β-D-galactopyranosyl}-(1→4)-β-D-glucopyranosyl(1→3)-β-D-glucopyranoside (2). To a solution of 3b (50 mg) in anhydrous pyridine (5 mL) was added lithium iodide (200 mg). The mixture was heated and stirred at reflux for 12 h under a N2 atmosphere, and then concentrated and co-evaporated with toluene under vacuum. After the 24 ACS Paragon Plus Environment

Page 24 of 30

Page 25 of 30 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


residue was dissolved in ethanol (5 mL), NH2-NH2•H2O (1 mL) was added. The mixture was stirred at reflux for 12 h and then concentrated and co-evaperated with toluene to afford a residue, which was purified on a Sephadex LH-20 gel column with CH3OH as the eluent to give a white solid. The solution of the resultant solid in pyridine (4 mL) and Ac2O (1 mL) was stirred at room teparature for 6 h and then concentrated under vacuum. The residue was dissolved in CH3OH, and to the solution was added CH3ONa (1M in CH3OH) until pH reached 11. The mixture was stirred at room temperature for 12 h and then neutrilized with Amberlyst 15 H+ resin, and filtered. The filtrate was concentrated under vacuum. The residue was purified on a Sephadex LH-20 gel column with CH3OH as the eluent to give a white solid. The solid was dissolved in CH3OH and H2O (V/V, 4:1, 5 mL) and then mixed with Pd(OH)2 (10 mg). After the mixture was stirred under a H2 atmosphere at 50 psi for 36 h, it was filtered. The filtrate was concentrated to give a residue, which was purified on a Sephadex G-25 gel column with H2O as the eluent to produce the synthetic target 2 (11 mg, 48%) as a white solid. 1H NMR (600 MHz, D2O) δ: 4.60 (d, J = 8.4 Hz, 1H), 4.51 (d, J = 8.4 Hz, 1H), 4.38 (d, J = 7.8 Hz, 1H), 4.34 – 4.29 (m, 2H), 4.26 (d, J = 7.8 Hz, 1H), 4.03 – 4.00 (m, 1H), 3.96 – 3.91 (m, 1H), 3.90 – 3.17 (m, 45H), 2.58 (dd, J = 12.6, 4.2 Hz, 1H), 1.85 (s, 3H), 1.82 (s, 3H), 1.64 (t, J = 12.6 Hz, 1H). 13C NMR (150 MHz, D2O) δ: 174.8, 174.24, 173.21, 103.16, 103.0, 102.7, 102.4, 102.3, 102.0, 99.7, 84.1, 81.8, 78.9, 77.9, 75.4, 75.3, 75.1, 75.0, 74.6, 74.4, 74.2, 73.5, 73.0, 72.7, 72.5, 72.0, 71.7, 71.4, 71.3, 70.7, 69.6, 69.3, 68.9, 68.5, 68.4, 68.2, 68.0, 60.9, 60.9, 60.5, 60.0, 59.8, 55.1, 39.2, 22.1, 21.7. HR ESI-TOF MS (m/z): calcd for C51H88N3O39 [M + H]+, 1366.4995; found, 1366.5010.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. 1D and 2D 1H and 13C NMR spectra and HR MS spectra of all new compounds (PDF) AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected] Notes The authors declare no competing financial interest. 25 ACS Paragon Plus Environment


ACKNOWLEDGMENT This work was supported in part by the National Natural Science Foundation of China (No. 21702124), the Shandong Provincial Natural Science Foundation (No. ZR2017MB013), and the Fundamental Research Funds of Shandong University (No. 2016TB004).

REFERENCES (1) Johri, A. K.; Paoletti, L. C.; Glaser, P.; Dua, M.; Sharma, P. K.; Grandi, G.; Rappuoli, R. Group B Streptococcus: global incidence and vaccine development. Nat. Rev. Microbiol. 2006, 4, 932-942. (2) Edmond, K. M.; Kortsalioudaki, C.; Scott, S.; Schrag, S. J.; Zaidi, A. K.; Cousens, S.; Heath, P. T. Group B streptococcal disease in infants aged younger than 3 months: systematic review and meta-analysis. Lancet 2012, 379, 547-556. (3) Doare, K. L.; Heath, P. T. An overview of global GBS epidemiology. Vaccine 2013, 31, D712. (4) Cieslewicz, M. J.; Chaffin, D.; Glusman, G.; Kasper, D.; Madan, A.; Rodrigues, S.; Fahey, J.; Wessels, M. R.; Rubens, C. E. Structural and genetic diversity of group B Streptococcus capsular polysaccharides. Infect. Immun. 2005, 73, 3096-3103. (5) Berti, F.; Campisi, E.; Toniolo, C.; Morelli, L.; Crotti, S.; Rosini, R.; Romano, M. R.; Pinto, V.; Brogioni, B.; Torricelli, G.; Janulczyk, R.; Grandi, G.; Margarit, I. Structure of the type IX group B Streptococcus capsular polysaccharide and its evolutionary relationship with types V and VII. J. Biol. Chem. 2014, 289, 23437-23448. (6) Baker, C. J.; Paoletti, L. C.; Rench, M. A.; Guttormsen, H. K.; Carey, V. J.; Hickman, M. E.; Kasper, D. L. Use of capsular polysaccharide-tetanus toxoid conjugate vaccine for type II group B Streptococcus in healthy women. J. Infect. Dis. 2000, 182, 1129-1138. (7) Baker, C. J.; Rench, M. A.; Fernandez, M.; Paoletti, L. C.; Kasper, D. L.; Edwards, M. S. Safety and immunogenicity of a bivalent group B streptococcal conjugate vaccine for serotypes II and III. J. Infect. Dis. 2003, 188, 66-73.


Page 26 of 30

Page 27 of 30 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


(8) Jennings, H. J.; Rosell, K. G.; Katzenellenbogen, E.; Kasper, D. L. Structural determination of the capsular polysaccharide antigen of type II group B Streptococcus. J. Biol. Chem. 1983, 258, 1793-1798. (9) Paoletti, L. C.; Madoff, L. C. Vaccines to prevent neonatal GBS infection. Semin. Neonatol. 2002, 7, 315-323. (10) Madhi, S. A.; Dangor, Z.; Heath, P. T.; Schrag, S.; Izu, A.; Sobanjo-Ter Meulen, A.; Dull, P. M. Considerations for a phase-III trial to evaluate a group B Streptococcus polysaccharide-protein conjugate vaccine in pregnant women for the prevention of early- and late-onset invasive disease in young-infants. Vaccine 2013, 31S, D52-57. (11) Astronomo, R. D.; Burton, D. R. Carbohydrate vaccines: Developing sweet solutions to sticky situations? Nat. Rev. Drug Discov. 2010, 9, 308-324. (12) Rappuoli, R.; Black, S.; Lambert, P. H. Vaccine discovery and translation of new vaccine technology. Lancet 2011, 378, 360-368. (13) Hecht, M. L.; Stallforth, P.; Silva, D. V.; Adibekian, A.; Seeberger, P. H. Recent advances in carbohydrate-based vaccines. Curr. Opin. Chem. Biol. 2009, 13, 354-359. (14) Seeberger, P. H.; Werz, D. B. Synthesis and medical applications of oligosaccharides. Nature 2007, 446, 1046-1051. (15) Seeberger, P. H.; Pereira, C. L.; Khan, N.; Xiao, G.; Diago-Navarro, E.; Reppe, K.; Opitz, B.; Fries, B. C.; Witzenrath, M. A semi-synthetic glycoconjugate vaccine candidate for carbapenem-resistant Klebsiella pneumoniae. Angew. Chem., Int. Ed. 2017, 56, 13973-13978. (16) Gao, J.; Guo, Z. Chemical synthesis of the repeating unit of type V group B Streptococcus capsular polysaccharide. Org. Lett. 2016, 18, 5552-5555. (17) Sun, B.; Srinivasan, B.; Huang, X. Pre-activation-based one-pot synthesis of an α-(2,3)sialylated core-fucosylated complex type bi-antennary N-glycan dodecasaccharide. Chem. – Eur. J. 2008, 14, 7072-7081. (18) Huang, X.; Huang, L.; Wang, H.; Ye, X. S. Iterative one-pot synthesis of oligosaccharide. Angew. Chem., Int. Ed. 2004, 43, 5221-5224. (19) Mondal, P. K.; Liao, G.; Mondal, M. A.; Guo, Z. Chemical synthesis of the repeating unit of type Ia group B Streptococcus capsular polysaccharide. Org. Lett. 2015, 17, 1102-1105. (20) Basu, N.; Maity, S. K.; Roy, S.; Singha S.; Ghosh, R. FeCl3 mediated arylidenation of carbohydrates. Carbohydr. Res. 2011, 346, 534-539. 27 ACS Paragon Plus Environment


(21) Ramadan, S.; Yang, W.; Zhang, Z.; Huang, X. Synthesis of chondroitin sulfate A bearing syndecan-1 glycopeptide. Org. Lett. 2017, 19, 4838-4841. (22) Xia, J.; Alderfer, J. L.; Piskorz, C. F.; Matta, K. L. Total synthesis of sialylated and sulfated oligosaccharide chains from respiratory mucins. Chem. – Eur. J. 2000, 6, 3442-3451. (23) Seifert, J.; Lergenmüller, M.; Ito, Y. Synthesis of an α-(2,3)-sialylated, complex-type undecasaccharide. Angew. Chem., Int. Ed. 2000, 39, 531-534. (24) Sherman, A. A.; Yudina, O. N.; Mironov, Y. V.; Sukhova, E. V.; Shashkov, A. S.; Menshov, V. M.; Nifantiev, N. E. Study of glycosylation with N-trichloroacetyl-D-glucosamine derivatives in the syntheses of the spacer-armed pentasaccharides sialyl lacto-N-neotetraose and sialyl lactoN-tetraose, their fragments, and analogues. Carbohydr. Res. 2001, 336, 13-46. (25) Gong, J. Z.; Liu, H.; Nicholls, J. M.; Li, X. C. Studies on the sialylation of galactoses with different C-5 modified sialyl donors. Carbohydr. Res. 2012, 361, 91-99. (26) Hsu, C. H.; Chu, K. C.; Lin, Y. S.; Han, J. L.; Peng, Y. S.; Ren, C. T.; Wu, C. Y.; Wong, C. H. Highly alpha-selective sialyl phosphate donors for efficient preparation of natural sialosides. Chem. - Eur. J. 2010, 16, 1754-1760. (27) Chuang, H. Y.; Ren, C. T.; Chao, C. A.; Wu, C. Y.; Shivatare, S. S.; Cheng, T. J.; Wu, C. Y.; Wong, C. H. Synthesis and vaccine evaluation of the tumor-associated carbohydrate antigen RM2 from prostate cancer. J. Am. Chem. Soc. 2013, 135, 11140-11150. (28) Crich, D.; Wu, B. Stereoselective iterative one-pot synthesis of N-glycolylneuraminic acidcontaining oligosaccharides. Org. Lett. 2008, 10, 4033-4035. (29) Demchenko, A. V.; Boons, G. J. A highly convergent synthesis of a complex oligosaccharide derived from group B type III Streptococcus. J. Org. Chem. 2001, 66, 2547-2554. (30) Eisele, T.; Toepfer, A.; Kretzschmar, G.; Schmidt, R. R. Synthesis of a thio-linked analogue of sialyl Lewis X. Tetrahedron Lett. 1996, 37, 1389-1392. (31) Hanashima, S.; Castagner, B.; Esposito, D.; Nokami, T.; Seeberger, P. H. Synthesis of a sialic acid α(2-3) galactose building block and its use in a linear synthesis of sialyl Lewis X. Org. Lett. 2007, 9, 1777-1779. (32) Hotta, K.; Ishida, H.; Kiso, M.; Hasegawa, A. Synthetic studies on sialoglycoconjugates 66: first total synthesis of a cholinergic neuron-specific ganglioside GQ1bα. J. Carbohydr. Chem. 1995, 14, 491-506. (33) Vohra. Y.; Buskas. T.; Boons. G. J. Rapid assembly of oligosaccharides: a highly convergent 28 ACS Paragon Plus Environment

Page 28 of 30

Page 29 of 30 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


strategy for the assembly of a glycosylated amino acid derived from PSGL-1. J. Org. Chem. 2009, 74, 6064-6071. (34) Seifert, J.; Ogawa, T.; Ito, Y. Syntheses of α-dystroglycan derived sialylated glycosyl amino acids carrying a novel mannosyl serine/threonine linkage. Tetrahedron Lett. 1999, 40, 6803-6807. (35) Cattaneo, V.; Carboni, F.; Oldrini, D.; Ricco, R. D.; Donadio, N.; Ros, I. M. Y.; Berti, F.; Adamo, R. Synthesis of group B Streptococcus type III polysaccharide fragments for evaluation of their interactions with monoclonal antibodies. Pure Appl. Chem. 2016, 89, 855-875. (36) Pozsgay, V.; Brisson, J.R.; Jennings, H. J.; Allen, S.; Paulson, J. C. Synthetic oligosaccharide related to group B streptococcal polysaccharide. 5. Combined chemical and enzymatic synthesis of a pentasaccharide repeating unit of the capsular polysaccharide of type III group B Streptococcus and one- and two-dimensional NMR spectroscopic studies. J. Org. Chem. 1991, 56, 3377-3385. (37) Pozsgay, V.; Gaudino, J.; Paulson, J. C.; Jennings, H. J. Chemo-enzymatic synthesis of a branching decasaccharide fragment of the capsular polysaccharide of type III group B Streptococcus. Bioorg. Med. Chem. Lett. 1991, 1, 391-394. (38) Zou, W.; Brisson, J. R.; Yang, Q. L.; Van der Zwan, M.; Jennings, H. J. Synthesis and NMR assignment of two repeating units (decasaccharide ) of the type III group B Streptococcus capsular polysaccharide and its

13

C-labeled and N-propionyl substituted sialic acid and analogues.

Carbohydr. Res. 1996, 295, 209-228. (39) Zou, W.; Jennings, H. J. Chemical-enzymatic synthesis of a branched hexasaccharide fragment of type I a group B Streptococcus capsular polysaccharide. J. Carbohydr. Chem. 1996, 15, 925-937.



TOC graphic


Page 30 of 30

Chemical Synthesis of the Repeating Unit of Type II Group B

Recommend Documents