A Synthetic Carbohydrate-Protein Conjugate Vaccine Candidate against Klebsiella pneumoniae Serotype K2

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A Synthetic Carbohydrate−Protein Conjugate Vaccine Candidate

against Klebsiella pneumoniae Serotype K2

Mettu Ravinder,^⊥Kuo-Shiang Liao,^⊥Yang-Yu Cheng, Sujeet Pawar, Tzu-Lung Lin, Jin-Town Wang,

and Chung-Yi Wu *

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ABSTRACT: Klebsiella pneumoniae causes pneumonia and liver

abscesses in humans worldwide and contains virulence factor

capsular polysaccharides and lipopolysaccharides linked to the cell

wall. Although capsular polysaccharides are good antigens for

vaccine production and capsular oligosaccharides conjugate

vaccines are proven eﬀective against infections caused by

encapsulated pathogens, there is still no Klebsiella pneumoniae

vaccine available. One obstacle is that the capsular polysaccharide

of a dominated Klebsiella pneumoniae serotype K2 is diﬃcult to

synthesize chemically due to the three 1,2-cis linkages in its

structure. In this study, we successfully synthesized K2 capsular

polysaccharides from tetra- to octasaccharides in highly a stereoselective manner. Subsequently, three synthesized glycans were

conjugated to DT protein to provide glycoconjugate vaccine candidates (DT-Hexa, DT-Hepta, and DT-Octa) that were used in in

vivo immunization experiments in mice. The results of immunized studies showed all three glycoconjugates elicited antibodies that

recognized all of the synthetic glycans at 1:200-fold dilution. Particularly, the DT-Hepta conjugate elicited a higher level of

antibodies that can recognize longer glycan (octasaccharide) even at 1:12800-fold dilution and exhibited good bactericidal activity.

Our results concluded that heptasaccharide is the minimal epitope and a potential candidate for the vaccine against the K2 sero

group of Klebsiella pneumoniae.

The structure of the K2 polysaccharide repeating unit was

characterized by Corsaro et al. in 2005 to be →3)-β-^D-Glcp-

(1→4)-β-^D-Manp-(1→4)-α-D-Glcp-(1→ as the backbone with

a chain carrying α-^D-GlcpA-(1→ branches at the 3 position of

the mannoses¹⁶(Figure 1). Notably, the K2 serotype does not

contain repeating sequences of D-mannose-α-2/3-D-mannose

or ^L-rhamnose-α-2/3-rhamnose units that are commonly found

in CPS structures of Klebsiella strains (K7, K14, K21, K24,

INTRODUCTION

■

Klebsiella pneumoniae (KP) is an opportunistic, rod-shaped,

Gram-negative bacterium, belonging to the Enterobacteriaceae

family,¹and causes both hospital-acquired and community-

acquired infections such as urinary tract infections, wound

infections, and bloodstream infections in addition to pneumo-

nia and meningitis, particularly in young children and

immunodeﬁcient adults.^2,3More than 50% of reported

pneumonia deaths were caused by KP.⁴Moreover, KP was

ranked second among all pathogens that cause a pyogenic liver

abscess (PLA) in humans.^5,6Initially, KP was geographically

restricted to some Asian countries, and the ﬁrst liver abscess

case caused by KP was reported in the 1980s in Taiwan.^7,8In

recent days, KP-associated PLA cases have been rapidly

increasing globally and is now considered as an endemic in

North America, Europe, and some Asian countries and regions

including Taiwan, Singapore, Korea, Hong Kong, China, and

Vietnam.⁹⁻¹¹Of the 78 serotypes of KP identiﬁed so far, K1

and K2 serotypes are reported as the most virulent and major

contributors for PLA disease.^12,13In general, members of the

genus Klebsiella express capsular polysaccharide (CPS, K-

antigen) and lipopolysaccharide (LPS, O-antigen) components

on their cell surface, and both CPS and LPS have virulent

factors that contribute to the pathogenicity in humans.^14,15

Figure 1. Structure of the tetrasaccharide repeating unit (K2

polysaccharide).

Special Issue: A New Era of Discovery in Carbohy-

drate Chemistry

Received: June 14, 2020

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K28, K74, K80, and others) and easily recognized by surface

lectin of macrophages to enable lectinophagocytosis. The

absence of these two sequences may be the cause for the more

virulent nature of K1 and K2 serotypes.^1,17

In developing countries, clinical diagnosis and treatment of

Klebsiella remain challenging and cause an economic burden.

Generally, various combinations of drugs with a prolonged

duration are needed to treat the bacterial infections caused by

Klebsiella.¹⁸Due to the emergence of hypervirulent and

multidrug-resistant KP strains,^19,20a safer and more eﬀective

alternate approach is urgently needed to treat or prevent the

infection. Over the last three decades, numerous eﬀorts have

focused on Klebsiella vaccine development by conducting

active and passive immunization trials; however, none has

successfully brought the vaccine into the global market so

far.²¹⁻²³Most of the clinical trials were executed on LPS-based

vaccines, which exhibited lots of issues during the active

immunization trials due to the presence of the endotoxic Lipid

A component in LPS.^21,24

Figure 2. Targeted K2 antigens for the synthesis and immunological

study.

In recent days, CPS-based glycoconjugate vaccines have

become very attractive alternatives due to a better immune

response, superior safety, and more success in preventing

infectious diseases caused by Haemophilus influenzae, Strepto-

coccus pneumonia, and Neisseria meningitides.²⁵⁻²⁸In order to

develop an eﬀective glycoconjugate vaccine against Klebsiella, it

is essential to identify the minimal, suitable length of the

saccharide that exhibits immunogenic nature. Although

oligosaccharide fragments can be isolated from natural sources,

the isolated products are often heterogeneous and may include

bacterial contaminants. Thus, chemical synthesis, which

provides pure polysaccharides with a deﬁned length and

preinstalled linker required for conjugation, is a preferable

approach to obtain oligosaccharide fragments for vaccine

development.²⁹To our knowledge, only one report exists on

the synthetic CPS repeating unit of the K2 serotype, and the

study was limited to the synthesis of the tetrasaccharide unit in

the form of methyl ester methyl glycoside,³⁰which may not be

suitable for further vaccination studies. Therefore, an

alternative synthetic strategy to prepare the desired compound

is required. In our continuing eﬀort toward the development of

synthetic glycoconjugate-based vaccines,³¹here we described

the chemical synthesis of the K2 antigen repeating unit and its

derivatives. We, then, coupled the synthetic products to carrier

proteins to study their immunological properties.

glycosylation of common tetrasaccharide acceptor 7 with

corresponding glycosyl ﬂuorides 13, 14, 15, and 16 via the

[1+4], [2 + 4], [3+4], and [4+4] glycosylation strategy,

respectively, and, then, global deprotection. On the other hand,

target compound 1 was obtained by complete deprotection of

6. The tetrasaccharides 6 was obtained by stereoselective β-

glycosylation of donor 9 with acceptor 8, and trisaccharide 8 is

assembled via β-mannosylation followed by selective α-

glucosylation on mannose 11 with corresponding glucose

building blocks 12 and 10, respectively. Our synthetic design is

to protect the 6-O-position in 10 with the TBDPS group,

which is bulky and can be selectively removable in the presence

of other functional groups, to accomplish stereoselective 1,2-cis

glycosylation. Moreover, the PMB group is used as a

temporary protection group at the 3-O-position in 9 and 11.

Synthesis of the Trisaccharide Acceptor. The synthesis of

target K2 antigens began with the preparation of required

monosaccharide building blocks 9−12. Among them, 9−11

were prepared based on the reported procedures with

modiﬁcations^32,33(Schemes S3 −S5), whereas building block

12 was prepared using a new synthetic strategy (Scheme S6).

Then, we focused on the synthesis of the trisaccharide linker

compound 8 by either β-mannosylation at C-1, followed by α-

glucosylation at C-3 of mannose, or vice versa. We chose the

ﬁrst strategy because the β-mannosylation is favored when the

aromatic groups (Bn, PMB) are at the C-3 position of

mannose; then, bulky groups such as sugar moieties at C-3 will

give poor selectivity.³⁴Donor 11 was glycosylated with

acceptor 12 using Prof. Crich’s reaction conditions,³⁵in

which BSP and TTBP were used as promoters to provide

desired disaccharide 17a in high anomeric selectivity (17a/

17b, β/α = 12:1) with a 62% yield. The compounds were

readily separable by silica gel chromatography. The stereo-

chemistry of the newly formed glycosidic bond was conﬁrmed

All of the target antigens, 1−5, were designed to have a

spacer aminopentyl group at the reducing end for further

conjugation to the carrier protein for immunological studies

(Figure 2). First, we constructed tetrasaccharide 1 and, then,

elongated the sugar chain via the [1+4], [2+4], [3+4], and

[4+4] glycosylation strategy to get 2−5 compounds,

respectively.

RESULTS AND DISCUSSION

■

Part 1: Chemical Synthesis. Retrosynthetic Analysis.

The chemical synthesis of the repeating CPS core

tetrasaccharide of KP is very challenging because the

tetrasaccharide unit contains three 1,2-cis glycosylation bonds

in their structure. The installation of glycosides in a

stereoselective fashion at the desired position is a diﬃcult

task, particularly at β-mannosylation and α-glucoronic acid

attachments, often resulting in the formation of an inseparable

mixture of anomers. We designed the retrosynthetic plan

(Scheme 1) to obtain the desired molecules 2−5 by

by their anomeric C−H coupling constants (β-anomer ¹J_C−H

=

1

156.5 Hz; α-anomer J_C−H= 172 Hz).

Next, the PMB ether of 17a was removed by DDQ

treatment, and the resulted alcohol 18 was treated with donor

10 in the presence of the NIS−TfOH system. Amazingly, this

reaction aﬀorded the desired trisaccharide 19 in only the α-

anomer with a good yield of 84%. The glycosidic linkage was

conﬁrmed to be α-linkage by its coupling constant values (³J_HH

1

= 3.5 Hz; J_C−H= 172.4 Hz). At this juncture, it became

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Scheme 1. Retrosynthetic Analysis of CPS Core Oligosaccharides 1−5

a

Scheme 2. Synthesis of Trisaccharide 22

a

Reagents and conditions: (a) BSP, TTBP, Tf₂O, CH₂Cl₂, 4 Å MS, −60 °C, 1.5 h, 62% (α/β = 1:12); (b) DDQ, CH₂Cl₂/phosphate buﬀer pH 7

(9:1), 0 °C to rt, 2 h, 81%; (c) 10, NIS, TfOH (0.5 M), 4 Å MS, CH₂Cl₂, −40 °C, 2 h, 84%; (d) HF·py, THF/pyridine (9:1), 0 °C to rt, overnight,

91%; (e) BAIB, TEMPO, CH₂Cl₂/H₂O, rt, 2.5 h, 92%; (f) MeI, K₂CO₃, CH₂Cl₂, rt, overnight, 92%.

a

Scheme 3. Synthesis of Tetrasaccharide Acceptor 23

a

Reagents and conditions: (a) Et₃SiH, TfOH, CH₂Cl₂, 4 Å MS, −78 °C, 1 h, 94%; (b) 9, NIS, TfOH, CH₂Cl₂, 4 Å MS, −20 °C, 1 h, 92%; (c)

DDQ, CH₂Cl₂/phosphate buﬀer, pH 7 (9:1), 0 °C, 1.5 h. 72%.

necessary to free compound 19 from the TBDPS group for

further manipulations. Hence, 19 was treated with HF·py to

aﬀord alcohol 20 in a 91% yield. The primary alcohol

functionality of 20 was converted into the corresponding

carboxylic acid moiety through 2,2,6,6-tetramethylpiperidine-

1-oxyl (TEMPO) free radical-mediated oxidation using

bis(acetoxy)iodo benzene (BAIB) as a co-oxidant to obtain

21, which was methylated with MeI and K₂CO₃to aﬀord the

trisaccharide ester 22 in a good yield (Scheme 2).

Synthesis of the Tetrasaccharide Acceptor. After the

successful synthesis of trisaccharide 22, we focused on the

preparation of tetrasaccharide 6 (Scheme 3) by regioselectively

opening the ring of the benzylidene group (22) in the Et₃SiH−

TfOH system to provide the secondary alcohol 8 that serves as

an acceptor for further reaction. Next, we examined the

coupling of acceptor 8 and donor 9 in the NIS−TfOH system.

Delightfully, we achieved the desired compound 6 in only the

β-anomer with a good yield of 92%. The glycosidic linkage was

conﬁrmed to be β-bonds based on its anomeric C−H coupling

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a

Scheme 4. Synthesis of Disaccharide Donors 14 and 25

a

Reagents and conditions: (a) PdCl₂, CH₃COONa, acetic acid (moist), rt, 24 h, 70%; (b) CCl₃CN, DBU, CH₂Cl₂, 0 °C to rt, 2 h, 65%; (c) DAST,

CH₂Cl₂, −20 °C, 1.5 h, 92%.

a

Scheme 5. Attempted Synthesis of Hexasaccharide 26

a

Reagents and conditions: (a) TMSOTf, CH₂Cl₂, 4 Å MS, −30 to −20 to −10 to 0 °C to rt; (b) Cp₂HfCl₂, AgOTf, Et₂O, 4 Å MS, −20 to 0 °C.

a

Scheme 6. Manipulation and Synthesis of Tetrasaccharide Acceptor 7

a

Reagents and conditions: (a) PTSA, MeOH/CH₂Cl₂(1:1), rt, overnight, 87%; (b) Ac₂O, pyridine, 0 °C to rt, 4 h, 99%; (c) DDQ, CH₂Cl₂/

phosphate buﬀer pH 7 (9:1), 0 °C to rt, 2 h, 48%; or SnCl₄, PhSH, −78 to −50 °C, 1 h, 93%.

constant (¹J_CH= 165 Hz). Finally, compound 6 was converted

into corresponding acceptor 23 by DDQ oxidation to remove

3-O-PMB ether. The same approach and reaction conditions

were used to prepare other structural units (S2a, S7, and S9)

that have an O-allyl group at the reducing end instead of the 5-

azidopentyl linker, and these compounds were used as a donor

source for chain extension (Scheme S1). During its synthesis,

we observed that all of the reactions gave good yields with high

anomeric selectivity at each glycosylation step.

Synthesis of Disaccharide Donor. With the tetrasaccharide

acceptor in hand, we turned our attention to chain elongation,

which is a challenging task because it requires selective α-

glycosidic bond linkage. In general, an increase in the length of

the donor or acceptor chain aﬀects its reactivity and anomeric

selectivity. Therefore, a study of optimization to access higher

α-selectivity is highly beneﬁcial. In this regard, we conducted a

model study on hexasaccharide construction through [2+4]

glycosylation. Disaccharide imidate donor 25 was prepared

from S2a via hydrolysis of the anomeric O-allyl group by the

treatment of PdCl₂and AcONa in AcOH,³⁶followed by

CCl₃CN and DBU (Scheme 4). Then, donor 25 and acceptor

23 were subjected to [2+4] glycosylation using TMSOTf at

various temperatures (30/−10/0 °C and rt). Unfortunately,

none has oﬀered satisfactory results due to the decomposition

of the donor (Scheme 5). Alternatively, we prepared glycosyl

ﬂuoride donor 14 from hemiacetal 24 by the treatment of

DAST (Scheme 4). The resulted glycosyl ﬂuoride, 14, was

treated with the same acceptor, 23, using the Cp₂HfCl₂−

AgOTf system³⁷at various temperatures. Disappointingly, this

reaction also did not proceed, and a slight decomposition of

the glycosyl donor was observed (Scheme 5).

e. Synthesis of a New Tetrasaccharide Acceptor. After

additional unsuccessful experiments, we suspected the problem

came from the structure of acceptor 23, in which the presence

of the benzylidene ring might cause steric hindrance to the

hydroxyl group. To increase reactivity and decrease the steric

hindrance, we modiﬁed acceptor 23 to remove the benzylidene

group and install acetyl groups at 4- and 6-OH by treating 6

with p-toluenesulfonic acid (PTSA) to produce diol 27, which

was later masked with acetyl groups by employing standard

conditions to give 28. The PMB ether was removed by

oxidation with DDQ in a mixture of CH₂Cl₂and neutral

phosphate buﬀer solution, and this reaction condition aﬀorded

compound 7 in a 48% yield; however, a better yield (93%) was

obtained when compound 28 was treated with SnCl₄and

thiophenol³⁸(Scheme 6).

Synthesis of K2 Penta to Octa Oligosaccharides. With

acceptor 7 in hand, we focused on screening for suitable

glycosylation conditions for the synthesis of hexasaccharide via

the [2+4] strategy. Glycosylation of imidate donor 25 with

acceptor 7 using catalytic TMSOTf resulted in the formation

of several products, which were not separable by column

chromatography. Alternatively, we used the Cp₂HfCl₂−AgOTf

system in toluene for glycosylation of glycosyl ﬂuoride 14 with

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a

Scheme 7. Synthesis of Hexasaccharide 26a

a

Reagents and conditions: (a) TMSOTf, CH₂Cl₂, 4 Å MS, −40 °C, 30 min; (b) Cp₂HfCl₂(4 equiv), AgOTf (8 equiv), toluene, 4 Å MS, −30 °C,

1.5 h, 64%.

a

Scheme 8. Synthesis of Penta-, Hepta-, and Octasaccharides 29−31

a

Reagents and conditions: (a) Cp₂HfCl₂, AgOTf, toluene, 4 Å MS, −30 °C, 90 min, 29 (70%), 30 (68%), and 31 (70%).

acceptor 7 and found this reaction aﬀorded fully protected

hexasaccharide 26 in only the α-isomer in a modest yield

(23%), which upon optimization was increased to 64% yield

(Scheme 7). The glycosidic bond in 26a was determined to be

α-linkage based on its anomeric C−H coupling constant value

(α-anomer ¹J_C−H= 172.6 Hz). After several attempts, we found

that 4 equiv of Cp₂HfCl₂and 8 equiv of AgOTf in toluene

solvent at −30 °C are the optimal conditions for glycosylation

of 14 with acceptor 7. Meanwhile, we have also screened this

glycosylation reaction using several promotors such as TfOH,

Tf₂O, BF₃(OEt)₂, SnCl₂−AgClO₄, and Cp₂HfCl₂₋AgOTf in

various mole ratios and solvent systems. All conditions either

led to a low yield of the desired compound or decomposition

of the donor (Table ST 1).

In order to identify the optimal length of the K2 antigen for

vaccine design, we prepared the remaining targeted penta-,

hepta-, and octasaccharides to complete the sequence for the

study. The required glycosyl ﬂuorides, 13, 15, and 16, were

prepared (Scheme S2) and coupled with acceptor 7

individually through the [1+4], [3+4], and [4+4] glycosylation

strategy using the optimized glycosylation conditions to aﬀord

29−31 in 68−70% yields (Scheme 8). The glycosidic bonds in

29−31 were determined to be α-linkage based on anomeric

C−H coupling constant values through 2D NMR analysis.

Finally, the synthesized oligosaccharides 6, 26a, and 29−31

were subjected to global deprotection through a two-step

procedure (Scheme 9). The ﬁrst saponiﬁcation of all esters was

carried out by the treatment of LiOH in a mixture of p-dioxane

and water. Then, Pd/C-catalyzed hydrogenolysis was carried

out in a mixture of MeOH, THF, and water with a catalytic

amount of formic acid; thus, all protecting groups were

removed, and the azido group was reduced to the amine group

in one pot to aﬀord antigens 1−5 in 39−60% yields. All

synthetic antigens, 1−5, were well characterized by 1D and 2D

NMR and HRMS spectroscopic analysis (Supporting In-

formation). Additionally, we compared the ¹H NMR spectrum

1

of the synthetic tetrasaccharide 1 with the reported H NMR

spectrum of the isolated tetrasaccharide fragment and found

both spectra are identical at all chemical shifts except in the

linker region.¹⁶

Part 2: Preparation and Characterization of Glyco-

conjugates. In order to study the immunological properties

of the synthesized oligosaccharides 1−5, the synthetic

compounds need to be coupled with the carrier protein at

the spacer arm because oligosaccharides are nonimmunogenic

by themselves. It should be noted that molecular weight is an

important factor for CPS-based vaccine preparation. Glyco-

conjugates of long-chain oligosaccharides are known to be

excellent immunogens in lower doses in clinical studies.³⁹

Short-length oligosaccharides generally do not induce anti-

bodies, so they usually are excluded from the preparation of

glycoconjugates.⁴⁰The repeating unit of K2 polysaccharide is

tetrasaccharide, which contains GlcA (Figure 1), a critical

component, because the carboxyl group of this GlcA provides a

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a

Scheme 9. Global Deprotection of Synthesized Oligosaccharides 6, 26a, and 29−31

a

Reagents and conditions: (a) (i) LiOH, p-dioxane/H₂O (3:1), 75 °C, overnight; (ii) Pd(OH)₂/H₂(balloons), MeOH/THF/H₂O (2:1:1),

HCOOH (cat.), rt, 36 h, 1 (58%), 2 (60%), 3 (39%), 4 (42%), and 5 (48%) over two steps.

Scheme 10. Oligosaccharides Conjugation to the Carrier Protein

negative charge that may lead to the change in glycan

conformation. However, one GlcA in the K2 oligosaccharide

does not demonstrate the charge eﬀect. Therefore, we chose to

use the hepta- and octamer with two GlcAs in order to provide

a preliminary charge eﬀect. Moreover, the glycan conformation

of the hepta- or octamer is closer to the real K2 polysaccharide.

Accordingly, we selected hexa-, hepta-, and octasaccharides 3−

5 for glycoconjugate vaccine preparation. We adopted a well-

known thiol-maleimide coupling method for carbohydrate−

proteins conjugation.⁴¹First, the amino-linker spacer was

converted into an amidothiol linker by treating the selected

oligosaccharides 3−5 with the commercially available reagent

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Figure 3. F635 total intensity of the synthesized oligosaccharides 1−5 with antibodies elicited in mice by glycoconjugates DT-Hexa 3aa, DT-Hepta

4aa, and DT-Octa 5aa. (a) Binding response of serum antibodies (4 mice, no. I, II, IV, and V) elicited by DT-Hexa 3aa to glycans 1−5. (b)

Binding response of serum antibodies (1 mouse, no. III) elicited by DT-Hexa 3aa to glycans 1−5. (c) Binding response of serum antibodies (5

mice) elicited by DT-Hepta 4aa to glycans 1−5. (d) Binding response of serum antibodies (5 mice) elicited by DT-Octa 5aa to glycans 1−5. RFU,

relative ﬂuorescence units. Error bars represent mean SD.

3,3-dithiobis(sulfosuccinimidylpropionate) (DTSSP) in PBS

buﬀer (pH 7.4) at 28 °C overnight. The disulﬁde bond was

then cleaved with dithiothreitol (DTT) by stirring at 40 °C for

2 h to obtain the free thiol products 3a−5a in 80−88% yields

after puriﬁcation on a size exclusion column, LH-20 (Scheme

10).

Moreover, the maleimide group was incorporated onto the

carrier protein DT (detoxiﬁed diphtheria toxin), and the

number of maleimide linkers on the protein was determined by

MALDI-TOF MS analysis, which revealed that 16 maleimide

linkers were coupled to one DT molecule on average. Finally,

the thiol-modiﬁed oligosaccharides 3a−5a were conjugated to

the maleimide-linked DT protein in PBS buﬀer (pH 7.4)

individually to obtain the glycoconjugate vaccine candidates

DT-Hexa 3aa, DT-Hepta 4aa, and DT-Octa 5aa with various

carbohydrate epitopes on the protein (Scheme 10). The

resulted oligosaccharide to DT molar ratio was determined by

MALDI-TOF MS spectrometry analysis (Figure SF1 −3 and

Table ST2).

Part 3: Immunological Studies. Immunization of Mice.

To test whether synthetic vaccine candidates DT-Hexa 3aa,

DT-Hepta 4aa, and DT-Octa 5aa are suﬃcient epitopes for

antibody recognition, we chose 6−8 week-old female BALB/c

mice (n = 5) and grouped them into four groups of ﬁve mice.

Mice were immunized three times with 2 μg of glycan and 2 μg

of adjuvant C34^42,43in 100 μL of PBS buﬀer at two week

intervals. PBS buﬀer alone was injected into the control group.

Antisera were collected one week after the third immunization,

the antibody response study, and bactericidal assay.

Identiﬁcation of an Oliogosaccharide Vaccine Candidate

by Glycan Microarray Studies. Carbohydrate antigens

(glycans) 1−5 were immobilized through their amine linker

on NHS-coated glass slides.⁴⁴The collected mice sera were

diluted to 1:200-fold, followed by 1:12800-fold and 1:25600-

fold and, then, added to antigen-bonded NHS glass slides. The

bound antibodies, after washing, were detected with a Cy5-

labeled goat antimouse IgG secondary antibody by scanning at

a 635 nm wavelength with a microarray ﬂuorescence chip

reader. The microarray results were compiled in the form of

line graphs plotted by taking the mean value of the intensity on

the x-axis against glycans 1−5 on the y-axis (Figure 3,

SF4−10).

The screening results of glycans 1−5 with sera from mice

immunized with DT-Hexa indicated (Figure 3a) very weak

binding signals from four mice sera out of ﬁve at 1:200-fold

dilution with respect to glycans 1−4 and a weak signal in

glycan 5. For the purpose of the detection study, the

concentration further reduced to 1:400-, 1:800-, and 1:1600-

fold, but the binding titers matches with the background at

these dilutions. The most surprising and signiﬁcant result was

that the serum of one mouse in this group showed a high

antibody titer against all glycans, 1−5, at 1:200-fold dilution,

and the mean intensity of all signals is around 2.5 × 10⁶

(Figure 3b). Upon dilution to 1:12800- and 1:25600-fold, no

signiﬁcant signals were observed. Interestingly, the DT-Hexa

vaccine candidate is immunogenic in one mouse but failed to

induce antibodies in four mice.

The microarray results of glycans 1−5 against the sera

collected from the group of mice immunized with DT-Hepta

4aa (Figure 3c) revealed that all ﬁve mice responded with high

antibody binding titers against glycans 1−3 (mean intensity

around 2.5 × 10⁵) and binding titers against glycans 4 and 5

were all very high (mean intensity around 3 × 10⁶) at 1:200-

fold dilution. Signiﬁcantly, at 1:12800-fold dilution, a near-

identical antibody titer was observed against glycans 4 and 5,

but no binding signal against the glycans 1−3 was observed.

The minimal antibody titer was observed against glycans 4 and

5 even when the concentration was further reduced to

1:25600-fold. The results clearly indicated that the DT-

Hepta 4aa vaccine candidate that could induce antibodies in all

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Figure 4. Serum bactericidal assay of DT-Hexa 3aa, DT-Hepta 4aa, and DT-Octa 5aa glycoconjugates. Error bars represent mean SD.

ﬁve mice is able to recognize all glycans with diﬀerent chain

lengths at 1:200-fold dilutions.

to kill the bacteria and a suitable epitope for the development

of a vaccine candidate to prevent the diseases caused by KP.

We then used a microarray to study glycans 1−5 with sera

from mice immunized with the DT-Octa 5aa vaccine (Figure

3d). Signiﬁcant antibody titers were observed against glycans

1−3, and identical high-intensity antibody titers were observed

in glycans 4−5 at 1:200-fold dilution. The observed antibody

titer against glycan 5 was higher than it against glycan 4, and

no signals were observed for glycans 1−3 at 1:12800 dilution.

When the concentration further reduced to 1:25600-fold,

signiﬁcant binding titers were observed for glycans 4 and 5,

and no binding signals were observed for glycans 1−3. The

microarray data demonstrated that the antibodies induced by

DT-Hepta 4aa and DT-Octa 5aa vaccine candidates were able

to recognize both short and long glycans. Because the antibody

responded to glycans 4 and 5 but not to glycan 3, which does

not have a glucuronic acid (GlcA) moiety at the nonreducing

end, we suspected that GlcA present at the nonreducing end

plays an important role in binding the antigen to the IgG

antibody. For economic considerations, we concluded that the

DT-Hepta 4aa glycoconjugate is a suﬃcient chain length for

the development of vaccine candidates against the K.

pneumonia K2 serotype.

Identiﬁcation of a Suitable Glycoconjugate Vaccine

Candidate by a Serum Bactericidal Assay. We further looked

into the serum bactericidal assay (SBA), which represents the

bactericidal ability of antibodies.⁴⁵The principle of serum

bactericidal assays is based on complement-mediated bacterial

killing by bactericidal antibodies. A detail of SBA was described

in the Experimental Section. SBA titers were deﬁned as the

reciprocal serum dilution at which 50% of cells are lysed,

compared to the number of cells prior to incubation.⁴⁶

Interestingly, our SBA results can be correlated to the

microarray results. Sera from mice immunized with DT-

Hepta 4aa showed a good bactericidal activity titer in 1/16

dilution compared to the results of DT-Octa 5aa immunized

sera, which showed a bactericidal activity titer in 1/8 dilution

(Figure 4). Therefore, we concluded that DT-Hepta 4aa is the

minimal length of a glycoconjugate vaccine candidate required

CONCLUSION

■

We have described the ﬁrst chemical syntheses of the K2

capsular polysaccharide of KP in various chain lengths in good

yields. All target molecules, 1−5, were furnished with an

aminopentyl linker at the reducing end for protein conjugation.

During the synthetic investigation, we attempted various

approaches and glycosylation methods to formulate an optimal

procedure to obtain the target molecules in a highly

stereoselective manner. The α-selectivity that is the key step

in chain elongation was successfully achieved using glycosyl

ﬂuorides with a tetrasaccharide acceptor by performing

Cp₂HfCl₂/AgOTf-mediated glycosylation in toluene. All

synthesized glycans were well characterized using NMR and

mass spectroscopic analysis.

We conjugated the synthesized glycans, 3−5, to carrier

protein DT to provide glycoconjugate vaccine candidates (DT-

Hexa 3aa, DT-Hepta 4aa, and DT-Octa 5aa) and immunized

mice with these vaccine candidates. Antigenicity and

immunogenicity of synthetic oligosaccharides and their

glycoconjugates were evaluated using glycan arrays and

serum bactericidal assay studies. The immunological results

demonstrated that two (4aa and 5aa) of the three

glycoconjugates are immunogenic and elicited antibodies that

recognized all synthetic glycans at 1:200-fold dilution. The

minimal immunogenic epitope of complex oligosaccharide

antigens is the DT-Hepta 4aa glycoconjugate that induced a

high level of antibodies in mice, and those antibodies were able

to recognize higher glycan (octasaccharide) and exhibited

good bactericidal activity. Thus, heptasaccharide 4 is a

promising vaccine candidate to be taken into the challenges

of live animal studies. We believe that the studies reported

herein will serve as a guideline for the future development of

the vaccine against the K2 sero group of KP.

Experimental Section. General Remarks. All chemical

reagents were obtained from commercial sources and used as

purchased. Anhydrous dichloromethane (CH₂Cl₂), acetonitrile

(CH₃CN), tetrahydrofuran (THF), N,N-dimethylformamide

H

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(DMF), toluene, and methanol (MeOH) were purchased from

a commercial source and used without further puriﬁcation. All

reactions were carried out under an argon atmosphere unless

mentioned otherwise, and standard syringe-septa techniques

were followed. Pulverized molecular sieves (4 Å MS, Aldrich)

for glycosylation were activated by heating at 350 °C for 3−4

h. Reactions were monitored by thin-layer chromatography

(TLC) analysis, which was performed on glass plates precoated

with Silica Gel 60 F254 (0.25 mm, Merck). The TLC was

stained with either acidic p-anisaldehyde or ceric ammonium

molybdate to be detected by UV light (254 nm). All products

prior to ﬁnal compounds were puriﬁed by ﬂash chromatog-

raphy with silica gel (Silicycle, 40−63 μm size, 230−400

mesh), whereas ﬁnal compounds were puriﬁed by gel

2H, Ar H ), 7.44 −7.25 (m, 25H, Ar H ), 6.89 (d, J = 8.5 Hz,

2H, Ar H ), 5.55 (s, 1H, Ar C H ), 5.09 (d, J = 10.6 Hz, 1H,

Ar C H_aH_b), 4.87−4.80 (m, 4H, 2 × Ar C H₂), 4.76 (d, J =

3.8 Hz, 1H, C1 H_{α‑glc‑linker}), 4.71 (d, J = 11.8 Hz, 1H, Ar 

CHa H_b), 4.66 (d, J = 12.4 Hz, 1H, Ar C H aH_b), 4.64 (d, J =

12.4 Hz, 1H, Ar CHa H_b), 4.57 (d, J = 11.8 Hz, 1H, Ar 

C H aH_b), 4.45 (s, 1H, C1−H_β‑man), 4.34 (d, J = 11.8 Hz, 1H,

Ar CHa H_b), 4.11−4.05 (m, 2H, C H_aH_b, C H_ring), 3.94−

3.89 (m, 2H, 2 × C H_ring), 3.81 (s, 3H, OC H₃), 3.71−3.66

(m, 3H, 2 × C H_ring, CH_aH_b), 3.60−3.45 (m, 5H, 

C H_ring, 2 × -CH₂), 3.38 (dd, J = 10.2, 3.1 Hz, 1H, C H_ring),

3.30 (t, J = 6.7 Hz, 2H,-CH_2linker), 3.11−3.07 (m, 1H, 

C H_ring), 1.74−1.70 (m, 2H, CH_2linker), 1.68−1.65 (m, 2H,

CH_2linker), 1.53−1.49 (m, 2H, CH_2linker). ¹³C{¹H} NMR

(150 MHz, CDCl₃, 298 K): δ 159.3 (Ar C ), 139.6 (Ar C ),

138.8 (Ar C ), 138.6 (Ar C ), 137.8 (Ar C ), 137.7 (Ar 

C ), 130.8 (Ar C ), 129.1 (Ar C ), 128.9 (Ar C ), 128.7

(Ar C ), 128.5 (Ar C ), 128.4 (Ar C ), 128.3 (Ar C ),

128.2 (Ar C ), 128.1 × 2(Ar C ), 127.9 (Ar C ), 127.7

(Ar C ), 127.6 (Ar C ), 127.3 (Ar C ), 126.2 (Ar C ),

1

chromatography (LH-20 or RP-18). H NMR, ¹³C NMR,

and 2D spectra were recorded on a Bruker AVANCE 600 (600

MHz) spectrometer at 298 K unless otherwise stated.

Chemical shifts on ¹H NMR were assigned according to

TMS (δ = 0 ppm, in CDCl₃) and D₂O (δ = 4.8 ppm).

Chemical shift measurements are reported in δ units, and

splitting patterns are described as singlet (s), doublet (d),

triplet (t), quartet (q), or multiplet (m). Coupling constants

(J) are reported in hertz (Hz). High-resolution ESI mass

spectra were recorded on a Bruker Daltonics or Bruker Bio-

TOF III spectrometer. MALDI-TOF spectra were recorded on

a Bruker Ultraﬂex II spectrometer. Alexa Fluor 647-conjugated

goat antimouse IgG antibody was purchased from JacksonIm-

munoresearch. Diphtheria toxoid cross-reactive protein ma-

terial DT was purchased from Merck. NHS-coated glass slides

were purchased from SCHOTT (Nexterion H). The micro-

array slides were scanned at a 635, 594, 532, or 488 nm

wavelength with a microarray ﬂuorescence chip reader

(ArrayWorx microarray reader). The ﬂuorescence data were

analyzed by GenePix Pro software (Axon Instruments).

Chemistry Experimental Procedures.

1

113.8 (Ar C ), 101.8 (C 1_β‑man, J_C−H= 156.5 Hz), 101.4

1

(Ar C H), 97.2 (C 1_{α‑glc‑linker}, J_C−H= 171.1 Hz), 80.3 (

C H_ring), 79.5 (C H_ring), 78.8 (C H_ring), 78.1 (C H_ring),

77.9 (C H_ring), 77.2 (C H_ring), 75.2 (C H₂), 75.0 (

C H₂), 73.7 (C H₂), 73.6 (C H₂), 72.4 (C H₂), 69.9 (

C H_ring), 68.7 (C H₂), 68.6 (C H₂), 68.1 (C H₂), 67.5 (

C H_ring), 55.4 (O C H₃), 51.4 (C H₂), 29.1 (C H₂), 28.8

(C H₂), 23.6 (C H₂). HRMS (ESI) m /z : [M + Na]⁺calcd

for C₆₀H₆₇N₃O₁₂Na, 1044.4617; found, 1044.4624.

5-Azidopentyl 2-O-Benzyl-3-O-p-methoxybenzyl-4,6-O-

benzylidene-α-^D-mannopyranosyl-(1→4)-2,3,6-tri-O-benzyl-

α-^D-glucopyranoside (17b). ¹H NMR (600 MHz, CDCl₃, 298

K): δ 7.46 (dd, J = 8.2, 1.7 Hz, 2H, ArH), 7.30−7.17 (m,

23H, ArH), 7.10 (dd, J = 7.2, 3.5 Hz, 2H, ArH), 6.80 (d, J

= 8.7 Hz, 2H, ArH), 5.56 (s, 1H, ArCH), 5.26 (s, 1H,

C1H_α‑man), 5.07 (d, J = 11.2 Hz, 1H, ArCH_aH_b), 4.72 (d,

J = 3.3 Hz, 1H, C1H_{α‑glc‑linker}), 4.69 (d, J = 11.7 Hz, 1H,

ArCH_aH_b), 4.63 (d, J = 12.0 Hz, 1H, ArCH_aH_b), 4.57−

4.46 (m, 5H, 5 × ArCH_aH_b), 4.38 (d, J = 11.8 Hz, 1H, Ar

CH_aH_b), 4.18 (d, J = 9.2 Hz, 1H, CH_ring), 4.17 (d, J = 11.8

Hz, 1H, ArCH_aH_b), 4.07 (dd, J = 9.9, 4.3 Hz, 1H, 

CH_aH_b), 3.90 (dd, J = 9.9, 3.0 Hz, 1H, CH_ring), 3.84−3.79

(m, 2H, 2 × CH_ring), 3.77−3.74 (m, 6H, OCH₃

(overlapped), 2 × CH_ring, CH_aH_b), 3.70−3.60 (m, 4H,

CH_ring, 3 × CH_aH_b), 3.53 (dd, J = 9.4, 3.5 Hz, 1H, 

CH_ring), 3.39−3.58 (m, 1H, CH_aH_b), 3.23 (t, J = 6.8 Hz, 2H,

CH_2linker), 1.66−1.62 (m, 2H, CH_2linker), 1.61−1.57 (m,

2H, CH_2linker), 1.45−1.41 (m, 2H, CH_2linker). ¹³C{¹H}

NMR (150 MHz, CDCl₃, 298 K): δ 159.2 (ArC), 138.9

(ArC), 138.5 (ArC), 138.2 (ArC), 138.1 (ArC),

137.9 (ArC), 131.0 (ArC), 129.9 (ArC), 128.9 (Ar

C), 128.6 (ArC), 128.5 (ArC), 128.3 (ArC), 128.2

(ArC), 128.1 × 2(ArC), 127.8 (ArC), 127.7 × 2 (Ar

C), 127.6, (ArC), 127.5 (ArC), (ArC), 126.9 (ArC),

126.2 (ArC), 113.86 (ArC), 100.57 (ArCH), 100.53

5-Azidopentyl-2-O-benzyl-3-O-p-methoxybenzyl-4,6-O-

benzylidene-β -^D-mannopyranosyl-(1→4)-2,3,6-tri-O-benzyl-

α -^D-glucopyranoside (17a ). The solution of thioglycoside 11

(4.2 g, 7.36 mmol, 1 equiv), BSP (1.66 g, 8.1 mmol, 1.1 equiv),

TTBP (3.66 g, 14.73 mmol, 2 equiv), and well-dried 4 Å

molecular sieves (10 g) in anhydrous CH₂Cl₂(175 mL) was

stirred at rt for 10 −15 min under an argon atmosphere and

cooled to −60 °C. Then, Tf₂O (1.5 mL, 8.8 mmol, 1.2 equiv)

was added into the mixture. After being stirred for 10 −15 min

at the same temperature, a solution of glycosyl acceptor 12

(3.1 g, 5.53 mmol, 0.75 equiv) in CH₂Cl₂(30 mL) was added

slowly at −60 °C. The reaction was further stirred for 90 min

at the same temperature and, then, allowed to reach room

temperature. After dilution with CH₂Cl₂, the mixture was

ﬁ ltered through a pad of Celite by repeatedly washing with

CH₂Cl₂. The organic layer was then washed with satd aq

NaHCO₃and later by brine, dried over MgSO₄, ﬁ ltered, and

evaporated to dryness. The resulting residue was puri ﬁ ed by

ﬂ ash chromatography over silica gel (EtOAc/hexanes, 1:4) to

a ﬀ ord the corresponding α -anomer 17b (272 mg) and β -

anomer 17a (3.23 g) compounds (α /β = 1:12) in viscous

colorless oil compounds with a 62% yield (combined yield).

¹H NMR (600 MHz, CDCl₃, 298 K): δ 7.52 (d, J = 7.3 Hz,

1

(C1_α‑man, J_C−H= 172.0 Hz), 96.7 (C1_{α‑glc‑linker}, J_C−H= 169.0

I

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Hz), 81.6 (CH_ring), 80.4 (CH_ring), 79.1 (CH_ring), 77.9

(CH_ring), 77.8 (CH_ring), 76.1 (CH_ring), 75.0 (CH₂),

73.7 (CH₂), 73.4 (CH₂), 73.1 (CH₂), 72.8 (CH₂),

69.9 (CH_ring), 69.2 (CH₂), 68.8 (CH₂), 68.1 (CH₂),

65.3 (CH_ring), 55.4 (OCH₃), 51.4 (CH₂), 29.1 (

CH₂), 28.8 (CH₂), 23.6 (CH₂). HRMS (ESI) m/z: [M +

Na]⁺calcd for C₆₀H₆₇N₃O₁₂Na, 1044.4617; found, 1044.4624.

dene-β-^D-mannopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-

glucopyranoside (19). A mixture of acceptor 18 (1.1 g, 1.22

mmol, 1 equiv), donor 10 (1.455 g, 1.83 mmol, 1.5 equiv),and

dried 4 Å molecular sieves (3 g) in anhydrous CH₂CL₂(75

mL) was stirred under an argon atmosphere for 1 h and cooled

to −40 °C. Then, NIS (535 mg, 2.38 mmol, 1.3 equiv with

respect to the donor) and TfOH (0.5 M in Et₂O, 730 μL, 0.2

equiv with respect to the donor) were added, and the stirring

was continued until TLC analysis indicated the disappearance

of the starting materials (90 min). Upon completion, the

reaction was quenched by the addition of NEt₃(200 μL), and

the reaction mixture was slowly warmed to room temperature

and ﬁltered through a pad of Celite. The ﬁltrate was washed

with satd aq Na₂S₂O₃, satd aq NaHCO₃, and brine. The

organic layer was dried over MgSO₄, ﬁltered, and concentrated.

The resulting residue was puriﬁed by silica gel column

chromatography (EtOAc/hexanes, 1:4) to aﬀord pure

5-Azidopentyl 2-O-Benzyl-4,6-O-benzylidene-β-D-manno-

pyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-glucopyranoside

(18). DDQ (533 mg, 2.35 mmol, 1.5 equiv) was added to

stirred solution of starting material 17a (1.6 g, 1.56 mmol, 1

equiv) in a mixture of DCM/phosphate buﬀer pH 7 (50 mL,

9:1 = v/v) at 0 °C. The reaction mixture was vigorously stirred

in the absence of light until TLC analysis indicated

disappearance of the starting material (180 min). Upon

completion, the reaction mixture was diluted with CH₂Cl₂and

washed with satd aq NaHCO₃and brine. The organic phase

was washed with water until the solution became colorless;

then, the organic solution was dried over MgSO₄and ﬁltered.

The ﬁltrate was concentrated under reduced pressure. The

resulting residue was puriﬁed by ﬂash chromatography over

silica gel (EtOAc/hexanes, 1:3) to aﬀord pure compound 18

1

compound 19 (1.61 g, 84%) as a white foam. H NMR (600

MHz, CDCl₃, 298 K): δ 7.77 (d, J = 6.6 Hz, 2H, ArH), 7.72

(d, J = 6.6 Hz, 2H, ArH), 7.53 (d, J = 7.2 Hz, 2H, ArH),

7.38−7.15 (m, 44H, ArH), 5.53 (d, J = 3.5 Hz, 1H, C1

H

_α‑glc), 5.35 (s, 1H, ArCH), 5.08 (d, J = 11.2 Hz, 1H, Ar

CH_aH_b), 5.01 (d, J = 11.2 Hz, 1H, ArCH_aH_b), 4.98 (d, J =

11.2 Hz, 1H, ArCH_aH_b), 4.88 (d, J = 11.2 Hz, 2H, Ar

CH₂), 4.83−4.75 (m, 4H, 3 × ArCHaH_b, C1H_{α‑glc‑linker}

(overlapped), 4.70−4.63 (m, 3H, 3 × ArCH_aH_b), 4.55−4.53

(m, 2H, C1H_β‑man(overlapped), ArCHaH_b), 4.43 (d, J =

12.3 Hz, 1H, ArCHaH_b), 4.31 (d, J = 11.7 Hz, 1H, Ar

CHaH_b), 4.22 (t, J = 9.3 Hz, 1H, CH_ring), 4.08 (t, J = 9.3 Hz,

1H, CH_ring), 4.03−3.93 (m, 4H, 2 × CH_ring, 2 × 

CH_aH_b), 3.90 (d, J = 11.2 Hz, 1H, CH_aH_b), 3.84 (dd, J =

9.9, 3.3 Hz, 1H, CH_ring), 3.81−3.74 (m, 3H, 3 × CH_ring),

3.71−3.67 (m, 2H, CH_aH_b, CH_ring), 3.58−3.45 (m, 6H, 2

× CH₂, 2 × CH_ring), 3.31 (t, J = 7.1 Hz, 2H, CH_2linker),

3.06−3.02 (m, 1H, CH_ring), 1.76−1.70 (m, 2H, CH_2linker),

1.69−1.65 (m, 2H, CH_2linker), 1.55−1.50 (m, 2H, 

CH_2linker), 1.17 (s, 9H, 3 × CH₃). ¹³C{¹H} NMR (150

MHz, CDCl₃, 298 K): δ 139.7 (ArC), 138.8 (ArC), 138.7

(ArC), 138.6 × 2 (ArC), 38.5 (ArC), 137.7 (ArC),

137.6 (ArC), 136.0 (ArC), 135.8 (ArC), 133.5 (Ar

C), 133.4 (ArC), 129.8 × 2 (ArC), 129.3 (ArC), 128.6

(ArC), 128.5 (ArC), 128.4 × 3 (ArC), 128.3 (ArC),

128.2 × 2 (ArC), 128.1 × 2 (ArC), 127.9 × 3 (ArC),

127.8 (ArC), 127.7 × 2 (ArC), 127.6 × 2 (ArC), 127.4

× 2 (ArC), 127.2 (ArC), 126.5 (ArC), 102.2 (Ph−

1

(1.14 g, 81%) as a viscus colorless oil. H NMR (600 MHz,

CDCl₃, 298 K): δ 7.50 (dd, J = 7.6, 1.9 Hz, 1H, ArH),

7.42−7.28 (m, 24H, ArH), 5.46 (s, 1H, ArCH), 5.07 (d, J

= 10.7 Hz, 1H, ArCH_aH_b), 4.99 (d, J = 11.8 Hz, 1H, Ar

CH_aH_b), 4.82 (s, 1H, ArCH_aH_b), 4.80 (s, 1H, ArCH_aH_b),

4.78 (d, J = 3.6 Hz, 1H, C1H_{α‑glc‑linker}), 4.76 (d, J = 11.6 Hz,

1H, ArCH_aH_b), 4.66 (d, J = 12.0 Hz, 1H, ArCH_aH_b), 4.62

(d, J = 11.4 Hz, 1H, ArCH_aH_b), 4.51 (s, 1H, C1H_β‑man),

4.42 (d, J = 12.0 Hz, 1H, ArCH_aH_b), 4.10 (dd, J = 10.4, 5.0

Hz, 1H, CH_aH_b), 3.98 (t, J = 9.1 Hz, 1H, CH_ring), 3.90 (t,

J = 9.1 Hz, 1H, CH_ring), 3.76−3.73 (m, 1H, CH_ring),

3.71−3.66 (m, 2H, CH_ring, CH_aH_b), 3.65−3.59 (m, 3H,

CH_ring, 2 × CH_aH_b), 3.58−3.56 (dd, J = 9.8, 3.1 Hz, 1H,

CH_ring), 3.54−3.45 (m, 3H, CH_ring, 2 × -CH_aH_b), 3.28

(m, 2H, CH_2linker), 3.07 (m, 1H, CH_ring), 1.74−1.63 (m,

4H, 2 × CH_2linker), 1.53−1.48 (m, 2H, CH_2linker). ¹³C{¹H}

NMR (150 MHz, CDCl₃, 298 K): δ 139.6 (ArC), 138.6

(ArC), 138.3 (ArC), 137.6 (ArC), 137.4 (ArC),

129.3 (ArC), 128.8 (ArC), 128.6 (ArC), 128.5 (Ar

C), 128.4 × 3 (ArC), 128.2 × 2 (ArC), 128.1 × 2(Ar

C), 127.9 (ArC), 127.7 (ArC), 127.4 (ArC), 126.5

(ArC), 102.1 (ArCH), 101.9 (C1_β‑man, ¹J_C−H= 156.6 Hz),

97.3 (C1_{α‑glc‑linker}¹J_C−H= 169.5 Hz), 80.4 (CH_ring), 79.4 (

CH_ring), 79.3 (CH_ring), 79.1 (CH_ring), 78.0 (CH_ring),

75.9 (CH₂), 75.3 (CH₂), 73.9 (CH₂), 73.6 (CH₂),

71.0 (CH_ring), 70.0 (CH_ring), 68.7 (CH₂), 68.4 (

CH₂), 68.2 (CH₂), 67.0 (CH_ring), 51.5 (CH₂), 29.1 (

CH₂), 28.8 (CH₂), 23.6 (CH₂). HRMS (ESI) m/z: [M +

Na]⁺calcd for C₅₂H₅₉N₃O₁₁Na, 924.4042; found, 924.4051.

1

CH), 101.2 (C1_β‑man, J_C−H= 156.7 Hz), 97.1 (C1_{α‑glc‑linker}

1

¹J_C−H= 171.4 Hz), 96.6 (C1_α‑glcJ_C−H= 172.1 Hz), 81.5 (

CH_ring), 80.3 (CH_ring), 79.7 × 2 (CH_ring), 79.6 (

CH_ring), 79.2 (CH_ring), 77.3 (CH_ring), 77.2 (CH_ring),

75.9 (CH₂), 75.8 (CH₂), 75.2 (CH₂), 74.9 × 2 (

CH_ring, CH₂), 73.6 (CH₂), 73.5 (CH₂), 72.5 (

CH_ring), 71.0 (CH₂), 69.8 (CH_ring), 68.7 (CH₂), 68.6

(CH₂), 68.1 (CH₂), 67.0 (CH_ring), 63.1 (CH₂), 51.4

(CH₂), 29.1 (CH₂), 28.8 (CH₂), 27.1 (3 × CH₃),

23.6 (CH₂), 19.5 (C). HRMS (ESI) m/z: [M + Na]⁺calcd

for C₉₅H₁₀₅N₃O₁₆SiNa, 1594.7156; found, 1594.7185.

5-Azidopentyl 2,3,4-Tri-O-benzyl-6-O-tert-butyldiphenyl-

silyl-α-^D-glucopyranosyl-(1→3)-2-O-benzyl-4,6-O-benzyli-

J

https://dx.doi.org/10.1021/acs.joc.0c01404

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

pubs.acs.org/joc

Article

5-Azidopentyl 2,3,4-Tri-O-benzyl-α-D-glucopyranosyl-(1→

3)-2-O-benzyl-4,6-O-benzylidene-β-^D-mannopyranosyl-(1→

4)-2,3,6-tri-O-benzyl-α-^D-glucopyranoside (20). To a Nal-

gene bottle containing a solution of silylether compound 19

(2.5 g, 1.59 mmol, 1 equiv) in a mixture of THF/pyridine (30

mL, 9:1 = v/v), the pyridinium hydorﬂuoride (HF·py) stock

solution (25 mL, 15 equiv; [stock solution was prepared from

commercially available Aldrich HF·py (70%) by dissolving 15

mL in 30 mL of pyridine, and 75 mL of THF]) was added with

the help of a plastic syringe over 5 min at 0 °C. The reaction

was allowed to warm to room temperature and stirred for

overnight, at which time all starting material disappeared

conﬁrmed by TLC. Upon completion, the reaction mixture

was poured into cold satd aq NaHCO₃(200 mL) and

extracted with CH₂Cl₂(2 × 50 mL). The combined organic

extracts were dried over MgsO₄, ﬁltered, and concentrated.

The resulting residue was puriﬁed by ﬂash chromatography

over silica gel (EtOAc/hexanes, 2:3) to get pure compound 20

5-Azidopentyl Methyl-2,3,4-tri-O-benzyl-α-D-glucopyra-

nosyluronate-(1→3)-2-O-benzyl-4,6-O-benzylidene-β-^D-

mannopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-glucopyra-

noside (22). Compound 20 (1.925 g, 1.443 mmol, 1 equiv)

was dissolved in a mixture of CH₂Cl₂/H₂O (20 mL, 1:1 = v/

v), and the reaction mixture was cooled to 0 °C. BAIB (1.395

g, 4.33 mmol, 3 equiv) and TEMPO (68 mg, 0.433 mmol, 0.3

equiv) were added, and the reaction mixture was allowed to

warm to rt and stirred for 3 h. Upon completion, the reaction

mixture was quenched by the addition of satd aq Na₂S₂O₃

solution (10 mL) and diluted with CH₂Cl₂(20 mL). The

organic layer was separated, the aqueous layer was extracted

with CH₂Cl₂(15 mL), and the combined organic layers were

dried over MgSO₄, ﬁltered, and concentrated. The resulting

residue was puriﬁed by ﬂash chromatography over silica gel

(CH₂Cl₂/MeOH, 98:2) to obtain acid compound 21 in 1.80 g

(92%) as a white foam.

The above puriﬁed compound was dissolved in dry DMF

(20 mL). Then, MeI (416 μL, 6.68 mmol, 5 equiv) and oven-

dried K₂CO₃(553 mg, 4 mmol, 3 equiv) were added at rt

under a nitrogen atmosphere, and the reaction mixture was

stirred overnight. Upon completion, the solvent was removed

under a high vacuum, and the remaining mixture was diluted

with CH₂Cl₂(30 mL) and washed with water (15 mL). The

aqueous layer was extracted with CH₂Cl₂(15 mL), and the

combined organic layers were dried over MgSO₄, ﬁltered, and

concentrated. The resulting residue was puriﬁed by ﬂash

chromatography over silica gel (EtOAc/hexanes, 1:3) to get

1

in 1.929 g (91%) as a white foam. H NMR (600 MHz,

CDCl₃, 298 K): δ 7.53 (d, J = 7.3 Hz, 2H, ArH), 7.41−7.18

(m, 36H, ArH), 7.04 (d, J = 7.3 Hz, 2H, ArH), 5.43 (d, J

= 3.6 Hz, 1H, C1H_α‑glc), 5.36 (s, 1H, ArCH), 5.09 (d, J =

10.4 Hz, 1H, ArCH_aH_b), 4.99 (d, J = 10.9 Hz, 1H, Ar

CH_aH_b), 4.93 (d, J = 8.8 Hz, 1H, ArCH_aH_b), 4.91 (d, J = 8.8

Hz, 1H, ArCH_aH_b), 4.87 (d, J = 11.4 Hz, 1H, ArCH_aH_b),

4.84 (s, 1H, ArCH_aH_b), 4.82 (s, 1H, ArCH_aH_b), 4.78 (d, J

= 3.6 Hz, 1H, C1H_{α‑glc‑linker}), 4.76 (d, J = 11.0 Hz, 1H, Ar

CH_aH_b), 4.72 (d, J = 12.1 Hz, 1H, ArCH_aH_b), 4.67 (d, J =

12.1 Hz, 1H, ArCH_aH_b), 4.63 (d, J = 11.0 Hz, 1H, Ar

CH_aH_b), 4.59 (d, J = 12.1 Hz, 1H, ArCH_aH_b), 4.58 (s, 1H,

C1H_β‑man), 4.43 (d, J = 12.4 Hz, 1H, ArCH_aH_b), 4.35 (d,

J = 12.4 Hz, 1H, ArCH_aH_b), 4.22 (t, J = 9.7 Hz, 1H, 

CH_ring), 4.08 (dd, J = 10.3, 4.7 Hz, 1H, CH_aH_b), 4.02 (m,

2H, 2 × CH_ring), 3.96 (t, J = 9.3 Hz, 1H, CH_ring), 3.80−

1

pure compound 22 (1.68 g, 92%) as a white foam. H NMR

(600 MHz, CDCl₃, 298 K): δ 7.53 (d, J = 7.2 Hz, 2H, ArH),

7.33−7.18 (m, 36H, ArH), 7.03 (d, J = 7.2 Hz, 2H, ArH),

5.56 (d, J = 3.5 Hz, 1H, C1H_{α‑glc‑COOMe}), 5.30 (s, 1H, Ar

CH), 5.08 (d, J = 10.8 Hz, 1H, ArCH_aH_b), 4.94 (d, J = 11.0

Hz, 1H, ArCH_aH_b), 4.88 (d, J = 11.5 Hz, 1H, ArCH_aH_b),

4.84−4.82 (m, 4H, 2 × ArCH₂), 4.78 (d, J = 3.6 Hz, 1H,

C1H_{α‑glc‑linker}), 4.73 (d, J = 11.6 Hz, 1H, ArCH_aH_b), 4.71

(d, J = 12.0 Hz, 1H, ArCH_aH_b), 4.67 (d, J = 12.0 Hz, 1H,

ArCH_aH_b), 4.59 (d, J = 11.0 Hz, 1H, ArCH_aH_b), 4.55 (d,

J = 12.0 Hz, 1H, ArCH_aH_b), 4.52 (s, 1H, C1H_β‑man), 4.42

(d, J = 12.0 Hz, 1H, ArCH_aH_b), 4.33 (d, J = 12.0 Hz, 1H,

ArCH_aH_b), 4.29 (d, J = 10.0 Hz, 1H, CH_ring), 4.19 (t, J =

9.3 Hz, 1H, CH_ring), 4.02−3.96 (m, 3H, 2 × CH_ring), 

CH_aH_b), 3.95 (t, J = 8.9 Hz, 1H, CH_ring), 3.82−3.74 (m, 4H,

4 × CH_ring), 3.72 (m, 1H, CH_aH_b), 3.69 (s, 3H, 

COOCH₃), 3.68−3.62 (m, 2H, CH_2linker), 3.58−3.54 (m,

2H, 2 × CH_ring), 3.53−3.45 (m, 2H, 2 × -CH_aH_b), 3.31 (t, J

= 6.6 Hz, 2H, CH_2linker), 3.05−3.06 (m, 1H, CH_ring),

1.75−1.70 (m, 2H, CH_2linker), 1.69−1.66 (m, 2H, 

CH_2linker), 1.56−1.49 (m, 2H, CH_2linker). ¹³C{¹H} NMR

(150 MHz, CDCl₃, 298 K): δ 170.06 (CO), 139.6 (ArC),

138.6 × 3 (ArC), 138.2 (ArC), 138.1 (ArC), 137.8

(ArC), 137.4 (ArC), 129.4 (ArC), 128.8 (ArC),

128.5 × 2 (ArC), 128.4 × 2 (ArC), 128.3 (ArC), 128.2

× 2 (ArC), 128.1(ArC), 128.0 (ArC), 127.9 × 2 (Ar

3.78 (m, 2H, 2 × CH_ring), 3.75−3.71 (m, 3H, 2 × CH_ring

,

CH_aH_b), 3.69−3.63 (m, 2H, CH_2linker), 3.60−3.55 (m,

4H, CH_ring, 3 × CH_aH_b), 3.49−3.44 (m, 3H, 2 × 

CH_ring, CH_aH_b), 3.30 (t, 2H, CH_2linker), 3.16−3.12 (m,

1H, CH_ring), 1.76−1.70 (m, 2H, CH_2linker), 1.69−1.65 (m,

2H, CH_2linker), 1.54−1.48 (m, 2H, CH_2linker). ¹³C{¹H}

NMR (150 MHz, CDCl₃, 298 K): δ 139.6 (ArC), 138.8

(ArC), 138.6 × 2 (ArC), 138.4 (ArC), 138.3 (ArC),

138.0 (ArC), 137.5 (ArC), 129.4 (ArC), 128.8 (Ar

C), 128.6 (ArC), 128.5 × 3 (ArC), 128.4 (ArC), 128.3

× 2 (ArC), 128.2 × 2 (ArC), 128.1 × 2 (ArC), 128.0

(ArC), 127.9 (ArC), 127.7 × 2 (ArC), 127.4 (ArC),

127.3 × 2 (ArC), 126.5 (ArC), 102.4 (ArCH), 101.4

1

(C1_β‑man, J_C−H= 160.3 Hz), 97.2 (C1_{α‑glc‑linker}, J_C−H= 171.0

1

Hz), 96.8 (C1_α‑glc, J_C−H= 171.0 Hz), 81.3 (CH_ring), 80.4

(CH_ring), 79.5 (CH_ring), 79.3 (CH_ring), 79.1 (CH_ring),

78.9 (CH_ring), 77.5 (CH_ring), 77.4 (CH_ring), 75.7 × 2

(CH₂), 75.3 (CH_ring), 75.2 (CH₂), 75.1 (CH₂), 73.8

(CH₂), 73.6 (CH₂), 71.6 (CH_ring), 70.8 (CH₂), 70.0

(CH_ring), 68.8 (CH₂), 68.6 (CH₂), 68.2 (CH₂), 67.2

(CH_ring), 62.3 (CH₂), 51.5 (CH₂), 29.1 (CH₂), 28.8

(CH₂), 23.6 (CH₂). HRMS (ESI) m/z: [M + Na]⁺calcd

for C₇₉H₈₇N₃O₁₆Na, 1356.5979; found, 1356.5994.

C), 127.7 × 3 (ArC), 127.5 (ArC), 127.3 × 2 (ArC),

1

126.5 (ArC), 102.3 (ArCH), 101.3 (C1_β‑man, J_C−H

=

155.4 Hz), 97.4 (C1_{α‑glc‑coome}

,

¹J_C−H= 172.2 Hz), 97.3

1

(C1_{α‑glc‑linker}, J_C−H= 171.4 Hz), 80.7 (CH_ring), 80.3 (

CH_ring), 79.5 (CH_ring), 79.3 (CH_ring), 79.2 (CH_ring),

79.1 (CH_ring), 78.6 (CH_ring), 77.5 (CH_ring), 75.9 (

CH₂), 75.8 (CH₂), 75.3 (CH_ring), 75.2 × 2 (CH₂), 73.8

(CH₂), 73.6 (CH₂), 71.2 (CH_ring), 71.0 (CH₂), 69.9

(CH_ring), 68.7 (CH₂), 68.6 (CH₂), 68.2 (CH₂), 67.0

K

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J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

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Article

(CH_ring), 52.6 (COOCH₃), 51.5 (CH₂), 29.1 (CH₂),

28.8 (CH₂), 23.6 (CH₂). HRMS (ESI) m/z: [M + Na]⁺

calcd for C₈₀H₈₇N₃O₁₇Na, 1384.5928; found, 1384.5940.

CH₂), 28.8 (CH₂), 23.6 (CH₂). HRMS (ESI) m/z: [M +

Na]⁺calcd for C₈₀H₈₉N₃O₁₇Na, 1386.6084; found, 1386.6084.

5-Azidopnetyl 2-O-Benzoyl-3-O-p-methoxybenzyl-4,6-O-

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

[methyl-2,3,4-tri-O-benzyl-α-^D-glucopyranosyluronate-(1→

3)]-β-D-mannopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-glu-

copyranoside (6). A mixture of acceptor 8 (1.05 g, 0.77

mmol), donor 9 (690 mg, 1.15 mmol, 1.5 equiv), and activated

4 Å molecular sieves (2 g) in anhydrous CH₂Cl₂(55 mL) was

stirred under an argon atmosphere for 30 min. Then, the

reaction mixture was cooled to −30 °C. NIS (310 mg, 1.38

mmol, 1.2 equiv with respect to the donor) and TfOH (0.5 M

in Et₂O, 690 μL, 0.34 mmol, 0.3 equiv with respect to the

donor) were added, and the mixture was stirred at −20 °C

until TLC analysis (EtOAc/toluene, 1:5) indicated the

complete disappearance of the starting materials (2 h). Upon

completion, the reaction mixture was quenched by the addition

of NEt₃(75 μL) and slowly warmed to room temperature. The

mixture was ﬁltered through a pad of Celite, and the ﬁltrate

was concentrated under reduced pressure. The resulting

residue was puriﬁed by ﬂash chromatography over silica gel

(EtOAc/hexanes, 3:7) to aﬀord pure product 6 (1.3 g, 92%) as

a white foam. ¹H NMR (600 MHz, CDCl₃, 298 K): δ 7.86 (d,

J = 7.2 Hz, 2H, ArH), 7.60−7.00 (m, 48H, ArH), 6.96 (d,

J = 8.6 Hz, 2H, ArH), 6.56 (d, J = 8.6 Hz, 2H, ArH), 5.19

(t, J = 8.3 Hz, 1H, CH_ring), 5.14 (d, J = 3.1 Hz, 1H, C1

5-Azidopentyl Methyl-2,3,4-tri-O-benzyl-α-D-glucopyra-

nosyluronate-(1→3)-2,6-di-O-benzyl-β-^D-mannopyranosyl-

(1→4)-2,3,6-tri-O-benzyl-α-^D-glucopyranoside (8). A mixture

of trisaccharide 22 (1.55 g, 1.14 mmol, 1 equiv) and activated

4 Å molecular sieves (3 g) in anhydrous CH₂Cl₂(50 mL) was

stirred under argon for 1 h. The reaction mixture was cooled to

−78 °C, Et₃SiH (545 μL, 3.41 mmol, 3 equiv) and TfOH (354

μL, 3.5 equiv) were added, and the stirring was continued at

−78 °C until TLC analysis (EtOAc/hexanes) indicated the

disappearance of the starting material (1 h). Upon completion,

the reaction mixture was quenched by the addition of satd aq

NaHCO₃(1 mL). The reaction mixture was slowly warmed to

room temperature. Then, the mixture was ﬁltered through a

pad of Celite, and the ﬁltrate was washed with satd aq

NaHCO₃(25 mL) and brine (25 mL). The organic phase was

dried over MgSO₄, ﬁltered, and concentrated under reduced

pressure. The resulting residue was puriﬁed by ﬂash

chromatography over silica gel (EtOAc/hexanes) to obtain

1

pure compound 8 (1.45 g, 94%) in a viscous colorless oil. H

NMR (600 MHz, CDCl₃, 298 K):δ 7.45 (d, J = 7.2 Hz, 2H,

ArH), 7.37−7.19 (m, 38H, ArH), 5.15 (d, J = 11.2 Hz,

1H, ArCH_aH_b), 5.12 (d, J = 3.5 Hz, 1H, C1H_{α‑glc‑COOMe}),

4.98 (d, J = 11.4 Hz, 1H, ArCH_aH_b), 4.93 (d, J = 10.9 Hz,

1H, ArCH_aH_b), 4.84−4.79 (m, 4H, 2 × ArCH₂), 4.78−

4.73 (m, 4H, C1H_{α‑glc‑linker}), 3 × ArCH_aH_b), 4.69 (d, J =

12.2 Hz, 1H, ArCH_aH_b), 4.63 (d, J = 12.2 Hz, 1H, Ar

CH_aH_b), 4.60 (d, J = 11.2 Hz, 1H, ArCH_aH_b), 4.50 (d, J =

11.8 Hz, 1H, ArCH_aH_b), 4.49 (s, 1H, C1H_β‑man), 4.45 (d,

J = 11.8 Hz, 1H, ArCH_aH_b), 4.41 (d, J = 9.9 Hz, 1H, 

CH_ring), 4.33 (d, J = 12.0 Hz, 1H, ArCH_aH_b), 4.06 (t, J = 9.2

Hz, 1H, CH_ring), 4.02 (d, J = 9.5 Hz, 1H, CH_ring), 3.97 (d,

J = 10.2 Hz, 1H, CH_ring), 3.92 (t, J = 9.2 Hz, 1H, CH_ring),

3.79−3.74 (m, 3H, 3 × CH_ring), 3.69−3.61 (m, 8H, 

CH_2linker, CH_ring, 2 × CHaH_b, COOCH₃), 3.51−3.45

(m, 3H, CH_ring, 2 × CH_aH_b), 3.28−3.25 (m, 4H, 2 × 

CH_ring, 2 × CH_aH_b), 1.73−1.67 (m, 2H, CH_2linker), 1.66−

1.63 (m, 2H, CH_2linker), 1.51−1.47 (m, 2H, CH_2linker).

¹³C{¹H} NMR (150 MHz, CDCl₃, 298 K): δ 170.2 (CO),

139.8 (ArC), 138.8 (ArC), 138.6 × 2 (ArC), 138.5

(ArC), 138.3 (ArC), 137.8 (ArC), 137.7 (ArC),

128.7 (ArC), 128.6 (ArC), 128.5 × 2 (ArC), 128.4 × 2

(ArC), 128.3 × 2 (ArC), 128.2 (ArC), 128.1 × 2 (Ar

C), 127.9 (ArC), 127.8 (ArC), 127.7 (ArC), 127.5

H

_{α‑glc‑COOMe}), 5.05 (d, J = 11.7 Hz, 1H, ArCH_aH_b), 4.97 (m,

2H, ArCH_aH_b, PhCH), 4.87−4.70 (m, 6H, 3 × ArCH₂),

4.69−4.67(m, 2H, C1H_{α‑glc‑linker}(overlapped), Ar

CH_aH_b), 4.65 (s, 1H, ArCH_aH_b), 4.63−4.60 (m, 2H,

C1H_β‑glc(overlapped) ArCH_aH_b), 4.56−4.42 (m, 6H, 2 ×

ArCH₂, ArCH_aH_b, CH_ring), 4.33 (t, J = 9.1 Hz, 1H, 

CH_ring), 4.28−4.26 (m, 2H, C1H_β‑man(overlapped), Ar

CH_aH_b), 4.14−4.09 (m, 2H, CH_ring, CH_aH_b), 4.02 (d, J = 12

Hz, 1H, ArCH_aH_b), 3.81−3.72 (m, 4H, 4 × CH_ring), 3.68

(m, 4H, OCH₃, CH_ring), 3.61−3.57 (m, 5H, COOCH₃,

2 × CH_aH_b), 3.56−3.48 (m, 5H, 2 × CH_ring, 3 × 

CH_aH_b), 3.45−3.40 (m, 2H, 2 × CH_ring), 3.38−3.34 (m,

1H, −CH_aH_b), 3.29 (d, J = 10.8 Hz, 1H, CH_aH_b), 3.22−

3.18 (m, 3H, CH_ring, CH_2linker), 3.12−3.07 (m, 1H, 

CH_ring), 2.75 (d, 1H, J = 9.3 Hz, CH_ring), 1.64−1.60 (m, 2H,

CH_2linker), 1.59−1.54 (m, 2H, CH_2linker), 1.42−1.38 (m,

2H, CH_2linker). ¹³C{¹H} NMR (150 MHz, CDCl₃, 298 K): δ

170.4 (CO), 164.8 (CO), 159.2 (ArC), 139.9 (ArC),

139.3 (ArC), 138.9 (ArC), 138.8 (ArC), 138.6 (Ar

C), 138.5 (ArC), 138.4 (ArC), 137.8 (ArC), 137.7

(ArC), 133.3 (ArC), 130.4(ArC), 130.0 (ArC),

129.9 (ArC), 129.6 (ArC), 129.0 (ArC), 128.7 (Ar

C), 128.6 × 2 (ArC), 128.5 × 2 (ArC), 128.4 (ArC),

128.3 (ArC), 128.2 × 2 (ArC), 128.1 × 2 (ArC), 128.0

× 2 (ArC), 127.9 × 2 (ArC), 127.8 × 2 (ArC), 127.7 ×

2 (ArC), 127.6 (ArC), 127.5 (ArC), 127.2 (ArC),

127.1 (ArC), 126.9 (ArC), 126.2 (ArC), 113.6 (Ar

C), 101.3 (C1_β‑man, ¹J_C−H= 155.6 Hz), 100.9 (Ph−CH), 100.3

(ArC), 127.4 (ArC), 127.1 (ArC), 100.8 (C1_β‑man

,

¹J_C−H= 156.8 Hz), 100.5 (C1_{α‑glc‑COOMe}, J_C−H= 171.0 Hz),

97.3 (C1_{α‑glc‑linker}, ¹J_C−H= 170.7 Hz), 83.8 (CH_ring), 81.5 (

CH_ring), 80.4 (CH_ring), 79.7 (CH_ring), 79.4 (CH_ring),

79.1 (CH_ring), 78.7 (CH_ring), 77.0 (CH_ring), 75.9 (

CH₂), 75.2 (CH₂), 75.1 (CH₂), 74.9 (CH₂), 73.8 × 2

(CH₂), 73.7 (CH₂), 73.6 (CH₂), 71.2 (CH_ring), 71.0

(CH₂), 70.1 (CH_ring), 69.0 (CH_ring), 68.6 (CH₂),

68.1 (CH₂), 52.6 (COOCH₃), 51.5 (CH₂), 29.1 (

1

(C1_β‑glc, J_C−H= 165.1 Hz), 97.6 (C1_{α‑glc‑COOMe}, ¹J_C−H= 171.4

1

Hz), 97.2 (C1_{α‑glc‑linker}, J_C−H= 171.3 Hz), 81.63 (CH_ring),

80.7 (CH_ring), 80.6 (CH_ring), 80.0 (CH_ring), 79.5 (

L

https://dx.doi.org/10.1021/acs.joc.0c01404

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

pubs.acs.org/joc

Article

CH_ring), 79.3 (CH_ring), 79.0 (CH_ring), 77.9 (CH_ring),

77.7 (CH_ring), 75.9 (CH_ring), 75.6 (CH₂), 75.0 (

CH₂), 74.9 (CH₂), 74.2 (CH₂), 73.8 (CH_ring), 73.6

(CH₂), 73.5 (CH₂), 73.4 (CH₂), 73.3 (CH₂), 72.6 (

CH₂), 72.1 (CH_ring), 71.6 (CH_ring), 70.0 (CH_ring), 68.7

(CH₂), 68.6 (CH_2linker), 68.3 (CH₂), 68.1 (CH₂),

66.2 (CH_ring), 55.3 (OCH₃), 52.5 (COOCH₃), 51.5

(CH_2linker), 29.1 (CH_2linker), 28.8 (CH_2linker), 23.6 (

CH_2linker). HRMS (ESI) m/z: [M + Na]⁺calcd for

C₁₀₈H₁₁₅N₃O₂₄Na, 1860.7763; found, 1860.7758.

79.5 (CH_ring), 79.3 (CH_ring), 79.2 (CH_ring), 77.9 (

CH_ring), 77.7 (CH_ring), 75.9 (CH_ring), 75.6 (CH₂), 75.0

× 2 (CH_ring, CH₂), 74.9 (CH₂), 74.2 (CH₂), 73.6

(CH₂), 73.5 × 2 (2 × CH₂), 72.6 × 2 (CH_ring, CH₂),

72.0 (CH_ring), 71.6 (CH_ring), 70.0 (CH_ring), 68.7 (

CH₂), 68.5 × 2 (CH₂, CH_2linker), 68.1 (CH₂), 66.1 (

CH_ring), 52.5 (COOCH₃), 51.5 (CH_{2 linker}), 29.1 (

CH_2linker), 28.8 (CH_2linker), 23.6 (CH_2linker). HRMS (ESI)

m/z: [M + Na]⁺calcd for C₁₀₀H₁₀₇N₃O₂₃Na, 1740.7188;

found, 1740.7182.

5-Azidopnetyl 2-O-Benzoyl-4,6-O-benzylidene-β-D-gluco-

pyranosyl-(1→4)-2,6-di-O-benzyl-[methyl-2,3,4-tri-O-ben-

zyl-α-^D-glucopyranosyluronate-(1→3)]-β-D-mannopyrano-

syl-(1→4)-2,3,6-tri-O-benzyl-α-^D-glucopyranoside (23).

DDQ (91 mg, 4.00 mmol, 1.75 equiv) was added to a stirred

solution of tetrasaccharide 6 (420 mg, 0.229 mmol) in DCM/

phosphate buﬀer pH 7 (10 mL, 9:1 = v/v) at 0 °C. The

reaction mixture was vigorously stirred in the absence of light

until TLC analysis indicated the disappearance of the starting

material (180 min). Upon completion, the reaction mixture

was diluted with CH₂Cl₂and washed with satd aq NaHCO₃

and brine. The organic phase was washed with water until the

solution becomes colorless; then, the organic solution was

dried over MgSO₄and ﬁltered. The ﬁltrate was concentrated

under reduced pressure, and the resulting residue was puriﬁed

by ﬂash chromatography over silica gel (EtOAc/hexanes, 3:7)

to aﬀord pure compound 23 (281 mg, 72%) as a white foam.

¹H NMR (600 MHz, CDCl₃, 298 K): δ 7.94 (d, J = 7.3 Hz,

2H, ArH), 7.35−7.00 (m, 48H, ArH), 5.12 (d, J = 3.0 Hz,

1H, C1H_{α‑glc‑COOMe}), 5.05−5.02 (m, 2H, ArCH_aH_b, 

CH_ring), 4.94 (d, J = 11.0 Hz, 1H, ArCH_aH_b), 4.89 (s, 1H,

PhCH), 4.87 (d, J = 11.8 Hz, 1H, ArCH_aH_b), 4.82 (d, J =

11.8 Hz, 2H, ArCH₂), 4.77 (d, J = 10.8 Hz, 1H, Ar

CH_aH_b), 4.73−4.62 (m, 6H, 2 × ArCH₂, C1H_{α‑glc‑linker}

(overlapped), C1H_β‑glc, (overlapped)), 4.55−4.47 (m, 4H, 2

× ArCH₂), 4.43 (d, J = 9.1 Hz, 1H, CH_ring), 4.34 (t, 1H, J

= 9.1 Hz, CH_ring), 4.28 (s, 1H, C1H_β‑man), 4.26 (d, J =

11.8 Hz, 1H, ArCH_aH_b), 4.10−4.06 (m, 3H, CH_ring, Ar

CH_aH_b, CH_aH_bOBn), 3.80−3.73 (m, 4H, 4 × CH_ring),

3.67−3.52 (m, 10H, COOCH₃, 1 × CH₂OBn, 

CH_aH_bOBn 1 × CH_aH_blinker3 × CH_ring), 3.48−3.32 (m,

5H, 1 × CH₂OBn, 2 × CH_ring, 1 × CH_aH_blinker), 3.18

(t, J = 6.8 Hz, 2H, CH_2linker), 3.08−3.04 (m, 1H, CH_ring),

2.88 (t, 1H, J = 9.3 Hz, CH_ring), 2.79 (d, 1H, J = 8.5 Hz, 

CH_ring), 1.62−1.52 (m, 4H, 2 × CH_2linker), 1.40−1.36 (m,

2H, CH_2linker). ¹³C{¹H} NMR (150 MHz, CDCl₃, 298 K): δ

170.4 (CO), 165.6 (CO), 139.9 (ArC), 139.3 (ArC),

139.0 (ArC), 138.8 (ArC), 138.6 (ArC), 138.5 (Ar

C), 138.3 (ArC), 137.8 (ArC), 137.3 (ArC), 133.6

(ArC), 130.0 (ArC), 129.6 (ArC), 129.4 (ArC),

128.7 × 2 (2 × ArC), 128.6 × 2 (2 × ArC), 128.5 × 3 3 ×

(ArC), 128.4 (ArC), 128.2 (ArC), 128.1 × 3 (ArC),

128.0 (ArC), 127.9 × 5 (ArC), 127.8 (ArC), 127.7

(ArC), 127.4 (ArC), 127.3 (ArC), 127.1 (ArC),

126.9 (ArC), 126.5 (ArC), 101.6 (C1_β‑man), 101.4 (Ph−

CH), 99.9 (C1_β‑glc), 97.8 (C1_{α‑glc‑COOMe}), 97.2 (C1_{α‑glc‑linker}),

80.7 × 2 (2 × CH_ring), 80.6 (CH_ring), 80.2 (CH_ring),

5-Azidopnetyl 2-O-Benzoyl-3-O-p-methoxybenzyl-β-D-

glucopyranosyl-(1→4)-2,6-di-O-benzyl-[methyl-2,3,4-tri-O-

benzyl-α-^D-glucopyranosyluronate-(1→3)]-β-D-mannopyra-

nosyl-(1→4)-2,3,6-tri-O-benzyl-α-^D-glucopyranoside (27).

To a stirred solution of tetrasaccharide 6 (1.175 g, 0.639

mmol) in CH₂Cl₂/MeOH (14 mL 1:1 = v/v), p-TsOH (120

mg, 0.639 mmol, 1 equiv) was added at rt. The reaction

mixture was allowed to stir overnight. Upon completion, the

reaction mixture was quenched with Et₃N (100 μL) and

concentrated by rotary evaporation. The obtained residue was

puriﬁed by ﬂash column chromatography (EtOAc/hexanes,

2:3) to give pure compound 27 (110 mg, 87%) as a white

foam. ¹H NMR (600 MHz, CDCl₃, 298 K): δ 7.96 (d, J = 7.1

Hz, 2H, ArH), 7.63−7.03 (m, 45H, ArH), 6.69 (d, J = 8.1

Hz, 2H, ArH), 5.24 (t, J = 8.4, CH_ring), 5.10 (dd, J = 11.8,

4.0 Hz, 2H, 2 × ArCH_aH_b), 5.02 (d, J = 10.7 Hz, 1H, Ar

CH_aH_b), 4.97 (d, J = 3.4 Hz, 1H, C1−H_{α‑glc‑COOMe}), 4.86 (d, J

= 12.9 Hz, 1H, ArCH_aH_b), 4.78−4.74 (m, 3H, 3 × Ar

CH_aH_b), 4.72−4.69 (m, 3H, ArCH₂, C1−H_{α‑glc‑linker}), 4.66

(s, 1H, C1H_β‑glc), 4.65−4.54 (m, 5H, 2 × ArCH₂, Ar

CH_aH_b), 4.52 (s, 1H, ArCH_aH_b), 4.47 (d, J = 11.8 Hz, 1H,

ArCH_aH_b), 4.37−4.33 (m, 2H, ArCH_aH_b, CH_ring),

4.29−4.27 (m, 2H, C1H_β‑man, ArCH_aH_b), 4.02−3.95 (m,

2H, CH_aH_b−CH_ring), 3.88−3.84 (m, 2H, CH_ring, 

CH_aH_b), 3.82−3.79 (m, 3H, 3 × CH_ring), 3.72−3.66 (m,

6H, OCH₃, 3 × CH_ring), 3.63−3.59 (m, 4H, CH_ring, 3

× CH_aH_b), 3.57 (s, 3H, COOCH₃), 3.56−3.51 (m, 2H,

CH_ring, CH_aH_b), 3.48 (dd, J = 9.1, 4.0 Hz, 1H, CH_ring),

3.43−3.35 (m, 3H, CH_ring, 2 × CH_aH_b), 3.22 (t, J = 7.0

Hz, 2H, CH_2linker), 3.11−3.06 (m, 2H, CH_ring, OH),

2.80 (d, J = 9.2 Hz, 1H, CH_ring), 2.25 (d, J = 36 Hz, 1H, 

OH), 1.65−1.61 (m, 2H, CH_2linker), 1.60−1.56 (m, 2H, 

CH_2linker), 1.45−1.34 (m, 2H, CH_2linker). ¹³C{¹H} NMR

(150 MHz, CDCl₃, 298 K): δ 170.0 (COOMe), 165.0

(COBz), 159.3 (ArC), 139.9 (ArC), 139.3 (ArC),

138.8 (ArC), 138.6 (ArC), 138.5 (ArC), 138.4 (Ar

C), 137.7 (ArC), 137.6 (ArC), 133.5 (ArC), 130.4

(ArC), 129.8 × 2 (ArC), 129.5 (ArC), 128.8 × 2 (Ar

C), 128.7 (ArC), 128.5 × 2 (ArC), 128.3 × 2 (ArC),

128.2 (ArC), 128.1 × 2 (ArC), 128.0 × 2 (ArC), 127.9

× 2 (ArC), 127.7 (ArC), 127.6 (ArC), 127.5 (ArC),

127.4 (ArC), 127.2 (ArC), 127.1 (ArC), 126.9 (Ar

1

C), 113.9 (ArC), 101.8 (C1_β‑man, J_C−H= 156.0 Hz), 100.2

(C1_{α‑glc‑COOMe}, J_C−H= 170.1 Hz), 99.9 (C1_β‑glc, ¹J_C−H= 163.6

1

Hz), 97.2 (C1_{α‑glc‑linker}, J_C−H= 171.6 Hz), 82.6 (CH_ring),

81.5 (CH_ring), 80.6 × 2 (CH_ring), 79.8 (CH_ring), 79.3

M

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Article

(CH_ring), 78.3 × 2 (CH_ring), 76.0 (CH_ring), 75.7 (

CH₂), 75.3 (CH_ring), 75.03 (CH₂), 74.6 (CH₂), 74.2

(CH₂), 73.9 (CH₂), 73.8 (CH_ring), 73.6 (CH₂), 73.5

× 2 (CH₂), 72.4 (CH₂), 71.4 (CH_ring), 70.9 (

CH_ring), 70.1 (CH_ring), 69.4 (CH_ring), 68.6 (CH₂), 68.1

(CH₂), 68.0 (CH₂), 60.7 (CH₂), 55.3 (OCH₃), 52.5

(COOCH₃), 51.4 (CH₂), 29.0 (CH₂), 28.8 (CH₂),

23.5 (CH₂). HRMS (ESI) m/z: [M + Na]⁺calcd for

127.9 (ArC), 127.7 × 2 (ArC), 127.6 (ArC), 127.1

(ArC), 126.9 (ArCH), 113.7 (ArC), 101.4 (C1_β‑man

,

1

¹J_C−H= 155.8 Hz), 99.8 (C1_β‑glc, J_C−H= 163.2 Hz), 98.6

1

(C1_{α‑glc‑COOMe}, J_C−H= 171.0 Hz), 97.1 (C1_{α‑glc‑linker}, J_C−H

=

172.5 Hz), 80.7 (CH_ring), 80.6 (CH_ring), 79.9 (CH_ring),

79.5 (CH_ring), 79.4 (CH_ring), 79.1 (CH_ring), 78.9 (

CH_ring), 78.0 (CH_ring), 77.4 (CH_ring), 75.9 (CH_ring),

75.3 (CH₂), 75.0 (CH₂), 74.7 (CH₂), 73.8 (CH₂),

73.6 (CH₂), 73.5 (CH₂), 73.4 (CH₂), 73.3 (CH₂),

72.3 (CH₂), 72.1 (CH_ring), 71.5 (CH_ring), 70.5 (

CH_ring), 70.0 (CH_ring), 68.7 (CH₂), 68.6 (CH₂), 68.1

(CH₂), 62.8 (CH₂), 55.3 (OCH₃), 52.3 (COOCH₃),

51.5 (CH₂), 29.1 (CH₂), 28.8 (CH₂), 23.6 (CH₂),

20.9 (COCH₃), 20.6 (COCH₃). HRMS (ESI) m/z: [M +

Na]⁺calcd for C₁₀₅H₁₁₅N₃O₂₆Na, 1856.7661; found,

1856.7662.

C₁₀₁H₁₁₁N₃O₂₄Na, 1772.7450; found, 1772.7460.

5-Azidopnetyl 4,6-Di-O-acetyl-2-O-benzoyl-3-O-p-me-

thoxybenzyl-β-^D-glucopyranosyl-(1→4)-2,6-di-O-benzyl-

[methyl-2,3,4-tri-O-benzyl-α-^D-glucopyranosyluronate-(1→

3)]-β-^D-mannopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-glu-

copyranoside (28). To a stirred solution of diol 27 (920 mg,

0.525 mmol) in a pyridine (10 mL) were added DMAP (7 mg,

0.1 mol %) and acetic anhydride (200 μL, 2.10 mmol) at 0 °C

under an argon atmosphere. The reaction mixture was stirred

at rt until TLC analysis indicated the disappearance of the

starting material (3 h). Upon completion, pyridine was

removed by rotary evaporation under a high vacuum. The

obtained residue was diluted with CH₂Cl₂(25 mL) and

washed with 1 N HCl (2 × 10 mL), satd aq NaHCO₃(10

mL), and water (10 mL). The organic solvent was dried over

anhydrous MgSO₄, and the residue was concentrated by rotary

evaporation. The concentrate was puriﬁed by ﬂash column

chromatography (EtOAc/hexanes, 2:3) to yield pure com-

5-Azidopnetyl 4,6-Di-O-acetyl-2-O-benzoyl-β-D-glucopyr-

anosyl-(1→4)-2,6-di-O-benzyl-[methyl-2,3,4-tri-O-benzyl-α-

D-glucopyranosyluronate-(1→3)]-β-D-mannopyranosyl-(1→

4)-2,3,6-tri-O-benzyl-α-^D-glucopyranoside (7). To a stirred

solution of tetrasaccharide 28 (953 mg, 0.520 mmol) and

thiophenol (75 μL, 0.676 mmol, 1.3 equiv) in dry CH₂Cl₂(10

mL) was added SnCl₄(162 mg, 0.624 mmol, 1.2 equiv) at −78

°C under an argon atmosphere. After 10 min, the resulting

mixture was stirred at −50 °C until TLC analysis indicated the

disappearance of the starting material (30−40 min). Upon

completion, the reaction mixture was quenched with satd aq

NaHCO₃(5 mL) and extracted with CH₂Cl₂(2 × 15 mL).

The combined organic layers were washed with brine, dried

over anhydrous MgSO₄, and concentrated by rotary evapo-

ration. The resulting residue was puriﬁed by ﬂash column

chromatography (EtOAc/Hexanes, 2:3) to give title com-

pound 7 (833 mg, 93%) as a white foam. ¹H NMR (600 MHz,

CDCl₃, 298 K): δ 7.95 (d, J = 7.3 Hz, 2H, ArH), 7.60−7.03

(m, 43H, ArH), 5.14 (d, J = 3.0 Hz, 1H, C1−H_{α‑glc‑coome}),

5.07 (d, J = 11.9 Hz, 1H, ArCH_aH_b), 4.94 (d, J = 11.9 Hz,

1H, ArCH_aH_b), 4.89−4.83 (m, 3H, ArCH₂, CH_ring),

4.76 (d, J = 11.4 Hz, 1H, ArCH_aH_b), 4.72 (d, J = 12.8 Hz,

1H, ArCH_aH_b), 4.71−4.62 (m, 6H, C1−H_{α‑glc‑link}, C1−

1

pound 28 (953 mg, 99%) as a white foam. H NMR (600

MHz, CDCl₃, 298 K): δ 7.92 (d, J = 7.3 Hz, 2H, ArH),

7.59−7.05 (m, 43H, ArH), 6.94 (d, J = 8.4 Hz, 2H, ArH),

6.64 (d, J = 8.7 Hz, 2H, ArH), 5.22 (t, J = 8.7 Hz, 1H, 

CH_ring), 5.12 (d, J = 3.1 Hz, 1H, C1−H_{α‑glc‑COOMe}), 5.07 (d, J =

11.7 Hz, 1H, ArCH_aH_b), 4.92 (s, 1H, ArCH_aH_b), 4.90 (s,

1H, ArCH_aH_b), 4.86 (d, J = 12.4 Hz, 1H, ArCH_aH_b),

4.80−4.71 (m, 4H, CH_ring, 3 × ArCH_aH_b), 4.69−4.68 (m,

4H, C1−H_{α‑glc‑linker}, C1−H_β‑glc, ArCH₂), 4.64 (d, J = 11.7

Hz, 1H, ArCH_aH_b), 4.58 (d, J = 11.7 Hz, 1H, ArCH_aH_b),

4.54−4.50 (m, 3H, 3 × ArCH_aH_b), 4.44 (d, J = 9.4 Hz, 1H,

CH_ring), 4.36−4.29 (m, 4H, C1−H_β‑man, ArCH₂, 

CH_ring), 4.28 (d, J = 11.7 Hz, 1H, ArCH_aH_b), 4.07−4.02 (m,

3H, CH_ring, ArCH_aH_b, CH_aH_b), 3.98 (dd, J = 12.1, 2.5

Hz, 1H, CH_aH_b), 3.82−3.72 (m, 4H, 4 × CH_ring), 3.68−

3.63 (m, 5H, OCH₃, CH_aH_b, CH_ring), 3.62−3.51 (m,

8H, 2 × CH_ring, COOCH₃, 3 × CH_aH_b), 3.46 (dd, J =

9.1, 3.5 Hz, 1H, CH_ring), 3.43 (d, J = 11.1, 1H, CH_aH_b),

3.38−3.34 (m, 2H, CH_ring, CH_aH_b), 3.32 (dd, J = 9.3, 2.3

Hz, 1H, CH_ring), 3.19 (t, J = 7.1 Hz, 2H, CH_2linker), 2.88

(d, 1H, J = 8.0 Hz, 1H, CH_ring), 1.93 (s, 3H, COCH₃),

1.69 (s, 3H, COCH₃), 1.62−1.58 (m, 2H, CH_2linker),

1.57−1.53 (m, 2H, CH_2linker), 1.41−1.37 (m, 2H, 

CH_2linker). ¹³C{¹H} NMR (150 MHz, CDCl₃, 298 K): δ

171.0 (CO), 170.4 (CO), 169.4 (CO), 164.6 (CO), 159.2

(ArC), 139.9 (ArC), 139.2 (ArC), 138.7 × 2 (ArC),

138.6 (ArC), 138.4 (ArC), 137.8 (ArC), 133.4 (Ar

C), 130.0 (ArC), 129.8 × 2 (ArC), 129.4 (ArC), 128.7

× 2 (ArC), 128.6 (ArC), 128.5 × 2 (ArC), 128.4 (Ar

C), 128.2 (ArC), 128.1 × 3 (ArC), 128.0 × 2 (ArC),

H

_β‑glc), 2 × ArCH₂), 4.58 (d, J = 11.6 Hz, 1H, ArCH_aH_b),

4.54−4.51 (m, 3H, ArCH₂, CH_ring), 4.47 (d, J = 9.4 Hz,

1H, CH_ring), 4.37−4.33 (m, 3H, C1−H_β‑man, 2 × CH_ring),

4.28 (d, J = 11.7 Hz, 1H, ArCH_aH_b), 4.08−4.00 (m, 3H, 

CH_ring, ArCH_aH_b, CH_aH_b), 3.94 (d, J = 11.9 Hz, 1H,

ArCH_aH_b), 3.80−3.74 (m, 4H, 4 × CH_ring), 3.67−3.65

(m, 2H, CH_ring, CH_aH_b), 3.64 (dd, 1H, J = 8.9, 3.1 Hz, 

CH_ring), 3.60−3.54 (m, 3H, CH₂, 1 × CHaH_b), 3.53 (s,

3H, COOCH₃), 3.47−3.44 (m, 3H, CH_ring, CH₂),

3.37−3.28 (m, 3H, 2 × CH_ring, CH_aH_b), 3.19 (t, J = 7.1

Hz, 2H, CH_2linker), 2.91 (d, J = 8.0 Hz, 1H, CH_ring), 2.07

(d 1H, J = 6.7, OH), 2.00 (s, 3H, COCH₃), 1.67 (s, 3H, 

COCH₃), 1.62−1.58 (m, 2H, CH_2linker), 1.56−1.53 (m, 2H,

CH_2linker), 1.41−1.36 (m, 2H,−CH_2linker). ¹³C{¹H} NMR

(150 MHz, CDCl₃, 298 K): δ 171.0 (CO), 170.7 (CO), 170.5

(CO), 165.8 (CO), 139.8 (ArC), 139.1 (ArC), 138.8

(ArC), 138.6 (ArC), 138.4 (ArC), 138.3 (ArC),

N

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137.6 (ArC), 133.7 (ArC), 129.9 (ArC), 129.4 (Ar

C), 128.8 (ArC), 128.7 (ArC), 128.6 × 2 (ArC), 128.5

× 2 (ArC), 128.4 (ArC), 128.2 (ArC), 128.1 × 2 (Ar

C), 128.0 × 2 (ArC), 127.9 × 2 (ArC), 127.8 × 2 (Ar

C), 127.7 (ArC), 127.5 (ArC), 127.3 (ArCH), 127.2

(ArCH), 127.0 (ArCH), 126.9 (ArCH), 101.5

2-O-Benzyl-3-O-p-methoxy bezyl-4,6-O-benzylidene-β-D-

mannopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-^D-glucopyra-

nosyl Fluoride (14). To a stirred solution of hemiacetal 24

(330 mg, 0.362 mmol) in anhydrous CH₂Cl₂(5 mL) was

added DAST (133 μL, 1.08 mmol) at −20 °C under an argon

atmosphere. The reaction mixture continued to stir until TLC

analysis (EtOAc/hexanes) indicated the disappearance of the

starting material (1.5 h). Upon completion, the mixture was

diluted with CH₂Cl₂(10 mL), washed with satd aq NaHCO₃

(3 mL) and brine (5 mL), and dried over MgSO₄. The solvent

was evaporated under reduced pressure to yield the crude

product, which was puriﬁed by ﬂash chromatography over

silica gel (EtOAc/hexanes, 1:5) to give pure compound 14

(302 mg, 92%, white foam) in an α,β-mixture (α/β = 1:2). ¹H

NMR (600 MHz, CDCl₃, 298 K): (major anomer) δ 7.52 (d, J

= 6.6 Hz, 2H, 2 × ArH), 7.43−7.26 (m, 25H, 25 × ArH),

6.89 (d, J = 8.5 Hz, 2H, 2 × ArH), 5.55 (s, 1H, Ph−CH),

5.35 (dd, J_HF= 53.1, 6.9 Hz, 1H, C1H_glc‑F), 5.06 (d, J = 10.9

Hz, 1H, ArCH_aH_b), 4.88−4.79 (m, 3H, 3 × ArCH_aH_b),

4.75−4.70 (m, 3H, 3 × ArCH_aH_b), 4.66 (d, J = 12.1 Hz, 1H,

ArCH_aH_b), 4.58 (d, J = 12.1 Hz, 1H, ArCH_aH_b), 4.53 (s,

1H, C1H_β‑man), 4.43 (m 1H, ArCH_aH_b), 4.12−4.06 (m,

3H, 2 × CH_ring, CH_aH_b), 3.81 (s, 3H, OCH₃), 3.71 (d,

J = 3.6 Hz, 1H, CH_ring), 3.68−3.48 (m, 6H, 3 × CH_ring, 3

× CH_aH_b), 3.44 (dd, J = 9.8, 3.1 Hz, 1H, CH_ring), 3.14−

3.08 (m, 1H, CH_ring). ¹³C{¹H} NMR (150 MHz, CDCl₃,

298 K): (major anomer) δ 159.3 (ArC), 138.9 (ArC),

138.7 (ArC), 137.8 × 2 (ArC), 137.7 (ArC), 130.7

(ArC), 129.2 (ArC), 129.0 (ArC), 128.7 (ArC),

128.6 (ArC), 128.5 (ArC), 128.3 × 2 (ArC), 128.2 × 3

(ArC), 128.0 (ArC), 127.9 (ArC), 127.8 (ArC),

126.2 (ArC), 113.9 (ArC), 110.4 (d, C1-F, J_C−F= 216

1

(C1_β‑man, J_C−H= 155.1 Hz), 99.3 (C1_β‑glc, J_C−H= 162.7

1

Hz), 98.6 (C1_{α‑glc‑coome}, J_C−H= 170.2 Hz), 97.1 (C1_{α‑glc‑linker}

,

¹J_C−H= 170.5 Hz), 80.7 (CH_ring), 80.5 (CH_ring), 79.8 (

CH_ring), 79.2 (CH_ring), 78.9 (CH_ring), 78.8 (CH_ring),

77.9 × 2 (CH_ring), 75.8 (CH_ring), 75.3 (CH₂), 75.0 (

CH₂), 74.8 (CH_ring), 74.6 (CH₂), 74.12 (CH_ring), 73.7

(CH₂), 73.5 (CH₂), 73.4 × 2 (CH₂), 72.1 (CH₂),

71.9 (CH_ring), 71.6 × 2 (CH_ring), 71.5 (CH_ring), 69.8

(CH_ring), 68.5 (CH₂), 68.4 (CH₂), 68.0 (CH₂), 62.7

(CH₂), 52.5 (COOCH₃), 51.4 (CH₂), 29.0 (CH₂),

28.8 (CH₂), 23.5 (CH₂), 21.0 (COCH₃), 20.5 (

COCH₃). HRMS (ESI) m/z: [M + Na]⁺calcd for

C₉₇H₁₀₇N₃O₂₅Na, 1736.7086; found, 1736.7130.

2-O-Benzyl-3-O-p-methoxybezyl-4,6-O-benzylidene-β-D-

mannopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-^D-glucopyra-

noside (24). To a stirred solution of disaccharide S2a (650 mg,

0.683 mmol) in moist acetic acid (10 mL, acetic acid/water,

10:1 = v/v) were added CH₃COONa (280 mg, 3.42 mmol, 5

equiv) and PdCl₂(242 mg, 1.36 mmol, 2 equiv) at rt. The

reaction mixture continued to stir until TLC analysis indicated

the disappearance of the starting material (16−20 h). Upon

completion, the reaction mixture was diluted with ethyl acetate

(20 mL) and poured into saturated NaHCO₃(50 mL). The

aqueous layer was extracted with ethyl acetate (2 × 25 mL),

and the combined organic phases were dried over MgSO₄,

ﬁltered, and concentrated. Flash chromatography over silica gel

(EtOAc/hexanes, 1:2) aﬀorded hemiacetal 24 (431 mg, 70%,

1

Hz), 101.7 (C-1_man, J_C−H= 159.7 Hz), 101.5 (Ph−CH), 80.8

(CH_ring), 79.8 (CH_ring), 78.8 (CH_ring), 78.1 (CH_ring),

77.2 (CH_ring), 77.1 (CH_ring), 75.1 (CH₂), 75.0 (

CH₂), 74.6 (CH₂), 73.8 (CH₂), 72.5 (CH₂), 72.4 (

CH_ring), 68.7 (CH₂), 68.3 (CH₂), 67.5 (CH_ring), 55.4

(OCH₃). HRMS (ESI) m/z: [M + Na]⁺calcd for

C₅₅H₅₇FO₁₁Na, 935.3777; found, 935.3783.

1

white foam) in an α,β-mixture. H NMR (600 MHz, CDCl₃,

298 K): (major anomer) δ 7.53 (d, J = 8.2 Hz, 2H, ArH),

7.45−7.26 (m, 25H, ArH), 6.89 (d, J = 8.6 Hz, 2H, ArH),

5.56 (s, 1H, Ph−CH), 5.24 (d, J = 3.5 Hz, 1H, C1H_α‑glc),

5.14 (d, J = 10.7 Hz, 1H, ArCH_aH_b), 4.84−4.80 (m, 4H, 2 ×

ArCH₂), 4.75 (d, J = 10.6 Hz, 1H, ArCH_aH_b), 4.71−4.68

(m, 2H, ArCH₂), 4.65 (d, J = 12.0 Hz, 1H, ArCH_aH_b),

4.57 (d, J = 12.0 Hz, 1H, ArCH_aH_b), 4.46 (s, 1H, C1

2-O-Benzyl-3-O-p-methoxy Bezyl-4,6-O-benzylidene-β-D-

mannopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-^D-glucopyra-

nosyl Trichloroacetimidate (25). To a stirred solution of

hemiacetal 24 (455 mg, 0.5 mmol) in anhydrous CH₂Cl₂(5

mL) were added trichloroacetonitrile (250 μL, 2.5 mmol, 5

equiv) and DBU (28 μL, 0.2 mmol, 0.4 equiv) at 0 °C under

an argon atmosphere. The reaction mixture was stirred at rt for

2 h. Upon completion, the solvent was removed under reduced

pressure, and the residue was puriﬁed by column chromatog-

raphy over neutral alumina (EtOAc/hexanes, 2:8) without

applying any pressure to aﬀord pure compound 25 (342 mg,

H

_β‑man), 4.36 (d, J = 11.8 Hz, 1H, CH_aH_b), 4.12−4.09 (m,

2H, CH_ring, CH_aH_b), 3.96−3.93 (m, 2H, 2 × CH_ring),

3.81 (s, 3H, OCH₃), 3.70−3.69 (m, 1H, CH_ring), 3.63−

3.52 (m, 4H, 2 × CH_ring, CH₂), 3.39−3.37 (m, 1H, 

CH_ring), 3.13−3.10 (m, 1H, CH_ring). ¹³C{¹H} NMR (150

MHz, CDCl₃, 298 K): (major anomer) δ 159.3 (ArC),

139.4 (ArC), 138.7 (ArC), 138.0 (ArC), 137.8 (Ar

C), 137.7 (ArC), 130.7 (ArC), 129.1 (ArC), 128.7

(ArC), 128.6 (ArC), 128.4 (ArC), 128.3 (ArC),

128.2 (ArC), 128.1 (ArC), 127.8 (ArC), 127.6 (Ar

C), 127.4 (ArC), 126.2 (ArC), 113.9 (ArC), 101.8 (C-

1

65%, white foam) in an α,β-mixture (α/β = 13:1). H NMR

1

_β‑man), 101.5 (PhCH), 91.5 (C-1_glc), 80.1 (CH_ring), 79.3 (

(600 MHz, CDCl₃, 298 K): (α-anomer) δ 8.58 (s, 1H, NH),

7.47−7.19 (d, m, 27H, ArH), 6.84 (d, J = 8.7 Hz, 2H, Ar

H), 6.44 (d, J = 3.5 Hz, 1H, C1H_α‑glc), 5.51 (s, 1H, Ph−

CH), 5.00 (d, J = 10.5 Hz, 1H, ArCH_aH_b), 4.85 (d, J =

11.8 H, 1H, ArCH_aH_b), 4.77 (m, 2H, ArCH₂), 4.68

(s, 2H, ArCH₂), 4.67 (d, J = 11.8, 1H, ArCH_aH_b),

CH_ring), 78.8 (CH_ring), 78.0 (CH_ring), 77.7 (CH_ring),

77.1 (CH_ring), 75.3 (CH₂), 75.1 (CH₂), 73.7 × 2 (

CH₂), 72.3 (CH₂), 70.1 (CH_ring), 68.7 (CH₂), 68.6 (

CH₂), 67.5 (CH_ring), 55.4 (OCH₃). HRMS (ESI) m/z:

[M + Na]⁺calcd for C₅₅H₅₈O₁₂Na, 933.3820; found, 933.3828.

O

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4.56 (d, J = 12.3, 1H, ArCH_aH_b), 4.52 (d, J = 11.6 H, 1H,

ArCH_aH_b), 4.43 (s, 1H, C1H_β‑man), 4.28 (d, J = 11.6 H,

1H, ArCH_aH_b), 4.08 (t, J = 9.1 Hz, 1H, CH_ring), 4.02−

3.92(m, 2H, CH_ring, CH_aH_b), 3.96 (t, J = 9.1 Hz, 1H,

CH_ring), 3.89 (m, 1H, CH_ring), 3.76 (s, 3H, OCH₃),

3.71 (dd, J = 9.3, 3.5 Hz, 1H, CH_ring), 3.64 (d, J = 3.0 Hz,

1H, CH_ring), 3.58 (t, J = 10.5 Hz, 1H, CH_aH_b), 3.54

(dd, J = 11.2, 2.0 Hz, 1H, CH_aH_b), 3.45 (dd, J = 11.2, 2.7

Hz, 1H, CH_aH_b), 3.35 (dd, J = 10.1, 3.0 Hz, 1H, 

CH_ring), 3.06−3.02 (m, 1H, CH_ring). ¹³C{¹H} NMR (150

MHz, CDCl₃, 298 K): (α-anomer) δ 161.4 (CNH), 159.3

(ArC), 139.3 (ArC), 138.7 (ArC), 138.2 (ArC),

137.8 × 2 (ArC), 130.7 (ArC), 129.2 (ArC), 129.0

(ArC), 128.7 (ArC), 128.5 × 2 (ArC), 128.4 (ArC),

128.3 (ArC), 128.2 × 2 (ArC), 128.8 × 3 (ArC), 127.7

(ArC), 127.5 (ArC), 126.3 (ArC), 113.9 (ArC),

101.9 (C-1_β‑man), 101.5 (PhCH), 94.6 (C-1_α‑glc), 91.5 (

CCl₃), 79.6 (CH_ring), 78.9 (CH_ring), 78.8 (CH_ring), 78.3

(CH_ring), 77.2 (CH_ring), 77.0 (CH_ring), 75.2 (CH₂),

75.0 (CH₂), 73.7 (CH₂), 73.3 (CH₂), 72.8 (CH_ring),

72.5 (CH₂), 68.7 (CH₂), 68.2 (CH₂), 67.6 (CH_ring),

5.4 (OCH₃).

(CH_ring), 79.7 (d, J_C3−F= 8.8 Hz, CH_ring−CH_ring), 74.7

=

(Ph−CH₂), 74.6 (Ph−CH₂), 68.7 (C−6CH₂), 65.6 (d, J_C5−F

3.6 Hz, CH_ring). HRMS (ESI) m/z: [M + H]⁺calcd for

C₂₇H₂₈FO₅, 451.1915; found, 451.1917.

Methyl-2,3,4-tri-O-benzyl-α-D-glucopyranosyluronate-

(1→3)-2-O-benzyl-4,6-O-benzylidene-β-^D-mannopyranosyl-

(1→4)-2,3,6-tri-O-benzyl-^D-glucopyranosyl Fluoride (15). To

a stirred solution of trisaccharide S7 (400 mg, 0.31 mmol, 1

equiv) in moist acetic acid (4.4 mL, acetic acid/water, 10:1 =

v/v), CH₃COONa (127 mg, 1.55 mmol, 5 equiv) and PdCl₂

(109 mg, 0.62 mmol, 2 equiv) were added at rt. The reaction

mixture was stirred until TLC analysis indicated disappearance

of starting material (16−20 h). Upon completion, the mixture

was diluted with ethyl acetate (15 mL) and poured into

saturated NaHCO₃(30 mL). The aqueous layer was extracted

with ethyl acetate (2 × 20 mL), and the combined organic

phases were dried over MgSO₄, ﬁltered, and concentrated.

Flash chromatography over silica gel (EtOAc/hexanes, 1:2)

aﬀorded hemiacetal S12 (285 mg, 74%, white foam) in an α,β-

mixture.

2,3-Di-O-benzyl-4,6-O-benzylidene-D-glucopyranosyl Flu-

oride (13). To a stirred solution of tetrasaccharide S10 (600

mg, 1.23 mmol) in moist acetic acid (11 mL, acetic acid/water,

10:1 = v/v) were added CH₃COONa (500 mg, 6.14 mmol)

and PdCl₂(435 mg, 2.45 mmol) at rt. The reaction mixture

was continued to stir until TLC analysis indicated the

disappearance of the starting material (12 h). Upon

completion, the mixture was diluted with ethyl acetate (20

mL) and poured into saturated NaHCO₃(30 mL). The

aqueous layer was extracted with ethyl acetate (2 × 25 mL),

and the combined organic phases were dried over MgSO₄,

ﬁltered, and concentrated. Flash chromatography over silica gel

(EtOAc/hexanes, 1:4) aﬀorded hemiacetal S11 (420 mg, 76%,

white foam) in an α,β-mixture.

A portion of the above dried hemiacetal S12 (260 mg, 0.21

mmol) was dissolved in anhydrous CH₂Cl₂(5 mL), and DAST

(77 μL, 0.62 mmol, 3 equiv) was added at −20 °C. The

reaction mixture was stirred until TLC analysis (EtOAc/

hexanes) indicated the disappearance of the starting material

(1.5 h). Upon completion, the mixture was diluted with

CH₂Cl₂(10 mL), washed with satd aq NaHCO₃(10 mL) and

brine, and dried over MgSO₄. The solvent was evaporated

under reduced pressure to yield the crude product, which was

puriﬁed by ﬂash chromatography over silica gel (EtOAc/

hexanes, 1:3) to aﬀord pure product 15 (249 mg, 96%, white

1

foam) in an α,β-mixture (α,β = 1:2). H NMR (600 MHz,

CDCl₃, 298 K): (major anomer) δ 7.52 (d, J = 7.2 Hz, 2H, 2 ×

ArH), 7.40−7.18 (m, 36H, 36 × ArH), 7.03 (d, J = 7.3

Hz, 2H, 2 × ArH), 5.56 (d, J = 3.6 Hz, 1H, C1

A portion of the above dried hemiacetal S11 (300 mg, 0.669

mmol) was dissolved in anhydrous CH₂Cl₂(5 mL), and DAST

(245 μL, 2 mmol, 3 equiv) was added at −20 °C. The reaction

mixture was stirred until TLC analysis indicated the

disappearance of the starting material (75 min). Upon

completion, the mixture was diluted with CH₂Cl₂(10 mL),

washed with satd aq NaHCO₃(5 mL) and brine, and dried

over MgSO₄. The solvent was evaporated under reduced

pressure to yield the crude product, which was puriﬁed by ﬂash

chromatography over silica gel (EtOAc/hexanes, 1:4) to aﬀord

pure product 13 (283 mg, 94%, white foam) in an α,β-mixture

(α/β = 1:4). NMR is in accordance with the literature.⁴⁷¹H

NMR (600 MHz, CDCl₃, 298 K): (major β-anomer) δ 7.52−

7.50 (m, 2H, 2 × ArH), 7.42−7.29 (m, 13H, 2 × ArH),

5.59 (s, 1H, PhCH−), 5.41 (d, J_HF= 53.2, 6.1 Hz, 1H, C1

H

_{α‑glc‑coome}), 5.37 (dd, J_HF= 53.3, 6.4 Hz, 1H, C1H_glc‑F),

5.30 (s, 1H, PhCH), 5.04 (d, J = 11.0 Hz, 1H, ArCH_aH_b),

4.95−4.89 (m, 2H, ArCH₂), 4.87- 4.81 (m, 3H, 3 × Ar

CH_aH_b), 4.77−4.68 (m, 5H, 5 × ArCH_aH_b), 4.62 (s, 1H,

C1H_β‑man), 4.58−4.53 (m, 2H, ArCH₂), 4.34 (d, J =

12.0 Hz, 1H, ArCH_aH_b), 4.28 (d, J = 10.0 Hz, 1H, 

CH_ring), 4.21 (t, J = 9.5 Hz, 1H, CH_ring), 4.15 (t, J = 9.2 Hz,

1H, CH_ring), 4.08−3.97 (m, 2H, 2 × CH_ring), 3.95−3.91

(m, 1H, CH_ring), 3.90−3.87 (m, 1H, CH_ring), 3.84−3.73

(m, 3H, 2 × CH_ring, CH_aH_b), 3.69−3.65 (m, 4H,

COOCH₃, CH_aH_b), 3.64−3.47 (m, 4H, 2 × CH_ring, 2

× CHaH_b), 3.10−3.05 (m, 1H, CH_ring). ¹³C{¹H} NMR

(150 MHz, CDCl₃, 298 K): (major anomer) δ 170.0 (ArC),

138.9 (ArC), 138.6 (ArC), 138.5 (ArC), 138.2 (Ar

C), 138.1 (ArC), 137.9 (ArC), 137.8 (ArC), 137.4

(ArC), 129.5 (ArC), 128.8 (ArC), 128.7 (ArC),

128.6 (ArC), 128.5 (ArC), 128.4 (ArC), 128.3 × 3

(ArC), 128.2 × 2 (ArC), 128.1 (ArC), 127.9 (ArC),

127.8 (ArC), 127.7 (ArC), 127.5 (ArC), 127.3 (Ar

C), 126.5 (ArC), 110.4 (d, C1_glc‑F, J_C−F= 218 Hz), 102.4

H

_glc‑F), 4.97 (d, J = 11.1 Hz, 1H, ArCH_aH_b), 4.80−4.73 (m,

3 H, ArCH_aH_b, ArCH₂), 4.42 (dd, 1H J = 5.0, 10.5 Hz,

CH_aH_b), 3.88−3.80 (m, 3 H, CH_aH_b, 2 × CH_ring),

3.67−3.58 (m, 2 H, 2 × CH_ring). ¹³C{¹H} NMR (150 MHz,

CDCl₃, 298 K): (major β-anomer) δ 138.5 (ArC), 137.8

(ArC), 137.2 (ArC), 129.2 (ArC), 128.6 (ArC),

128.5 (ArC), 128.4 (ArC), 128.3 (ArC), 128.2 (Ar

C), 128.1(ArC), 127.9(ArC), 126.1 (ArC), 110.3 (d,

C1_glc‑F, J_C−F= 217 Hz), 101.5 (Ph−CH), 81.1 (CH_ring), 80.8

(Ph−CH), 101.3 C1_β‑man

,

¹J_C−H= 157.5 Hz), 97.4

1

(C1_{α‑glc‑COOMe}, J_C−H= 172.2 Hz), 81.8 × 2 (CH_ring), 80.8

P

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J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

pubs.acs.org/joc

Article

(CH_ring), 80.7 (CH_ring), 79.8 (CH_ring), 79.3 (CH_ring),

78.2 (CH_ring), 78.9 (CH_ring), 78.6 (CH_ring), 76.0

(CH₂), 75.8 (CH₂), 75.3 (CH_ring), 75.3 (CH₂), 74.9

(CH₂), 74.6 (CH₂), 73.8 (CH₂), 71.3 (CH_ring), 71.1

(CH₂), 68.7 (CH₂), 68.2 (CH₂), 67.1 (CH_ring), 52.6 (

COOCH₃). HRMS (ESI) m/z: [M + Na]⁺calcd for

C₇₅H₇₇FO₁₆Na, 1275.5088; found, 1275.5094.

× 2 (ArC), 127.9 (ArC), 127.7 (ArC), 127.6 (ArC),

127.4 (ArC), 126.2 (ArC), 113.6 (ArC), 110.2 (d, C1-

F_glc‑F, J_C−F= 216 Hz), 100.9 (C1_β‑man, ¹J_C−H= 160.9 Hz), 100.8

(Ph−CH), 100.4 (C1_β‑glc,

¹J_C−H= 166.2 Hz), 97.6

1

(C1_{α‑glc‑COOMe}, J_C−H= 172.6 Hz), 81.6 (CH_ring), 80.8 (

CH_ring), 80.1 (CH_ring), 79.3 (CH_ring), 78.8 (CH_ring),

77.7 (CH_ring), 77.4 (CH_ring), 77.0 (CH_ring), 76.7 (

CH_ring), 75.7 (CH_ring), 75.6 (CH₂), 75.0 (CH₂), 74.5 (CH₂),

74.4 (CH₂), 74.2 (CH₂), 73.8 (CH_ring), 73.6 (CH₂), 73.5 ×

2 (CH₂), 73.4 (CH₂), 72.7 (CH₂), 72.3 (CH_ring), 71.5 (

CH_ring), 71.6 (CH_ring), 68.6 (CH₂), 68.4 (CH₂), 66.3 × 2

(CH_ring), 55.3 (OCH₃), 52.5 (COOCH₃). HRMS

(ESI) m/z: [M + Na]⁺calcd for C₁₀₃H₁₀₅FO₂₃Na,

1751.6923; found, 1751.6929.

2-O-Benzoyl-3-O-p-methoxy-benzyl-4,6-O-benzylidene-β-

D-glucopyranosyl-(1→4)-2,6-di-O-benzyl-[methyl-2,3,4-tri-

O-benzyl-α-^D-glucopyranosyluronate-(1→3)]-β-D-manno-

pyranosyl-(1→4)-2,3,6-tri-O-benzyl-^D-glucopyranosyl Fluo-

ride (16). To a stirred solution of tetrasaccharide S9 (300

mg, 0.17 mmol) in moist acetic acid (4.4 mL, acetic acid/

water, 10:1 = v/v) were added CH₃COONa (70 mg, 0.85

mmol) and PdCl₂(60 mg, 0.34 mmol) at rt. The reaction

mixture was stirred until TLC analysis indicated the

disappearance of the starting material (20 h). Upon

completion, the mixture was diluted with ethyl acetate (20

mL) and poured into saturated NaHCO₃(30 mL). The

aqueous layer was extracted with ethyl acetate (2 × 20 mL),

and the combined organic phases were dried over MgSO₄,

ﬁltered, and concentrated. Flash chromatography over silica gel

(EtOAc/hexanes, 2:3) aﬀorded hemiacetal S13 (207 mg, 71%,

white foam) in an α,β-mixture.

5-Azidopnetyl 4,6-O-Benzylidine-2,3-di-O-benzyl-α-D-glu-

copyranosyl-(1→3)-4,6-di-O-acetyl-2-O-benzoyl-β-^D-gluco-

pyranosyl-(1→4)-2,6-di-O-benzyl-[methyl-2,3,4-tri-O-ben-

zyl-α-^D-glucopyranosyluronate-(1→3)-]-β-D-mannopyrano-

syl-(1→4)-2,3,6-tri-O-benzyl-α-^D-glucopyranoside (29). A

mixture of silver triﬂate (AgOTf) (114 mg, 0.44 mmol, 8

equiv with respect to the donor), bis (cyclopentadienyl)

hafnium dichloride (CP₂HfCl₂) (85 mg, 0.22 mmol, 4 equiv

with respect to the donor), and freshly activated 4 Å molecular

sieves (200 mg) in dry toluene (2 mL) was stirred at room

temperature under an argon atmosphere for 30 min. Then the

reaction mixture was cooled to −30 °C, and a solution of

donor 13 (25 mg, 0.055 mmol, 1 equiv) and acceptor 7 (47.5

mg, 0.027 mmol, 0.5 equiv) in dry toluene (1.5 mL) was

added. Stirring was continued at the same temperature for 2 h.

Then, the reaction mixture was quenched with Et₃N (100 μL),

diluted with EtOAc, and ﬁltered through a pad of Celite. The

ﬁltrate was washed twice with satd aq NaHCO₃and brine

solution. The organic layer was dried over MgSO₄and

concentrated in vacuo. The obtained residue was puriﬁed by

open column chromatography over silica gel (EtOAc/hexanes,

1:2) to aﬀord pure pentasaccharide 29 (42 mg, 70%) as a

white foam. ¹H NMR (600 MHz, CDCl_3β298 K): δ 7.89 (d, J

= 7.2 Hz, 2H, ArH), 7.62−7.05 (m, 58H, ArH), 5.38 (s,

1H, Ph−CH), 5.15 (d, J = 3.3 Hz, 1H, C1H_{α‑glc‑COOMe}12 (d,

1H, J = 11.8 Hz, −ArCH_aH_b), 5.03−4.98 (m, 4H, ArCH₂,

CH_aH_b, CH_ring), 4.83 (d, J = 8.9 Hz, 1H, ArCH_aH_b),

4.82 (d, J = 8.9 Hz, 1H, ArCH_aH_b), 4.79−4.70 (m, 7H, 2 ×

ArCH₂, C1H_{α‑glc‑linker}, CH_aH_b, CH_ring), 4.69−4.61

(m, 5H, 2 × ArCH₂, C1H_β‑glc), 4.59−4.57 (m, 3H, Ar

CH₂, ArCH_aH_b), 4.52 (d, J = 3.4 Hz, 1H, C1H_α‑glc), 4.50

(d, J = 9.6 Hz, 1H, ArCH_aH_b), 4.39 (t, J = 9.3 Hz, 1H, 

CH_ring), 4.32 (d, J = 11.6 Hz, 1H, ArCH_aH_b), 4.29 (s, 1H,

C1H_β‑man), 4.17−4.13 (m, 2H, CH₂), 4.11−4.06 (m, 2H,

CH_aH_b, CH_ring), 4.05 (d, J = 11.6 Hz, 1H, ArCH_aH_b),

3.86−3.79 (m, 5H, 5 × CH_ring), 3.77−3.73 (m, 1H, CH_ring),

3.72−3.67 (m, 3H, 2 × CH_ring, CH_aH_b), 3.66−3.22 (m,

2H, CH₂), 3.60−3.57 (m, 4H, COOMe, CH_aH_b), 3.56 (t,

J = 8.9 Hz, 1H, CH_ring), 3.51−3.45 (m, 2H, 2 × CH_ring),

3.43−3.39 (m, 2H, CH₂), 3.35 (t, J = 9.3 Hz, 1H, CH_ring),

3.30 (dd, J = 3.1, 9.2 Hz, 1H, CH_ring), 3.25−3.22 (m, 3H, 

CH₂, CH_aH_b), 3.19 (dd, J = 2.4, 9.2 Hz, 1H, CH_ring), 2.86

(d, J = 7.9 Hz, 1H, CH_ring), 1.78 (s, 3H, COCH₃), 1.72

A portion of the above dried hemiacetal S13 (115 mg, 0.066

mmol) was dissolved in anhydrous CH₂Cl₂(3 mL), and DAST

(25 μL, 0.2 mmol, 3 equiv) was added at −20 °C. The reaction

mixture was stirred until TLC analysis indicated the

disappearance of the starting material (1.5 h). Upon

completion, the mixture was diluted with CH₂Cl₂(10 mL),

washed with satd aq NaHCO₃(10 mL) and brine, and dried

over MgSO₄. The solvent was evaporated under reduced

pressure to yield the crude product, which was puriﬁed by ﬂash

chromatography over silica gel (EtOAc/hexanes, 1:2) to aﬀord

pure product 16 (108 mg, 94%, white foam) in an α,β-mixture

1

(α/β = 1:2). H NMR (600 MHz, CDCl₃, 298 K): (major

anomer) δ 7.91−7.89 (m, 2H, ArH), 7.65−7.10 (m, 48H,

48 × ArH), 7.02 (d, J = 8.6 Hz, 2H, 2 × ArH), 6.61 (d, J

= 8.6 Hz, 2H, 2 × ArH), 5.27−5.17 (m, 3H, C1

H

_{α‑glc‑COOMe}, CH_ring, C1H_glc‑F, overlapped), 5.03−5.00

(m, 3H, PhCH, ArCH₂), 4.90−4.83 (m, 4H, 2 × ArCH₂),

4.80−4.68 (m, 4H, 3 × ArCH_aH_b, C1−H_β‑glc(overlapped),

4.66−4.59 (m, 4H, 2 × ArCH₂), 4.56−4.46 (m, 4H, 2 ×

ArCH₂), 4.40 (s, 1H, C1H_β‑man), 4.38−4.34 (m, 1H, 

CH_ring), 4.32 (m, 1H, CH_ring), 4.20−4.13 (m, 2H, CH_ring

,

CH_aH_b), 4.08 (d, J = 11.8 Hz, 1H, ArCH_aH_b), 3.97 (t, J =

9.1 Hz, 1H, CH_ring), 3.90−3.81 (m, 3H, 3 × CH_ring), 3.73

(s, 3H, OCH₃), 3.70−3.45 (m, 12H, COOCH₃, 5 × CH_ring, 2

× CH₂), 3.39 (t, 1H, J = 12.2 Hz, CH_aH_b), 3.31−3.27 (m,

1H, CH_ring), 3.19−3.15 (m, 1H, CH_ring), 2.85−2.84(m,

1H, CH_ring). ¹³C{¹H} NMR (150 MHz, CDCl₃, 298 K): δ

170.3 (CO), 164.8 (CO), 159.2 ArC), 139.1 × 2 (ArC),

138.9 (ArC), 138.8 (ArC), 138.4 (ArC), 138.3 (Ar

C), 137.8 × 2 (ArC), 137.6 (ArC), 133.4 (ArC), 130.4

(ArC), 129.9 × 2 (ArC), 129.6 × 2 (ArC), 128.7 (Ar

C), 128.6 × 3 (ArC), 128.5 × 2 (ArC), 128.4 (ArC),

128.3 (ArC), 128.2 × 3 (ArC), 128.1 × 3 (ArC), 128.0

Q

https://dx.doi.org/10.1021/acs.joc.0c01404

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

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Article

(s, 3H, COCH₃), 1.67−1.60 (m, 4H, 2 × CH_2linker),

1.47−1.41 (m, 2H,−CH_2linker). ¹³C{¹H} NMR (150 MHz,

CDCl₃, 298 K): δ 171.1 (CO), 170.6 (CO), 169.5 (CO), 164.5

(CO), 140.0 (ArC), 139.2 (ArC), 138.9 (ArC), 138.8

(ArC), 138.7 × 2 (ArC), 138.6 (ArC), 138.5 × 2 (Ar

C), 137.8 (ArC), 137.7 (ArC), 133.3 (ArC), 130.1

(ArC), 129.5 (ArC), 128.9 (ArC), 128.8 × 2 (ArC),

128.7 × 2 (ArC), 128.6 × 2 (ArC), 128.5 × 3 (ArC),

128.4 × 4 (ArC), 128.3 (ArC), 128.2 × 2 (ArC), 128.1

× 2 (ArC), 128.0 (ArC), 127.9 × 2 (ArC), 127.8 × 2

(ArC), 127.7 × 2 (ArC), 127.6 (ArC), 127.2 (ArC),

CH_ring), 4.68−4.63 (m, 4H, C1H_{α‑glc‑linker}, ArCH₂,

ArCH_aH_b), 4.62−4.58 (m, 5H, C1H_α‑glc, C1H_β‑glcAr

CH₂, ArCH_aH_b), 4.56−4.49 (m, 5H, 2 × ArCH₂, Ar

CH_aH_b), 4.47−4.42 (m, 3H, 3 × ArCH_aH_b), 4.34−4.31 (m,

1H, CH_ring), 4.27−4.25 (m, 2H, C1−H_β‑man, ArCH_aH_b),

4.23 (s, 1H, C1−H_β‑man), 4.16 (d, J = 11.9 Hz, 1H, Ar

CH_aH_b), 4.07−3.94 (m, 7H, 3 × CH_ring, ArCH_aH_b3 × 

CH_aH_b), 3.78−3.74 (m, 10H, 6 × CH_ring, OCH₃, Ar

CH_aH_b), 3.66−3.54 (m, 9H, 5 × CH_ring, 2 × CH₂), 3.53−

3.51 (m 4H, CH_ring, COOCH₃), 3.44 (dd, 1H, J = 9.3, 3.8

Hz, CH_ring), 3.41−3.58 (m, 4H, 2 × CH₂), 3.26−3.19 (m,

4H, 3 × CH_ring, CH_aH_b), 3.18 (t, J = 6.9 Hz, 2H, 

CH_2linker), 2.98−2.94 (m, 1H, CH_ring), 2.84 (d, 1H, J = 9.0,

CH_ring), 1.69 (s, 3H, COCH₃), 1.65 (s, 3H, COCH₃),

1.60−1.53 (m, 4H, 2 × CH_2linker), 1.40−1.36 (m, 2H,−

CH_2linker). ¹³C{¹H} NMR (150 MHz, CDCl₃, 298 K): δ 171.0

(CO), 170.5 (CO), 169.5 (CO), 164.8 (CO), 159.3 (ArC),

139.9 (ArC), 139.7 (ArC), 139.1 (ArC), 138.8 × 2

(ArC), 138.7 (ArC), 138.6 (ArC), 138.5 (ArC),

138.4 (ArC), 137.9 × 2 (ArC), 137.7 (ArC), 133.5

(ArC), 130.9 (ArC), 130.2 (ArC), 129.8 × 2 (ArC),

129.1 (ArC), 128.8 (ArC), 128.7 × 2 (ArC), 128.6

(ArC), 128.5 × 3 (ArC), 128.4 (ArC), 128.3 × 2 (Ar

C), 128.2 × 2 (ArC), 128.1 × 4 (ArC), 128.0 × 2 (Ar

C), 127.9 × 2 (ArC), 127.8 (ArC), 127.7 × 2 (ArC),

127.6 (ArC), 127.5 (ArC), 127.4 (ArC), 127.2 (Ar

C), 127.1 (ArC), 126.9 (ArC), 126.3 (ArC), 113.9

127.0 (ArC), 126.9 (ArC), 126.4 (ArC), 101.7

1

(C1_β‑man, J_C−H= 156.6 Hz), 101.5 (Ph−CH), 100.6 (C1_α‑glc

,

1

¹J_C−H= 171.0 Hz), 99.7 (C1_β‑glc, J_C−H= 160.2 Hz), 99.3

1

(C1_{α‑glc‑COOMe}, J_C−H= 169.8 Hz), 97.2 (C1_{α‑glc‑linker}, J_C−H

=

168.8 Hz), 82.6 (CH_ring), 82.2 (CH_ring), 80.8 (CH_ring),

80.7 (CH_ring), 80.6 (CH_ring), 80.5 (CH_ring), 79.3 × 2

(CH_ring), 79.1 (CH_ring), 78.2 (CH_ring), 78.1 (CH_ring),

77.9 (CH_ring), 76.1 (CH_ring), 75.4 (CH₂), 75.3 (

CH₂), 75.0 (CH₂), 74.7 (CH₂), 74.2 (CH₂), 73.7 (

CH₂), 73.6 (CH₂), 73.5 × 3 (2 × CH₂, CH_ring), 71.8 ×

2 (CH_ring), 71.7 (CH₂), 71.4 (CH_ring), 70.0 (CH_ring),

69.1 (CH_ring), 68.6 (CH₂), 68.4 (CH₂), 68.2 (CH₂),

63.7 (CH_ring), 62.9 (CH₂), 52.4 (COOCH₃), 51.5 (

CH₂), 29.1 (CH₂), 28.8 (CH₂), 23.6 (CH₂), 20.8 (

COCH₃), 20.6 (COCH₃). HRMS (ESI) m/z: [M + Na]⁺

calcd for C₁₂₄H₁₃₃N₃O₃₀Na, 2166.8866; found, 2166.8917.

1

(ArC), 101.5 (Ph−CH), 101.4 (C1_β‑man, J_C−H= 156.5 Hz,

1

C1_β‑man, J_C−H= 156.1 Hz), 100.1 (C1_β‑glc, J_C−H= 163.5 Hz),

1

99.8 (C1_α‑glc, J_C−H= 172.6 Hz), 99.0 (C1_{α‑glc‑COOMe}, J_C−H

=

1

172.3 Hz), 97.2 (C1_{α‑glc‑linker}, J_C−H= 171.1 Hz), 82.0 (

CH_ring), 80.7 (CH_ring), 80.2 (CH_ring), 79.9 (CH_ring),

79.5 (CH_ring), 79.3 (CH_ring), 79.2 × 2 (CH_ring), 78.8

(CH_ring), 78.0 (CH_ring), 77.6 (CH_ring), 77.4 (CH_ring),

76.9 (CH_ring), 76.0 (CH_ring), 75.4 × 2 (CH₂), 75.3 (

CH₂), 75.0 (CH₂), 74.7 (CH₂), 74.1 (CH₂), 73.7 × 2

(CH₂, CH_ring), 73.6 × 2 (CH₂), 73.5 × 2 (CH₂),

72.1 (CH₂), 72.0 (CH₂), 71.6 (CH_ring), 71.4 (

CH_ring), 70.0 (CH_ring), 69.2 (CH_ring), 68.7 (CH₂), 68.5

× 2 (CH₂), 68.1 (CH₂), 67.9 (CH₂), 67.4 (CH_ring),

62.8 (CH₂), 55.4 (OCH₃), 52.4 (COOCH₃), 51.5 (

CH₂), 29.1 (CH₂), 28.8 (CH₂), 23.6 (CH₂), 20.9 (

COCH₃), 20.6 (COCH₃). HRMS (ESI) m/z: [M + 2Na/

2]⁺calcd for C₁₅₂H₁₆₃N₃O₃₆Na₂, 1326.0400; found,

1326.0439.

5-Azidopnetyl 2-O-Benzyl-3-O-p-methoxy-4,6-O-benzyli-

dine-β-mannopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-^D-glu-

copyranosyl-(1→3)-4,6-di-O-acetyl-2-O-benzoyl-β-^D-gluco-

pyranosyl-(1→4)-2,6-di-O-benzyl-[methyl-2,3,4-tri-O-ben-

zyl-α-^D-glucopyranosyluronate-(1→3)-]-β-D-mannopyrano-

syl-(1→4)-2,3,6-tri-O-benzyl-α-^D-glucopyranoside (26a). A

mixture of silver triﬂate (AgOTf) (170 mg, 0.66 mmol, 8

equiv with respect to the donor), bis (cyclopentadienyl)

hafnium dichloride (CP₂HfCl₂) (125 mg, 0.33 mmol, 4 equiv

with respect to the donor), and freshly activated 4 Å molecular

sieves (300 mg) in dry toluene (4 mL) was stirred at room

temperature under an argon atmosphere for 30 min. Then the

reaction mixture was cooled to −30 °C, and a solution of

donor 14 (75 mg, 0.082 mmol, 1 equiv) and acceptor 7 (70

mg, 0.041 mmol, 0.5 equiv) in dry toluene (3 mL) was added.

Stirring was continued at the same temperature for 2 h,

quenched with Et₃N (150 μL), diluted with EtOAc, and

ﬁltered through a pad of Celite. The ﬁltrate was washed twice

with satd aq NaHCO₃and brine solution. The organic layer

was dried over MgSO₄and concentrated in vacuo. The

obtained residue was puriﬁed by open column chromatography

over silica gel (EtOAc/hexanes, 1:2) to aﬀord pure

5-Azidopnetyl Methyl-2,3,4-tri-O-benzyl-α-D-glucopyra-

nosyluronate-(1→3)-2-O-benzyl-4,6-O-benzylidine-β-man-

nopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-^D-glucopyranosyl-

(1→3)-4,6-di-O-acetyl-2-O-benzoyl-β-^D-glucopyranosyl-(1→

4)-2,6-di-O-benzyl-[methyl-2,3,4-tri-O-benzyl-α-^D-glucopyr-

anosyluronate-(1→3)]-β-^D-mannopyranosyl-(1→4)-2,3,6-

tri-O-benzyl-α-^D-glucopyranoside (30). A mixture of silver

triﬂate (262 mg, 1.02 mmol, 8 equiv with respect to the

donor), bis (cyclopentadienyl) hafnium dichloride (CP₂HfCl₂)

(193 mg, 0.51 mmol, 4 equiv with respect to the donor), and

freshly activated 4 Å molecular sieves (500 mg) in dry toluene

1

hexasaccharide 26a (69 mg, 64%) as a white foam. H NMR

(600 MHz, CDCl₃, 298 K): δ 7.89 (d, J = 7.2 Hz, 2H, ArH),

7.43−7.39 (m, 5H, ArH), 7.35−7.14 (m, 56H, ArH),

7.09−7.07 (m, 5H, ArH), 7.04−7.00 (m, 4H, ArH), 6.82

(d, J = 8.8 Hz, 2H, ArH), 5.43 (s, 1H, Ph−CH), 5.11−5.10

(m, 2H, C1H_{α‑glc‑COOMe}, CH_ring), 5.05 (d, J = 12.0 Hz,

1H, ArCH_aH_b), 4.94−4.91 (m, 2H, ArCH₂), 4.90 (d, J =

12.2 Hz, 1H, ArCH_aH_b), 4.77−4.70 (m, 5H, 2 × ArCH₂,

R

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J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

pubs.acs.org/joc

Article

(7 mL) was stirred at room temperature under an argon

atmosphere for 30 min. Then the reaction mixture was cooled

to −30 °C, and a solution of donor 15 (160 mg, 0.127 mmol, 1

equiv) and acceptor 7 (109 mg, 0.064 mmol, 0.5 equiv) in dry

toluene (3 mL) was added. Stirring was continued at the same

temperature for 2 h, and the reaction mixture was quenched

with Et₃N (160 μL), diluted with EtOAc, and ﬁltered through

a pad of Celite. The ﬁltrate was washed twice with satd aq

NaHCO₃and brine solution. The organic layer was dried over

MgSO₄and concentrated in vacuo. The obtained residue was

puriﬁed by open column chromatography over silica gel

(EtOAc/hexanes, 1:2) to aﬀord pure heptasaccharide 30 (127

(CH₂), 67.9 (CH₂), 66.8 (CH_ring), 62.9 (CH₂), 52.7

(COOCH₃), 52.4 (COOCH₃), 51.5 (CH₂), 29.1 (

CH₂), 28.8 (CH₂), 23.6 (CH₂), 20.9 (COCH₃), 20.6

(COCH₃). HRMS (ESI) m/z: [M + Na]⁺calcd for

C

₁₇₂H₁₈₃N₃O₄₁Na, 2969.2219; found, 2969.2276.

5-Azidopnetyl 2-O-Benzoyl-3-O-p-methoxybenzyl-4,6-O-

1

mg, 68%) as a white foam. H NMR (600 MHz, CDCl₃, 298

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

[methyl-2,3,4-tri-O-benzyl-α-^D-glucopyranosyluronate-(1→

3)]-β-^D-mannopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-glu-

copyranosyl-(1→3)-4,6-di-O-acetyl-2-O-benzoyl-β-^D-gluco-

pyranosyl-(1→4)-2,6-di-O-benzyl-[methyl-2,3,4-tri-O-ben-

zyl-α-^D-glucopyranosyluronate-(1→3)]-β-D-mannopyrano-

syl-(1→4)-2,3,6-tri-O-benzyl-α-D-glucopyranoside (31). A

mixture of silver triﬂate (118 mg, 0.462 mmol, 8 equiv with

respect to the donor), bis (cyclopentadienyl) hafnium

dichloride (C_P2HfCl₂) (87.7 mg, 0.231 mmol, 4 equiv with

respect to the donor), and freshly activated 4 Å molecular

sieves (300 mg) in dry toluene (4 mL) was stirred at room

temperature under an argon atmosphere for 30 min. Then the

reaction mixture was cooled to −30 °C, and a solution of

donor 16 (100 mg, 0.058 mmol, 1 equiv) and acceptor 7 (49.5

mg, 0.029 mmol, 0.5 equiv) in dry toluene (3 mL) was added.

Stirring was continued at the same temperature for 2 h, and the

reaction mixture was quenched with Et₃N (125 μL), diluted

with EtOAc, and ﬁltered through a pad of Celite. The ﬁltrate

was washed twice with satd aq NaHCO₃and brine solution.

The organic layer was dried over MgSO₄and concentrated in

vacuo. The residue was puriﬁed by open column chromatog-

raphy over silica gel (EtOAc/hexanes, 1:2) to aﬀord pure

K): δ 7.91 (d, J = 7.1 Hz, 2H, ArH), 7.47−7.41 (m, 6H,

ArH), 7.32−7.08 (m, 73H, ArH), 7.05 (t, 2H, J = 7.3 Hz,

ArH), 6.98 (d, 2H, J = 7.5 Hz, ArH), 5.49 (d, J = 3.6 Hz,

1H, C1H_{α‑glcCOOMe}), 5.19 (s, 1H, Ph−CH), 5.18 (t, J = 8.9

Hz, 1H, CH_ring), 5.11 (d, J = 3.3 Hz, 1H, C1H_{α‑glcCOOMe}),

5.08 (d, J = 11.7 Hz, 1H, ArCH_aH_b), 4.96−4.86 (m, 5H, 5 ×

ArCH_aH_b), 4.80−4.72 (m, 6H, 3 × ArCH₂), 4.70−4.61

(m, 11H, C1H_{α‑glc‑linker}, C1H_α‑glc, C1H_β‑glc, 4 × Ar

CH₂), 4.56−4.43 (m, 8H, 3 × ArCH₂, 2 × CH_ring), 4.37

(t, J = 9.0 Hz, 1H, CH_ring), 4.28−4.24 (m, 4H, 2 × C1

H_β‑man, ArCH₂), 4.22 (d, J = 9.8 Hz, 1H, CH_ring), 4.10−

4.00 (m, 5H, 2 × CH_ring, ArCH_aH_b−CH₂), 3.96−3.88

(m, 3H, CH_ring, ArCH_aH_b−CH_aH_b), 3.81−3.73 (m, 5H,

5 × CH_ring), 3.70−3.62 (m, 11H, 7 × CH_ring, 

COOCH₃, CH_aH_b), 3.60−3.52 (m, 7H, CH_ring, 

COOCH₃, 3 × CH_aH_b), 3.50 (dd, 1H, J = 9.7, 3.5 Hz, 

CH_ring), 3.47 (dd, 1H, J = 9.3, 4.1 Hz, CH_ring), 3.44−3.29

(m, 6H, CH_ring, 5 × CH_aH_b), 3.27 (dd, 1H, J = 9.5, 3.1

Hz, CH_ring), 3.20−3.17 (m, 3H, CH_ring, CH_2linker),

2.94−2.90 (m, 1H, CH_ring), 2.85 (d, 1H, J = 8.8, CH_ring),

1.72 (s, 3H, COCH₃), 1.67 (s, 3H, COCH₃), 1.63−1.54

(m, 4H, 2 × CH_2linker), 1.42−1.38 (m, 2H,−CH_2linker).

¹³C{¹H} NMR (150 MHz, CDCl₃, 298 K): δ 171.0 (CO),

170.5 (CO), 170.2 (CO), 169.5 (CO), 164.8 (CO), 140.0

(ArC), 139.7 (ArC), 139.2 (ArC), 138.8 × 3 (ArC),

138.7 × 3 (ArC), 138.6 (ArC), 138.5 (ArC), 138.3

(ArC), 138.2 (ArC), 138.0 (ArC), 137.8 (ArC),

137.5 (ArC), 133.7 (ArC), 130.1 (ArC), 129.8 (Ar

C), 129.4 (ArC), 128.9 (ArC), 128.8 × 2 (ArC), 128.7

× 2 (ArC), 128.6 × 2 (ArC), 128.5 × 2 (ArC), 128.4 ×

3 (ArC), 128.3 × 2 (ArC), 128.2 × 3 (ArC), 128.1

(ArC), 128.0 (ArC), 127.9 × 3 (ArC), 127.8 × 3 (Ar

C), 127.7 × 2 (ArC), 127.6 (ArC), 127.5 (ArC), 127.4

(ArC), 127.3 (ArC), 127.2 × 2 (ArC), 127.1 (ArC),

126.9 (ArC), 126.5 (ArC), 102.2 (Ph−CH), 101.6

1

heptasaccharide 31 (70 mg, 70%) as a white foam. H NMR

(600 MHz, CDCl₃, 298 K): δ 7.88 (d, J = 7.3 Hz, 2H, ArH),

7.85 (d, J = 7.5 Hz, 2H, ArH), 7.61−7.55 (m, 2H, ArH),

7.44−7.15 (m, 97H, ArH), 6.55 (d, 2H, J = 8.4 Hz, ArH),

5.18 (t, 1H, J = 8.7 Hz, CH_ring), 5.10 (d, J = 3.4 Hz, 1H,

C1H_{α‑glcCOOMe}), 5.09 (d, J = 3.5 Hz, 1H, C1H_{α‑glcCOOMe}),

5.07−5.00 (m, 3H, CH_ring, ArCH₂), 4.97.4.89 (m, 6H,

Ph−CH, 2 × ArCH₂, CH_ring), 4.86−4.71 (m, 7H, 3 ×

ArCH₂, CH_ring), 4.69−4.66 (m, 4H, C1H_{α‑glclinker}, 

CH_ring, ArCH₂), 4.64−4.59 (m, 5H, C1H_β‑glc, 2 × Ar

CH₂), 4.59−4.50 (m, 7H, C1H_α‑glc(overlapped), C1

H_β‑glc(overlapped), 2 × ArCH₂, ArCH_aH_b), 4.49−4.36

(m, 6H, 2 × ArCH₂, 2 × CH_ring), 4.33−4.25 (m, 4H,

C1H_β‑man(overlapped), CH₂, CH_aH_b), 4.14−4.06 (m,

4H, C1H_β‑man(overlapped), 2 × CH_ring, CH_aH_b),

4.05−3.97 (m, 5H, 2 × ArCH₂, CH_ring), 3.80−3.73 (m,

5H, 5 × CH_ring), 3.72−3.66 (m, 6H, COOCH₃, 2 × 

CH_ring, ArCH_aH_b), 3.64−3.45 (m, 20H, 3 × CH₂, 2 × 

COCH₃, 8 × CH_ring), 3.41−3.29 (m, 5H, 2 × CH₂, 

CH_ring), 3.22−3.15 (m, 6H, 2 × CH₂, 2 × CH_ring), 3.13−

3.06 (m, 2H, 2 × CH_ring), 2.84 (d, 1H, J = 8.8, CH_ring),

2.71 (d, 1H, J = 8.5, CH_ring), 1.65 (s, 3H, COCH₃), 1.64

(s, 3H, COCH₃), 1.61−1.54 (m, 4H, 2 × CH_2linker),

1.42−1.37 (m, 2H,−CH_2linker). ¹³C{¹H} NMR (150 MHz,

CDCl₃, 298 K): δ 171.0 (CO), 170.5 (CO), 170.4 (CO), 169.5

(CO), 164.9 (CO), 164.8 (CO), 159.1 (ArC), 140.0 (Ar

C), 139.9 (ArC), 139.2 (ArC), 139.1 (ArC), 139.0

1

(C1_β‑man, J_C−H= 157.1 Hz), 100.8 (C1_β‑man, J_C−H= 158.8

Hz), 99.8 × 2 (C1_α‑glc, ¹J_C−H= 169.1 Hz, C1_β‑glc, ¹J_C−H= 158.4

Hz), 99.1 (C1_{α‑glcCOOMe}

(C1_{α‑glc‑COOMe}, J_C−H= 171.0 Hz), 97.2 (C1_{α‑glclinker}, J_C−H

,

¹J_C−H= 171.9 Hz), 97.3

1

=

174.0 Hz), 81.3 (CH_ring), 80.8 × 2 (CH_ring), 80.2 (

CH_ring), 79.6 (CH_ring), 79.5 (CH_ring), 79.4 × 2 (

CH_ring), 79.3 × 2 (CH_ring), 79.2 (CH_ring), 79.1 (

CH_ring), 78.7 (CH_ring), 78.1 × 2 (CH_ring), 77.4 (

CH_ring), 76.6 (CH_ring), 76.4 (CH₂), 76.1 (CH_ring), 75.9

(CH₂), 75.4 (CH₂), 75.3 (CH₂), 75.2 (CH₂), 75.1

(CH₂), 74.7 (CH₂), 74.1 (CH₂), 73.7 × 2 (CH₂),

73.6 × 2 (CH₂), 73.5 (CH₂), 72.1 (CH₂), 72.0 (

CH_ring), 71.9 (CH_ring), 71.6 (CH_ring), 71.4 (CH_ring),

71.3 (CH_ring), 71.0 (CH₂), 70.1 (CH_ring), 69.7 (

CH_ring), 68.7 (CH₂), 68.6 (CH₂), 68.5 (CH₂), 68.2

S

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(ArC), 138.9 (ArC), 138.8 × 2 (ArC), 138.7 (ArC),

138.6 × 3 (ArC), 138.5 × 2 (ArC), 137.8 (ArC), 137.7

× 2 (ArC), 133.5 (ArC), 133.3 (ArC), 130.5 (ArC),

130.1 (ArC), 130.0 (ArC), 129.8 (ArC), 129.7 (Ar

C), 129.6 (ArC), 129.0 (ArC), 128.9 (ArC), 128.8

(ArC), 128.7 × 2 (ArC), 128.6 (ArC), 128.5 × 4 (Ar

C), 128.4 × 3 (ArC), 128.3 × 3 (ArC), 128.2 × 2 (Ar

C), 128.1 × 2 (ArC), 128.0 × 2 (ArC), 127.9 × 4 (Ar

C), 127.8 (ArC), 127.7 × 3 (ArC), 127.6 (ArC), 127.5

(ArC), 127.4 × 2 (ArC), 127.3 × 2 (ArC), 127.2 × 2

(ArC), 127.1 × 2 (ArC), 127.1 (ArC), 127.0 (ArC),

126.9 (ArC), 126.8 (ArC), 126.7 (ArC), 126.2 (Ar

CH_ring), 4.20 (t, J = 9.8 Hz, 1H, CH_ring), 4.17−4.12 (m,

2H, 2 × CH_aH_b), 4.00−3.94 (m, 4H, 2 × CH_aH_b, 2 × 

CH_ring), 3.93−3.85 (m, 5H, 3 × CH_aH_b, 2 × CH_ring), 3.81

(t, J = 9.0 Hz, 1H, CH_ring), 3.76−3.63 (m, 7H, 6 × CH_ring

,

1 × CH_aH_b), 3.46 (d, J = 10.0 Hz, 1H, CH_ring), 3.43 (d, J

= 9.0 Hz, 1H, CH_ring), 3.16 (t, J = 7.8 Hz, 2H, CH₂),

1.86−1.78 (m, 4H, 2 × CH₂), 1.63−1.59 (m, 2H, CH₂).

¹³C{¹H} NMR (150 MHz, D₂O, 313 K): δ 176.4 (CO), 102.4

(C1_β‑glc, ¹J_C−H= 163.5 Hz), 101.7 (C1_α‑glcA, ¹J_C−H= 172.0 Hz),

1

99.9 (C1_β‑man, J_C−H= 163.7 Hz), 98.0 (C1_{α‑glc‑linker}, J_C−H

=

172.1 Hz), 81.1 (CH_ring), 79.0 (CH_ring), 76.8 (CH_ring),

75.5 (CH_ring), 73.7 (CH_ring), 73.5 (CH_ring), 73.0 (

CH_ring), 72.3 (CH_ring), 72.0 (CH_ring), 71.9 (CH_ring),

71.8 (CH_ring), 71.0 (CH_ring), 70.9 (CH_ring), 70.5 (

CH_ring), 70.1 (CH_ring), 68.1 (CH_2linker), 61.3 (CH₂),

60.2 (CH₂), 60.0 (CH₂), 39.5 (CH_2linker), 28.2 (

CH_2linker), 26.6 (CH_2linker), 22.6 (CH_2linker). HRMS (ESI)

m/z: [M + H]⁺calcd for C₂₉H₅₂NO₂₂, 766.2975; found,

766.2984.

1

C), 113.6 (ArC), 101.6 (C1_β‑man, J_C−H= 159.5 Hz), 101.3

1

(C1_β‑man, J_C−H= 158.6 Hz), 100.9 (Ph−CH), 100.2 × 2

1

(C1_β‑glc, J_C−H= 162.1 Hz, C1_β‑glc, J_C−H= 162.1 Hz), 99.8

1

(C1_α‑glc, J_C−H= 168.3 Hz), 99.2 (C1_{α‑glcCOOMe}, J_C−H= 171.1

1

Hz), 98.1 (C1_{α‑glcCOOMe}, J_C−H= 173.6 Hz), 97.2 (C1_{α‑glclinker}

,

¹J_C−H= 171.1 Hz), 82.2 (CH_ring), 81.5 (CH_ring), 80.7 × 3

(CH_ring), 80.2 (CH_ring), 80.1 (CH_ring), 80.0 (CH_ring),

79.9 (CH_ring), 79.5 × 2 (CH_ring), 79.3 × 2 (CH_ring),

79.2 (CH_ring), 78.3 (CH_ring), 78.1 (CH_ring), 77.7 (

CH_ring), 77.4 × 2 (CH_ring), 76.2 (CH_ring), 76.1 (

CH_ring), 75.6 (CH₂), 75.4 (CH₂), 75.3 (CH₂), 75.0 (

CH₂), 74.9 (CH₂), 74.7 (CH₂), 74.4 (CH₂), 74.1 (

CH₂), 73.8 (CH_ring), 73.7 (CH₂), 73.6 (CH_ring), 73.5 ×

2 (CH₂), 73.4 (CH₂), 73.3 (CH₂), 72.4 (CH₂), 72.1

(CH_ring), 72.0 (CH_ring), 71.9 (CH_ring), 71.8 (CH₂),

71.7 (CH_ring), 71.4 (CH_ring), 71.3 (CH_ring), 70.1 (

CH_ring), 69.3 (CH_ring), 68.6 (CH₂), 68.5 (CH₂), 68.4

(CH₂), 68.2 × 2 (CH₂), 67.8 (CH₂), 66.1 (CH_ring),

62.9 (CH₂), 55.3 (OCH₃), 52.5 (COOCH₃), 52.4 (

COOCH₃), 51.5 (CH₂), 29.9 (CH₂), 29.5 (CH₂), 29.1

(CH₂), 28.8 (CH₂), 23.6 (CH₂), 20.7 (COCH₃),

20.5 (COCH₃). HRMS (ESI) m/z: [M + 2Na/2]⁺calcd for

C₂₀₀H₂₁₁N₃O₄₈Na₂, 1734.1973; found, 1734.2006.

5-Aminopnetyl α-D-Glucopyranosyl-(1→3)-β-D-glucopyr-

anosyl-(1→4)-β-^D-mannopyranosyl-[α-D-glucopyranosylur-

onate-(1→3)]-(1→4)-α-^D-glucopyranoside (2). To a stirred

solution of pentasaccharide 29 (29 mg, 0.0135 mmol) in p-

dioxane and H₂O (2 mL, p-dioxane/H₂O, 3:1 = v/v) was

added LiOH·H₂O (68 mg, 0.162 mmol, 12 equiv). The

reaction mixture was heated to 75 °C overnight. Upon

completion, the mixture was neutralized with IR-120, ﬁltered,

and concentrated. The obtained residue was dissolved in 4 mL

of a MeOH/THF/H₂O mixture (2:1:1), and a drop of

HCOOH and 20% Pd(OH)₂/C (29 mg) were added. The

reaction mixture was stirred under a H₂atmosphere for 36 h at

rt, ﬁltered over a Whatman ﬁlter paper, and concentrated

under reduced pressure. The residue was puriﬁed by reverse-

phase column chromatography (Bond Eluct-C18) by elution

with H₂O. Fractions containing the product were pooled and

lyophilized to give free pentasaccharide 2 (7.5 mg, 60% over

5-Aminopnetyl β-D-Glucopyranosyl-(1→4)-β-D-manno-

pyranosyl-[α-D-glucopyra-nosyluronate-(1→3)]-(1→4)-α-D-

glucopyranoside (1). To a well-stirred solution of tetrasac-

charide 6 (90 mg, 0.049 mmol) in a mixture of p-dioxane and

H₂O (4 mL, p-dioxane/H₂O, 3:1 = v/v) was added LiOH·H₂O

(123 mg, 0.29 mmol, 6 equiv). The reaction mixture was

heated to 75 °C overnight. Upon completion, the reaction

mixture was neutralized with IR-120, ﬁltered, and concen-

trated. The obtained residue was dissolved in 4 mL of MeOH/

THF/H₂O (2:1:1). Then, a drop of HCOOH and 20%

Pd(OH)₂/C (90 mg, 100 wt %) were added. The reaction

mixture was stirred under a H₂atmosphere for 36 h at rt. Upon

completion, it was ﬁltered over a Whatman ﬁlter paper and

concentrated under reduced pressure. The obtained residue

was puriﬁed by reverse-phase column chromatography (Bond

Eluct-C18) by elution with H₂O. Fractions containing the

product were pooled and lyophilized to give the free

tetrasaccharide 1 (21.6 mg, 58% over two steps) in a white

powder. ¹H NMR (600 MHz, D₂O, 313 K): δ 5.30 (d, J = 3.8

Hz, 1H, C1H_α‑glcA), 5.05 (d, J = 3.6 Hz, 1H, C1

1

two steps) in a white powder. H NMR (600 MHz, D₂O, 313

K): δ 5.46 (d, J = 3.9 Hz, 1H, C1H_α‑glc), 5.30 (d, J = 3.9 Hz,

1H, C1H_α‑glc‑A), 5.05 (d, J = 3.7 Hz, 1H, C1H_{α‑glc‑linker}),

4.91 (s, 1H, C1H_β‑man), 4.69 (d, J = 7.9 Hz, 1H, C1

H

_β‑glc), 4.37 (d, J = 2.9 Hz, 1H, CH_ring), 4.26 (d, J = 10.1 Hz,

1H, CH_ring), 4.20 (t, J = 9.9 Hz, 1H, CH_ring), 4.17−4.12

(m, 3H, CH_ring, 2 × CH_aH_b), 4.00−3.87 (m, 12H, 3 × 

CH₂, 1 × CH_aH_b, 5 ×CH_ring), 3.81−3.59 (m, 10H, 9 × CH_ring

,

1 × CH_aH_b), 3.63 (t, J = 9.6 Hz, 1H, CH_ring), 3.55 (t, J = 8.6

Hz, 1H, CH_ring), 3.17 (t, J = 7.5 Hz, 2H, CH_2linker), 1.87−

1.79 (m, 4H, 2 × CH_2linker), 1.63−1.59 (m, 2H, CH_2linker).

¹³C{¹H} NMR (150 MHz, D₂O, 313 K): δ 176.5 (CO), 102.5

(C1_β‑glc, ¹J_C−H= 165.1 Hz), 101.7 (C1_α‑glcA, ¹J_C−H= 172.8 Hz),

99.9 (C1_β‑man, ¹J_C−H= 163.4 Hz), 99.4 (C1_{− α‑glc}, ¹J_C−H= 172.0

1

Hz), 98.0 (C1_{α‑glc‑linker}, J_C−H= 172.1 Hz), 82.5 (CH_ring),

81.2 (CH_ring), 79.0 (CH_ring), 76.4 (CH_ring), 75.6 (

CH_ring), 73.7 (CH_ring), 73.0 × 2 (CH_ring), 72.4 (

CH_ring), 72.3 (CH_ring), 72.0 (CH_ring), 71.9 × 2 (2 × 

CH_ring), 71.8 × 2 (2 × CH_ring), 71.1 (CH_ring), 70.9 (

CH_ring), 70.5 (CH_ring), 70.4 (CH_ring), 69.5 (CH_ring),

68.1 (CH_2linker), 61.1 (CH₂), 60.4 (CH₂), 60.3 (

H

_{α‑glc‑linker}), 4.90 (s, 1H, C1H_β‑man), 4.65 (d, J = 7.9 Hz, 1H,

C1H_β‑glc), 4.37 (s, 1H, CH_ring), 4.26 (d, J = 10.0 Hz, 1H,

T

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J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

pubs.acs.org/joc

Article

CH₂), 60.1 (CH₂), 39.5 (CH_2linker), 28.2 (CH_2linker),

26.6 (CH_2linker), 22.6 (CH_2linker). HRMS (ESI) m/z: [M +

H]⁺calcd for C₃₅H₆₂NO₂₇, 928.3504; found, 928.3537.

syluronate-(1→3)]-(1→4)-α-^D-glucopyranoside (4). To a

stirred solution of heptasaccharide 30 (96 mg, 0.0325 mmol)

in a mixture of p-dioxane and H₂O (4 mL, p-dioxane/H₂O, 3:1

= v/v) was added LiOH·H₂O (20.5 mg, 0.487 mmol, 15

equiv). The reaction mixture was heated to 75 °C overnight.

Upon completion, the mixture was neutralized with IR-120,

ﬁltered, and concentrated. The residue was dissolved in 4 mL

of MeOH/THF/H₂O (2:1:1), and a drop of HCOOH and

20% Pd(OH)₂/C (96 mg) were added. The reaction mixture

was stirred under a H₂atmosphere for 36 h at rt, ﬁltered over a

Whatman ﬁlter paper, and concentrated under reduced

pressure. The obtained residue was puriﬁed by reverse-phase

column chromatography (Bond Eluct-C18) by elution with

H₂O. Fractions containing the product were pooled and

lyophilized to give free hexasaccharide 4 (17.5 mg, 42% over

5-Aminopnetyl β-D-Mannopyranosyl-(1→4)-α-D-gluco-

pyranosyl-(1→3)-β-^D-glucopyranosyl-(1→4)-β-D-mannopyr-

anosyl-[α-D-glucopyranosyluronate-(1→3)]-(1→4)-α-D-glu-

copyranoside (3). To a stirred solution of hexasaccharide 26

(37 mg, 0.0142 mmol) in a mixture of p-dioxane and H₂O (2

mL, p-dioxane/H₂O, 3:1 = v/v) was added LiOH·H₂O (71.5

mg, 0.17 mmol, 12 equiv). The reaction mixture was heated to

75 °C overnight. Upon completion, the mixture was

neutralized with IR-120, ﬁltered, and concentrated. The

residue was dissolved in 4 mL of MeOH/THF/H₂O (2:1:1),

and a drop of HCOOH and 20% Pd(OH)₂/C (37 mg) were

added. The reaction mixture was stirred under a H₂

atmosphere for 36 h at rt, ﬁltered over a Whatman ﬁlter

paper, and concentrated under reduced pressure. The obtained

residue was puriﬁed by reverse-phase column chromatography

(Bond Eluct-C18) by elution with H₂O. Fractions containing

the product were pooled and lyophilized to give free

pentasaccharide 3 (6 mg, 39% over two steps) in a white

powder. ¹H NMR (600 MHz, D₂O, 313 K): δ 5.45 (d, J = 3.9

1

two steps) in a white powder. H NMR (600 MHz, D₂O, 313

K): δ 5.44 (d, J = 3.6 Hz, 1H, C1H_α‑glc), 5.41 (d, J = 4.0 Hz,

1H, C1H_α‑glcA), 5.32 (d, J = 4.0 Hz, 1H, C1H_α‑glc‑A), 5.05

(d, J = 4.1 Hz, 1H, C1H_{α‑glc‑linker}), 4.92 (s, 1H, C1H_β‑man),

4.91 (s, 1H, C1H_β‑man), 4.68 (d, J = 8.1 Hz, 1H, C1

H

_β‑glc), 4.36−4.32 (m, 4H, 4 × CH_ring), 4.23−4.20 (m, 2H, 2 ×

CH_ring), 4.19−4.07 (m, 4H, 2 × CH₂), 4.05−3.93 (m,

9H, 2 × CH₂, 5 × CH_ring), 3.92−3.81 (m, 9H, 3 × 

CH_aH_b, 6 × CH_ring), 3.79−3.72 (m, 5H, 5 × CH_ring), 3.71−

3.67 (m, 5H, CH_aH_b, 4 × CH_ring), 3.61 (t, J = 7.1 Hz, 1H,

CH_ring), 3.54 (t, J = 8.6 Hz, 1H, CH_ring), 3.17 (t, J = 7.6

Hz, 2H, CH_2linker), 1.87−1.79 (m, 4H, 2 × CH_2linker),

1.64−1.59 (m, 2H, CH_2linker). ¹³C{¹H} NMR (150 MHz,

D₂O, 313 K): δ 175.4 (CO), 175.1 (CO), 102.5 (C1_β‑glc, ¹J_C−H

Hz, 1H, C1H_α‑glc), 5.30 (d, J = 4.0 Hz, 1H, C1H_α‑glcA

,

5.05 (d, J = 3.8 Hz, 1H, C1H_{α‑glc‑linker}), 4.90 (s, 2H, 2 × C1-

= 162.8 Hz), 101.7 (C1_α‑glcA

,

¹J_C−H= 172.7 Hz), 100.8

H

_β‑man), 4.68 (d, J = 7.9 Hz, 1H, C1H_β‑glc), 4.37 (d, J = 3.1

1

(C1_α‑glc‑A, J_C−H= 173.1 Hz), 100.0 (C1_β‑man, J_C−H= 162.2

Hz, 1H, CH_ring), 4.26−4.18 (m, 4H, 4 × CH_ring), 4.17−4.11

(m, 2H, CH₂), 4.10 (dd, J = 12.2, 2.0 Hz, 1H, CH_aH_b),

4.06 (t, J = 9.3 Hz, 1H, CH_ring), 4.01−3.95 (m, 5H, 2 × 

CH_ring, CH_aH_b, CH₂), 3.93−3.87 (m, 7H, CH_aH_b, 2 ×

CH_ring, 2 × CH₂), 3.85−3.77 (m, 4H, 4 × CH_ring), 3.76−

3.71 (m, 4H, 4 × CH_ring), 3.70−3.63 (m, 5H, 4 × CH_ring, 

CH_aH_b), 3.58−3.55 (m, 1H, CH_ring), 3.55 (t, J = 8.3 Hz, 1H,

CH_ring), 3.17 (t, J = 7.6 Hz, 2H, CH_2linker), 1.87−1.77 (m,

4H, 2 × CH_2linker), 1.63−1.58 (m, 2H, CH_2linker). ¹³C{¹H}

1

Hz), 99.8 (C1_β‑man, J_C−H= 162.2 Hz), 99.2 (C1_α‑glc, J_C−H

=

1

172.7 Hz), 98.1 (C1_{α‑glc‑linker}, J_C−H= 168.7 Hz), 82.6 (

CH_ring), 81.2 (CH_ring), 78.9 (CH_ring), 78.7 (CH_ring),

76.4 (CH_ring), 76.3 (CH_ring), 75.6 (CH_ring), 73.6 (

CH_ring), 72.7 (CH_ring), 72.3 (CH_ring), 72.1 (CH_ring),

71.9 (CH_ring), 71.8 × 2 (CH_ring), 71.7 (CH_ring), 71.6

(CH_ring), 71.5 (CH_ring), 71.1 (CH_ring), 70.9 (CH_ring),

70.7 (CH_ring), 70.5 (CH_ring), 70.4 (CH_ring), 68.1 (

CH_2linker), 66.0 (CH_ring), 61.1 (CH₂), 61.0 (CH₂), 60.3

(CH₂), 60.0 (CH₂), 59.9 (CH₂), 39.5 (CH_2linker),

28.2 (CH_2linker), 26.6 (CH_2linker), 22.6 (CH_2linker).

HRMS (ESI) m/z: [M + H]⁺calcd for C₄₇H₈₀NO₃₈,

1266.4353; found, 1266.4379.

NMR (150 MHz, D₂O, 313 K): δ 176.5 (CO), 102.5 (C1_β‑glc

,

¹J_C−H= 162.9 Hz), 101.7 (C1_α‑glc‑A, ¹J_C−H= 173.6 Hz), 100.13

(C1_β‑man, ¹J_C−H= 162.5 Hz), 99.9 (C1_β‑man, ¹J_C−H= 162.5 Hz),

1

99.2 (C1_α‑glc, J_C−H= 173.6 Hz), 98.0 (C1_{α‑glc‑linker}, J_C−H

=

173.6 Hz), 82.7 (CH_ring), 81.1 (CH_ring), 79.0 (CH_ring),

78.6 (CH_ring), 76.5 (CH_ring), 76.4 (CH_ring), 75.6 (

CH_ring), 73.7 (CH_ring), 73.0 × 2 (CH_ring), 72.3 × 2 (

CH_ring), 72.0 (CH_ring), 71.9 (CH_ring), 71.8 (CH_ring),

71.6 (CH_ring), 71.5 (CH_ring), 71.0 (CH_ring), 70.9 (

CH_ring), 70.7 (CH_ring), 70.5 (CH_ring), 70.4 (CH_ring),

68.1 (CH_2linker), 66.8 (CH_ring), 61.1 × 2 (CH₂), 60.2

(CH₂), 60.0 (CH₂), 59.9 (CH₂), 39.5 (CH_2linker),

28.2 (CH_2linker), 26.6 (CH_2linker), 22.6 (CH_2linker).

HRMS (ESI) m/z: [M + H]⁺calcd for C₄₁H₇₂NO₃₂,

1090.4032; found, 1090.4084.

5-Aminopnetyl-β-D-glucopyranosyl-(1→4)-β-D-manno-

pyranosyl-[α-^D-glucopyranosyl uronate-(1→3)]-(1→4)-α-D-

glucopyranosyl-(1→3)-β-^D-glucopyranosyl-(1→4)-β-D-man-

nopyranosyl-[α-^D-glucopyranosyluronate-(1→3)]-(1→4)-α-

D-glucopyranoside (5). To a stirred solution of octasaccharide

31 (35 mg, 0.010 mmol) in a mixture of p-dioxane and H₂O (2

mL, p-dioxane/H₂O, 3:1 = v/v) was added LiOH·H₂O (77

mg, 0.18 mmol, 18 equiv). The reaction mixture was heated to

75 °C overnight. Upon completion, the mixture was

neutralized with IR-120, ﬁltered, and concentrated. The

residue was dissolved in 4 mL of MeOH/THF/H₂O (2:1:1),

and a drop of HCOOH and 20% Pd(OH)₂/C (35 mg) were

added. The reaction mixture was stirred under a H₂

5-Aminopnetyl α-D-Glucopyranosyl Uronate-(1→3)-β-D-

mannopyranosyl-(1→4)-α-D-glucopyranosyl-(1→3)-β-D-glu-

copyranosyl-(1→4)-β-D-mannopyranosyl-[α-D-glucopyrano-

U

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J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

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Article

atmosphere for 36 h at rt, ﬁltered over a Whatman ﬁlter paper,

and concentrated under reduced pressure. The residue was

puriﬁed by reverse-phase column chromatography (Bond

Eluct-C18) by elution with H₂O. Fractions containing the

product were pooled and lyophilized to give free hexasacchar-

ide 5 (7 mg, 48% over two steps) in a white powder. ¹H NMR

(600 MHz, D₂O, 313 K): δ 5.45 (d, J = 3.8 Hz, 1H, C1

CH_ring, 2 × CH₂), 3.84−3.76 (m, 6H, 4 × CH_ring), 3.72−

3.66 (m, 5H, 4 × CH_ring), 3.61−3.55 (m, 1H, CH_ring), 3.41

(t, J = 6.5 Hz, 1H, CH_ring), 2.97 (t, J = 6.6 Hz, 2H, CH₂),

2.73 (t, J = 6.6 Hz, 2H, CH₂), 1.85−1.80 (m, 2H, 

CH_2linker), 1.75−1.71 (m, 2H, CH_2linker)1.62−1.52 (m, 2H,

CH_2linker), ¹³C{¹H} NMR (150 MHz, D₂O, 313 K): δ 176.4

1

(CO), 174.2 (CONH), 102.4 (C1_β‑glc, J_C−H= 162.9 Hz),

1

H

_{α‑glc‑linker}), 5.31 (t, J = 4.1 Hz, 2H, 2 × C1H_α‑glcA

,

101.6 (C1_α‑glc‑A, J_C−H= 173.6 Hz), 99.9 (C1_β‑man, J_C−H

=

,

1

overlapped), 5.05 (d, J = 3.8 Hz, 1H, C1H_α‑glc), 4.93 (s, 1H,

C1H_β‑man), 4.92 (s, 1H, C1H_β‑man), 4.68 (t, J = 8.2 Hz,

2H, 2 × C1H_β‑glc, overlapped), 4.38 (t, J = 3.0 Hz, 2H, 2 ×

CH_ring), 4.27−4.12 (m, 9H, 5 × CH_ring, 2 × CH₂OH),

4.06−3.84 (m, 17H, 8 × CH_ring, 4 × CH₂OH, CH_aH_blinker),

3.82−3.63 (m, 15H, 14 × CH_ring, CH_aH_blinker), 3.52 (t, J =

8.5 Hz, 1H, CH_ring), 3.47 (d, J = 10.2 Hz, 1H, CH_ring),

3.43 (d, J = 9.1 Hz, 1H, CH_ring), 3.18 (t, J = 7.7 Hz, 2H, 

CH_2linker), 1.86−1.77 (m, 4H, 2 × CH_2linker), 1.67−1.58 (m,

2H, CH_2linker). ¹³C{¹H} NMR (150 MHz, D₂O, 313 K): δ

162.5 Hz), 99.7 (C1_β‑man, J_C−H= 162.5 Hz), 99.1 (C1_α‑glc

1

¹J_C−H= 173.6 Hz), 97.8 (C1_{α‑glc‑linker}, J_C−H= 173.6 Hz), 82.4

(CH_ring), 80.9 (CH_ring), 78.8 (CH_ring), 78.4 (CH_ring),

76.4 (CH_ring), 76.3 (CH_ring), 75.4 (CH_ring), 73.5 (

CH_ring), 72.9 (CH_ring), 72.8 (CH_ring), 72.2 (CH_ring),

71.9 (CH_ring), 71.8 (CH_ring), 71.6 (CH_ring), 71.4 (

CH_ring), 71.3 (CH_ring), 70.9 (CH_ring), 70.8 (CH_ring),

70.5 (CH_ring), 70.3 (CH_ring), 70.2 (CH_ring), 68.2 (

CH_2linker), 66.6 (CH_ring), 60.9 × 2 (CH₂), 60.0 (CH₂),

59.9 (CH₂), 59.7 (CH_2linker), 39.4 (CH_2linker), 39.2 (

CH_2linker), 28.2 (CH_2linker), 28.05 (CH_2linker), 22.7 (

CH_2linker), 20.0 (CH_2linker). HRMS (ESI) m/z: [M + Na]⁺

calcd for C₄₄H₇₅NO₃₃SNa, 1200.3834; found, 1200.3860.

1

176.5 × 2 (CO), 102.6 (C1_β‑glc, J_C−H= 163.7 Hz), 102.4

(C1_β‑glc, ¹J_C−H= 163.7 Hz), 101.7 (C1_α‑glcA, ¹J_C−H= 171.4 Hz),

1

101.6 (C1_α‑glc‑A, J_C−H= 171.4 Hz), 99.9 (C1_β‑man, J_C−H

=

,

1

160.2 Hz), 99.7 (C C1_β‑man, J_C−H= 160.2 Hz), 99.2 (C1_α‑glc

¹J_C−H= 173.2 Hz), 98.0 (C1_{α‑glc‑linker}, J_C−H= 170.3 Hz), 82.5

(CH_ring), 81.3 (CH_ring), 81.0 (CH_ring78.9 (CH_ring),

78.8 (CH_ring), 76.8 (CH_ring), 76.4 (CH_ring), 75.6 × 3

(CH_ring), 73.7 (CH_ring), 73.6 (CH_ring), 73.0 × 2 (

CH_ring), 72.4 (CH_ring), 72.3 (CH_ring), 72.1 (CH_ring),

72.0 (CH_ring), 71.9 × 2 (CH_ring), 71.7 (CH_ring), 71.6

(CH_ring), 71.5 (CH_ring), 71.1 (CH_ring), 70.9 × 2 (

CH_ring), 70.5 × 2 (CH_ring), 70.4 (CH_ring), 70.1 (

CH_ring), 68.1 (CH_2linker), 61.3 (CH₂), 61.2 (CH₂), 60.3

(CH₂), 60.1 × 2 (CH₂), 59.9 (CH₂), 39.5 (

CH_2linker), 28.2 (CH_2linker), 26.6 (CH_2linker), 22.6 (

CH_2linker). HRMS (ESI) m/z: [M + H]⁺calcd for C₅₃H₉₀NO₄₃,

1428.4881; found, 1428.4924.

1

3-Mercapto-N-propanamidopentyl-α-D-glucopyranosy-

luronate-(1→3)-β-^D-mannopyranosyl-(1→4)-α-D-glucopyra-

nosyl-(1→3)-β-^D-glucopyranosyl-(1→4)-β-D-mannopyrano-

syl-[α-^D-glucopyranosyluronate-(1→3)]-(1→4)-α-D-gluco-

pyranoside (4a). To a solution of free amine 4 (15 mg, 0.0118

mmol, 1 equiv) in PBS buﬀer (pH 7.4, 1 mL) was added 3,3′-

dithiobis(sulfosuccinimidylpropionate (DTSSP) (29 mg,

0.0475 mmol, 4 equiv) at room temperature. The pH value

of the reaction mixture was adjusted to 7 by adding 1 N NaOH

(aq) dropwise every 20 min up to 1 h, and the mixture was

stirred overnight. Then, dithiothreitol (DTT) (2.7 mg, 0.177

mmol, 15 equiv) was added to the reaction mixture, which was

stirred at 40 °C for another 2 h. The solvent was evaporated

under reduced pressure, and the residue was puriﬁed by

Sephadex LH-20 column chromatography using H₂O as an

eluent. Fractions containing the product were pooled and

lyophilized to give the desired thiolate compound 4a (14 mg,

88%) in a white powder. ¹H NMR (600 MHz, D₂O, 313 K): δ

5.44 (d, J = 3.6 Hz, 1H, C1H_α‑glc), 5.40 (d, J = 3.6 Hz, 1H,

C1H_α‑glcA), 5.30 (d, J = 3.8 Hz, 1H, C1H_α‑glc‑A), 5.04 (d, J

= 3.5 Hz, 1H, C1H_{α‑glclinker}), 4.92 (s, 1H, C1H_β‑man), 4.91

(s, 1H, C1H_β‑man), 4.68 (d, J = 8.2 Hz, 1H, C1H_β‑glc),

4.37 (s, 1H, CH_ring), 4.33 (s, 1H, CH_ring), 4.25−4.20 (m,

4H, 4 × CH_ring), 4.18−4.13 (m, 2H), 4.11−4.02 (m, 3H),

4.01−3.82 (m, 17H), 3.82−3.72 (m, 7H), 3.68−3.65 (m, 6H),

3.63 (t, J = 9.4 Hz, 1H, CH_ring), 3.54 (t, J = 8.2 Hz, 1H, 

CH_ring), 3.37 (t, J = 6.9 Hz, 2H, CH_2linker), 2.94 (t, J = 6.6

Hz, 2H, CH_2linker), 2.69 (t, J = 6.6 Hz, 2H, CH_2linker),

1.83−1.75 (m, 2H, CH_2linker), 1.72−1.67 (m, 2H, 

CH_2linker), 1.60−1.52 (m, 2H, CH_2linker). ¹³C{¹H} NMR

(150 MHz, D₂O, 313 K): δ 176.4 (CO), 176.3 (CO), 174.1

(CONH), 102.4 (C1_β‑glc), 101.6 (C1_α‑glcA), 100.6 (C1_α‑glc‑A),

99.8 (C1_β‑man), 99.6 (C1_β‑man), 99.1 (C1_α‑glc), 97.7

(C1_{α‑glc‑linker}), 82.3 (CH_ring), 81.1 (CH_ring), 8.06 (

CH_ring), 78.7 (CH_ring), 78.4 (CH_ring), 76.3 (CH_ring),

76.1 (CH_ring), 75.4 (CH_ring), 73.5 (CH_ring), 72.9 (

3-Mercapto-N-propanamidopentyl-β-D-mannopyranosyl-

(1→4)-α-^D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→

4)-β-^D-mannopyranosyl-[α-D-glucopyranosyluronate-(1→

3)]-(1→4)-α-D-glucopyranoside (3a). To a solution of free

amine 3 (3.4 mg, 0.0031 mmol, 1 equiv) in PBS buﬀer (pH

7 . 4 ,

1

m L ) w a s a d d e d 3 , 3 ′ - d i t h i o b i s -

(sulfosuccinimidylpropionate (DTSSP) (7.6 mg, 0.0124

mmol, 4 equiv) at room temperature. The pH value of the

reaction mixture was adjusted to 7 by adding 1 N NaOH (aq)

dropwise every 20 min up to 1 h, and the mixture was stirred

overnight. Then, dithiothreitol (DTT) (7.2 mg, 0.0465 mmol,

15 equiv) was added to the reaction mixture, which was stirred

at 40 °C for another 2 h. The solvent was evaporated under

reduced pressure, and the residue was puriﬁed by Sephadex

LH-20 column chromatography using H₂O as an eluent.

Fractions containing the product were pooled and lyophilized

to give the desired thiolate compound 3a (2.94 mg, 80%) in a

white powder. ¹H NMR (600 MHz, D₂O, 313 K): δ 5.48 (d, J

= 3.1 Hz, 1H, C1H_α‑glc), 5.34 (d, J = 2.9 Hz, 1H, C1

H

_α‑glcA, 5.08 (d, J = 3.0 Hz, 1H, C1H_{α‑glc‑linker}), 4.94 (s, 2H, 2

× C1H_β‑man), 4.72 (d, J = 7.7 Hz, 1H, C1H_β‑glc), 4.41 (s,

1H, CH_ring), 4.28−4.23 (m, 4H, 4 × CH_ring), 4.19−4.06 (m,

4H), 4.05−3.99 (m, 5H), 3.97−3.87 (m, 7H, CH_aH_b, 2 ×

V

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CH_ring), 72.6 × 2 (CH_ring), 72.2 (CH_ring), 72.2 (

CH_ring), 71.9 (CH_ring), 71.9 (CH_ring), 71.8 (CH_ring),

71.6 (CH_ring), 71.5 (CH_ring), 71.4 (CH_ring), 71.2 (

CH_ring), 70.9 (CH_ring), 70.7 (CH_ring), 70.5 (CH_ring),

70.3 (CH_ring), 70.2 (CH_ring), 70.1 (CH_ring), 68.2 (

CH_2linker), 65.9 (CH_ring), 60.9 (CH₂), 60.8 (CH₂), 59.9

(CH₂), 59.8 (CH₂), 59.6 (CH₂), 39.4 (CH_2linker),

39.1 (CH_2linker), 28.2 (CH_2linker), 28.0 (CH_2linker), 22.7

(CH_2linker), 20.03 (CH_2linker). HRMS (ESI) m/z: [M +

Na]⁺calcd for C₅₀H₈₃NO₃₉SNa, 1376.4155; found, 1376.4184.

Phenyl 2-O-Benzyl-4,6-O-benzylidene-3-p-methoxyben-

zyl-1-thio-α-^D-mannopyranoside (11). To a suspension of

NaH (0.9 g, 22.5 mmol, 1.75 equiv) in dry DMF (60 mL) was

added alcohol compound S19 (6 g, 12.86 mmol) dissolved in

dry DMF (35 mL) at 0 °C under an argon atmosphere. The

reaction mixture was stirred for 10 min; then, benzyl bromide

(2.3 mL, 19.2 mmol, 1.5 equiv) was added dropwise. The

mixture was stirred for another 2 h at room temperature. Upon

completion, the reaction was carefully quenched with MeOH

at 0 °C, and the mixture was concentrated in vacuo. The

residue was diluted with ice-cold water, and the organic

material was extracted with Et₂O (3 × 60 mL). The combined

organic layers were dried over MgSO₄, ﬁltered, and

concentrated in vacuo. The resulting residue was puriﬁed by

ﬂash column chromatography on silica gel (EtOAC/hexanes,

100% hexane followed by1:5) to yield compound 11 (7.25 g,

99%) in a viscus oily compound. ¹H NMR (600 MHz, CDCl₃,

298 K): δ 7.55 (dd, J = 7.9, 1.4 Hz, 2H, ArH), 7.43−7.28

(m, 15H, ArH), 6.90 (d, J = 8.7 Hz, 2H, ArH), 5.67 (s,

1H), 5.52 (brd, J = 1.3 Hz, 1H), 4.78 (d, J = 11.7 Hz, 1H),

4.78 (s, 2H), 4.63 (d, J = 11.7 Hz, 1H), 4.33−4.28 (m, 2H),

4.25−4.23 (m, 1H), 4.03 (dd, J = 3.2, 1.2 Hz, 1H), 3.98 (dd, J

= 9.6, 3.2 Hz, 1H), 3.92 (m, 1H), 3.84 (s, 3H, OCH₃).

¹³C{¹H} NMR (150 MHz, CDCl₃, 298 K): δ 159.4 (ArC),

137.9 (ArC), 137.8 (ArC), 133.9 (ArC), 131.8 (Ar

C), 130.6 (ArC), 129.6 (ArC), 129.3 (ArC), 129.1

(ArC), 128.6 (ArC), 128.4 (ArC), 128.3 (ArC),

128.1 (ArC), 127.8 (ArC), 126.3 (ArC), 113.9 (Ar

C), 101.7 (PhCH), 87.3 (C1_{α‑man‑SPh}), 79.2 (CH_ring), 78.3

(CH_ring), 76.0 (CH_ring), 73.2 (CH₂), 72.9 (CH₂),

68.7 (CH₂), 65.7 (CH_ring), 55.5 (OCH₃). HRMS (ESI)

m/z: [M + Na]⁺calcd for C₃₄H₃₄O₆SNa, 593.1968; found,

593.2000.

3-Mercapto-N-propanamidopentyl-β-D-glucopyranosyl-

(1→4)-β-^D-mannopyranosyl-[α-D-glucopyranosyl uronate-

(1→3)]-(1→4)-α-^D-glucopyranosyl-(1→3)-β-D-glucopyrano-

syl-(1→4)-β-^D-mannopyranosyl-[α-D-glucopyranosyluro-

nate-(1→3)]-(1→4)-α-D-glucopyranoside (5a). To a solution

of free amine 5 (9 mg, 0.0063 mmol, 1 equiv) in PBS buﬀer

(pH 7. 4,

1 mL) was added 3, 3 ′-dithiobis-

(sulfosuccinimidylpropionate (DTSSP) (15.3 mg, 0.025

mmol, 4 equiv) at room temperature. The pH value of the

reaction mixture was adjusted to 7 by adding 1 N NaOH (aq)

dropwise every 20 min up to 1 h, and the mixture was stirred

overnight. Then, dithiothreitol (DTT) (14.5 mg, 0.094 mmol,

15 equiv) was added to the reaction mixture, which was stirred

at 40 °C for another 2 h. The solvent was evaporated under

reduced pressure, and the residue was puriﬁed by Sephadex

LH-20 column chromatography using H₂O as an eluent.

Fractions containing the product were pooled and lyophilized

to give the desired thiolate compound 5a (7 mg, 84%) in a

white powder. ¹H NMR (600 MHz, D₂O, 313 K): δ 5.45 (d, J

= 3.3 Hz, 1H, C1H_α‑glc), 5.31 (br s, J = 3.5 Hz, 2H, 2 ×

C1H_α‑glcA, overlapped), 5.05 (d, J = 3.5 Hz, 1H, C1

H_α‑glc), 4.93 (s, 1H, C1H_β‑man), 4.92 (s, 1H, C1H_β‑man),

4.68 (like t, J = 8.3 Hz, 2H, 2 × C1H_β‑glc, overlapped), 4.38

(s, 2H, 2 × CH_ring), 4.26−4.12 (m, 9H, 5 × CH_ring, 2 × 

CH₂OH), 4.07−3.85 (m, 17H, 8 × CH_ring, 4 × CH₂OH, 

CH_aH_blinker), 3.83−3.74 (m, 6H, CH_aH_blinker, 5 × CH_ring),

3.78−3.64 (m, 8H, 8 × CH_ring), 3.54 (t, J = 8.8 Hz, 1H, 

CH_ring), 3.47−3.42 (m, 2H, 2 × CH_ring), 3.38 (t, J = 6.9 Hz,

2H, CH_2linker), 2.95 (t, J = 6.6 Hz, 2H, CH_2linker), 2.71 (t, J

= 6.6 Hz, 2H, CH_2linker), 1.83−1.77 (m, 2H, CH_2linker),

1.73−1.68 (m, 2H, CH_2linker), 1.607−1.52 (m, 2H, 

CH_2linker). ¹³C{¹H} NMR (150 MHz, D₂O, 313 K): δ 176.5 ×

2 (CO), 174.3 (CONH), 102.5 (C1_β‑glc), 102.4 (C1_β‑glc), 101.7

(C1_α‑glcA), 101.6 (C1_α‑glc‑A), 99.9 (C1_β‑man), 99.8 (C1_β‑man),

99.2 (C1_α‑glc), 98.0 (C1_{α‑glc‑linker}), 82.6 (CH_ring), 81.1 (

CH_ring), 80.9 (CH_ring79.0 (CH_ring), 78.7 (CH_ring), 76.8

(CH_ring), 76.4 (CH_ring), 75.6 × 3 (CH_ring), 73.7 (

CH_ring), 73.6 (CH_ring), 73.5 (CH_ring), 73.0 × 2 (

CH_ring), 72.4 (CH_ring), 72.3 × 2 (CH_ring), 72.1 × 3 (

CH_ring), 71.9 × 3 (CH_ring), 71.8 (CH_ring), 71.6 (

CH_ring), 71.5 (CH_ring), 70.9 (CH_ring), 70.5 (CH_ring),

70.4 (CH_ring), 70.1 (CH_ring), 68.4 (CH_2linker), 61.3 (

CH₂), 61.2 (CH₂), 60.3 (CH₂), 60.1 × 2 (CH₂), 59.9

(CH₂), 39.4 (CH_2linker), 39.6 (CH_2linker), 28.3 (

CH_2linker), 28.2 (CH_2linker), 22.9 (CH_2linker), 20.2 (

CH_2linker). HRMS (ESI) m/z: [M + Na]⁺calcd for

C₅₆H₉₃NO₄₄SNa, 1538.4683; found, 1538.4717.

p-Methylphenyl 2-Benzoyl-4,6-O-benzylidene-3-p-me-

thoxybenzyl-1-thio-β-^D-glucopyranoside (9). Compound

S21 (6.74 g, 13.63 mmol) was dissolved in pyridine (60

mL), and DMAP (167 mg, 0.1 equiv) was added. Then,

benzoyl chloride (3.17 mL, 27.25 mmol, 2 equiv) was added

dropwise over 5 min at 0 °C. The reaction mixture was

warmed to rt and stirred for 1 h. Upon completion, methanol

was added at 0 °C to decompose the excess BzCl, and the

reaction mixture was concentrated in vacuo. The obtained

white solid was dissolved in CH₂Cl₂(100 mL) and washed

with 1 N HCl (2 × 30 mL), satd aq NaHCO₃(2 × 25 mL),

and water (25 mL). The organic layer was dried over MgSO₄

and concentrated. The obtained white solid was suspended in

CH₂Cl₂/hexane (1:3), and the title compound 9 was obtained

1

after ﬁltration in a white solid (7.7 g, 94%). H NMR (600

MHz, CDCl₃, 298 K): δ 7.99 (d, J = 7.5 Hz, 2H, ArH),

7.65−7.29 (m, 10H, ArH), 7.06 (d, J = 7.8 Hz, 2H, ArH),

7.00 (d, J = 8.5 Hz, 2H, ArH), 6.55 (d, J = 8.5 Hz, 2H, Ar

H), 5.57 (s, 1H), 5.21 (t, J = 9.1 Hz, 1H), 4.75 (d, J = 10.1 Hz,

1H), 4.71 (d, J = 11.6 Hz, 1H), 4.57 (d, J = 11.6 Hz, 1H), 4.40

(dd, J = 10.6, 5.0 Hz, 1H), 3.84−3.79 (m, 2H), 3.77 (t, J = 9.3

Hz, 1H), 3.66 (s, 3H, OCH₃), 3.53−3.49 (m, 1H), 2.30 (s,

3H, CH₃). ¹³C{¹H} NMR (150 MHz, CDCl₃, 298 K): δ

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165.1 (CO), 159.3 (ArC), 138.7 (ArC), 137.4 (Ar

C), 133.8 (ArC), 133.3 (ArC), 130.1 × 2 (ArC), 130.0

(ArC), 129.9 (ArC), 129.8 (ArC), 129.2 (ArC),

128.5 × 2 (ArC), 128.4 (ArC), 126.2 (ArC), 113.7

(ArC), 101.4 (PhCH), 87.3 (C1_β‑glc), 81.6 (CH_ring),

78.9 (CH_ring), 74.0 (CH₂), 72.2 (CH_ring), 70.8 (

CH_ring), 68.8 (CH₂), 55.3 (OCH₃), 21.3 (CH₃).

HRMS (ESI) m/z: [M + Na]⁺calcd for C₃₅H₃₄O₇SNa,

621.1917; found, 621.1937.

S1 (2.71 g, 5.53 mmol, 0.75 equiv) in CH₂Cl₂(25 mL) was

added slowly at the same temperature. The reaction was stirred

for another 3 h at the same temperature and, then, allowed to

reach room temperature before it was diluted with CH₂Cl₂,

ﬁltered, and washed with excess CH₂Cl₂. The organic layer was

washed with saturated sodium bicarbonate solution and brine,

then dried over sodium sulfate, evaporated to dryness, and

puriﬁed by ﬂash chromatography over silica gel (EtOAc/

hexanes, 1:5) to aﬀorded the corresponding α-anomer S2b

(365 mg, viscous oil) and β-anomer S2a (3.653 g, viscous oil)

compounds in 77% yield (yields based on the acceptor used).

¹H NMR (600 MHz, CDCl₃, 298 K): δ 7.51 (d, J = 7.2 Hz,

2H, ArH), 7.43−7.25 (m, 25H, ArH), 6.88 (d, J = 7.7 Hz,

2H, ArH), 6.02−5.97 (m, 1H, CHCH₂), 5.55 (s, 1H,

PhCH), 5.39 (d, J = 17.1 Hz, 1H, CH_aH_b), 5.25 (d, J = 10.5

Hz, 1H, CH_aH_b), 5.07 (d, J = 10.8 Hz, 1H, ArCH_aH_b),

4.94 (d, J = 10.8 Hz, 1H, ArCH_aH_b), 4.88 (d, J = 11.9 Hz,

1H, ArCH_aH_b), 4.83 (d, J = 11.9 Hz, 1H, ArCH_aH_b), 4.76

(d, J = 10.6 Hz, 2H, ArCH₂), 4.69 (d, J = 11.9 Hz, 1H, Ar

CH_aH_b), 4.65 (d, J = 11.9 Hz, 1H, ArCH_aH_b), 4.55−4.53

(m, 2H, C1−H_β‑man(overlapped), ArCH_aH_b), 4.48−4.42

(m, 3H, C1−H_β‑glc, Ar−CH_aH_b, CH_aH_b), 4.20 (dd, J = 12.7,

5.6 Hz, 1H, CH_aH_b), 4.12−4.07 (m, 2H, CH_ring, 

CH_aH_b), 3.97 (t, J = 9.2 Hz, 1H, CH_ring), 3.81 (s, 3H, 

OCH₃), 3.73 (s, 1H, CH_ring), 3.68 (d, J = 10.7 Hz, 1H, 

CH_aH_b), 3.62−3.56 (m, 3H, CH_ring, CH₂), 3.50 (t, J = 8.3

Hz, 1H, CH_ring), 3.43 (d, J = 9.7 Hz, 1H, CH_ring), 3.38 (d,

J = 9.7 Hz, 1H, CH_ring), 3.14 (m, 1H, CH_ring). ¹³C{¹H}

NMR (150 MHz, CDCl₃, 298 K): δ 159.3 (ArC), 139.3

(ArC), 138.8 (ArC), 138.6 (ArC), 138.0 (ArC),

137.8 (ArC), 134.2 (CH), 130.7 (ArC), 129.2 (Ar

C), 129.0 (ArC), 128.6 (ArC), 128.5 (ArC), 128.4

(ArC), 128.3 (ArC), 128.2 (ArC), 128.1 (ArC),

129.0 (ArC), 127.9 (ArC), 127.8 × 2 (2 × ArC), 127.6

(ArC), 127.4 (ArC), 126.3 (ArC), 117.4 (=CH₂),

5-Azidopentyl 2,3,6-Tri-O-benzyl-α-D-glucopyranoside

(12). A mixture of starting material S24 (6.5 g, 11.62 mmol,

1 equiv) and 4 Å molecular sieves (7 g) in CH₂Cl₂(150 mL)

was stirred under argon for 1 h. The reaction was cooled to

−78 °C, and triethylsilane (5.6 mL, 34.86 mmol, 3 equiv) and

triﬂuoroacetic acid (3.6 mL, 40.67 mmol, 3.5 equiv) were

added. The mixture was stirred at −78 °C until TLC analysis

indicated the disappearance of the starting material (1 h). The

reaction mixture was quenched by the addition of satd aq

NaHCO₃(10 mL) and, then, slowly warmed to rt. The

mixture was ﬁltered, and the ﬁltrate was washed with saturated

NaHCO₃(50 mL) and brine. The organic phase was dried

over MgSO₄, ﬁltered, and concentrated under reduced

pressure. The resulting residue was puriﬁed by ﬂash

chromatography over silica gel (EtOAc/hexanes, 1:4) to give

pure product 12 (5.07 g, 78%) as a white solid. ¹H NMR (600

MHz, CDCl₃, 298 K): δ 7.36−7.23 (m, 15H, ArH), 5.00 (d,

J = 11.4 Hz, 1H, ArCH_aH_b), 4.74−4.71 (m, 3H, 2 × Ar

CH_aH_b, C1H (overlapped), 4.62 (d, J = 12.0 Hz, 1H, Ar

CH_aH_b), 4.57 (d, J = 12.2 Hz, 1H, ArCH_aH_b), 4.53 (d, J =

12.2 Hz, 1H, ArCH_aH_b), 3.79 (t, J = 9.2 Hz, 1H, CH_ring),

3.72−3.70 (m, 1H, CH_ring), 3.65−3.61 (m, 3H, 

CH_aH_blinker, CH₂OBn), 3.60 (t, J = 9.2 Hz, 1H, CH_ring),

3.52 (dd, J = 9.6, 3.6 Hz, 1H, CH_ring), 3.41 (dt, J = 9.7, 6.2

Hz, 1H, CH_aH_blinker), 3.23 (t, J = 6.9 Hz, 2H, CH_2linker),

2.36 (brs, 1H,-OH), 1.67−1.57 (m, 4H, 2 × CH_2linker),

1.46−1.41 (m, 2H, CH_2linker). ¹³C{¹H} NMR (150 MHz,

CDCl₃, 298 K): δ 139.1 (ArC), 138.4 (ArC), 138.2 (Ar

C), 128.7 (ArC), 128.6 (ArC), 128.5 (ArC), 128.2

(ArC), 128.1 × 2 (ArC), 128.0 (ArC), 127.8 × 2 (Ar

C), 97.2 (C1_{α‑glc‑linker}), 81.6 (CH_ring), 79.9 (CH_ring), 75.5

(ArCH₂), 73.7(ArCH₂), 73.1 (ArCH₂), 71.0 (

CH_ring), 70.2 (CH_ring), 69.7 (CH₂OBn), 68.1 (

CH_2linker), 51.5 (CH_2linker), 29.1 (CH_2linker), 28.8 (

CH_2linker), 23.6 (CH_2linker). HRMS (ESI) m/z: [M + Na]⁺

calcd for C₃₂H₃₉N₃O₆Na, 584.2764; found, 584.2731.

113.9 (ArC), 102.9 (C1_β‑glc

,

¹J_C−H= 160.1 Hz), 101.9

1

(C1_β‑man, J_C−H= 157.8 Hz), 101.5 (PhCH), 83.1 (CH_ring),

81.9 (CH_ring), 78.8 (CH_ring), 78.2 (CH_ring), 78.1 (

CH_ring), 77.2 (CH_ring), 75.4 (CH₂), 75.2 (CH₂), 75.1

(CH₂), 74.8 (CH_ring), 73.7 (CH₂), 72.4 (CH₂), 70.5

(CH₂), 68.9 (CH₂), 68.7 (CH₂), 67.5 (CH_ring), 55.4

(OCH₃). HRMS (ESI) m/z: [M + Na]⁺calcd for

C₅₈H₆₂O₁₂Na, 973.4133; found, 973.4139.

Allyl 2-O-Benzyl-3-O-p-methoxybezyl-4,6-O-benzylidene-

α-^D-mannopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-gluco-

1

pyranoside (S2b). H NMR (600 MHz, CDCl₃, 298 K): δ

7.53 (d, J = 6.6 Hz, 2H, ArH), 7.40−7.13 (m, 25H, ArH),

6.86 (d, J = 8.3 Hz, 2H, ArH), 6.02−5.96 (m, 1H, CH

CH₂), 5.63 (s, 1H, PhCH), 5.39 (d, J = 15.9 Hz, 1H, 

CHaH_b), 5.31 (s, 1H, C1H_α‑man), 5.25 (d, J = 9.26 Hz, 1H,

CH_aH_b), 5.07 (d, J = 11.4 Hz, 1H, ArCH_aH_b), 4.96 (d, J

= 10.8 Hz, 1H, ArCH_aH_b), 4.75 (d, J = 11.8 Hz, 1H, Ar

CH_aH_b), 4.67 (dd, J = 12.6, 8.6 Hz, 2H, ArCH₂), 4.60 (d, J

= 12.1 Hz, 1H, ArCH_aH_b), 4.58 (d, J = 11.9 Hz, 1H, Ar

CH_aH_b), 4.55 (d, J = 11.7 Hz, 1H, ArCH_aH_b), 4.49 (d, J =

7.5 Hz, 1H, C1H_β‑glc), 4.46 (dd, J = 12.6, 7.7 Hz, 1H, Ar

CH_aH_b), 4.41 (d, J = 11.9 Hz, 1H, ArCH_aH_b), 4.24−4.13

(m, 4H, 3 × CH_aH_b, CH_ring), 3.92 (dd, J = 9.8, 3.5 Hz,

Allyl 2-O-Benzyl-3-O-p-methoxybezyl-4,6-O-benzylidene-

β-^D-mannopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-D-gluco-

pyranoside (S2a). To a stirred solution of thioglycoside 11

(4.2 g, 7.36 mmol, 1 equiv) were added BSP (1.69 g, 8.1 mmol,

1.1 equiv), TTBP (3.66 g, 14.73 mmol, 2 equiv), and activated

4 Å molecular sieves (9 g) in CH₂Cl₂(120 mL). The reaction

mixture was stirred at rt for 10−15 min under an argon

atmosphere and, then, cooled to −60 °C before Tf₂O (1.49

mL, 8.84 mmol, 1.2 equiv) was added. After stirring for

another 10−15 min at −60 °C, a solution of glucosyl acceptor

X

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1H, CH_ring), 3.88−3.85 (m, 1H, CH_ring), 3.83−3.78 (m,

6H, OCH₃, CH_ring, OCH₂), 3.74−3.71 (m, 2H, 

CH_ring, CH_aH_b), 3.58 (t, J = 9.0 Hz, 1H, CH_ring), 3.53 (t,

1H, CH_ring), 3.47 (m, 1H, CH_ring). ¹³C{¹H} NMR (150

MHz, CDCl₃, 298 K): δ 159.3 (ArC), 138.6 (ArC), 138.5

(ArC), 138.4 (ArC), 138.3 (ArC), 137.9 (ArC),

134.2 (CH), 131.0 (ArC), 129.3 (ArC), 128.9 (Ar

C), 128.6 (ArC), 128.5 × 3 (ArC), 128.3 × 2 (ArC),

127.9 (ArC), 127.8 (ArC), 127.7 × 2 (ArC), 127.5

(ArC), 126.9 (ArC), 126.3 (ArC), 117.4 (=CH₂),

CH₂), 67.10 (CH_ring). HRMS (ESI) m/z: [M + Na]⁺calcd

for C₅₀H₅₄O₁₁Na, 853.3558; found, 853.3563.

Allyl 2,3,4-Tri-O-benzyl-6-O-tert-butyldiphenylsilyl-α-D-

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

mannopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-^D-glucopyra-

noside (S4). A mixture of the acceptor S3 (1.21 g, 1.45 mmol),

donor 10 (1.622 g, 2.04 mmol, 1.5 equiv), and activated 4 Å

molecular sieves (3 g) in anhydrous CH₂Cl₂(75 mL) was

stirred under an argon atmosphere for 1 h. The reaction

mixture was cooled to −40 °C; then, NIS (550 mg, 2.45 mmol,

1.2 equiv with respect to the donor) and TfOH (0.5 M in

Et₂O, 1.25 mL, 0.61 mmol, 0.3 equiv with respect to the

donor) were added into the mixture. The stirring continued

until TLC analysis (EtOAc/hexanes) indicated the disappear-

ance of the starting materials (2 h). Upon completion, the

reaction was quenched by the addition of NEt₃(100 μL), and

the reaction mixture was slowly warmed to room temperature

before it was ﬁltered through a pad of Celite, and the ﬁltrate

was concentrated under reduced pressure. The resulting

residue was puriﬁed by ﬂash chromatography over silica gel

(EtOAc/hexanes 1:5) to obtain pure product S4 (2.05 g, 95%)

113.8 (ArC), 102.7 (C1_β‑glc

,

¹J_C−H= 160.1 Hz), 101.6

1

(PhCH), 101.2 (C1_α‑man, J_C−H= 173.6 Hz), 84.5 (CH_ring),

82.3 (CH_ring), 79.1 (CH_ring), 77.9 (CH_ring), 76.7 (

CH_ring), 76.1 (CH_ring), 74.9 (CH₂), 74.8 (CH₂), 74.6

(CH_ring), 73.7 (CH₂), 73.4 (CH₂), 72.8 (CH₂), 70.5

(CH₂), 69.5 (CH₂), 68.8 (CH₂), 65.4 (CH_ring), 55.4

(OCH₃). HRMS (ESI) m/z: [M + Na]⁺calcd for

C₅₈H₆₂O₁₂Na, 973.4133; found, 973.4141.

Allyl 2-O-Benzyl-4,6-O-benzylidene-β-D-mannopyranosyl-

(1→4)-2,3,6-tri-O-benzyl-α-^D-glucopyranoside (S3). To a

stirred solution of starting material S2a (1.67 g, 1.83 mmol,

1 equiv) in CH₂Cl₂/Phosphate buﬀer (pH = 7, 60 mL, 9:1 =

v/v), DDQ (625 mg, 2.75 mmol, 1.5 equiv) was added at 0 °C.

The reaction mixture was vigorously stirred until TLC analysis

(EtOAc/hexanes) indicated the disappearance of starting

material (2 h). Upon completion, the reaction mixture was

diluted with CH₂Cl₂(30 mL) and washed with satd aq

NaHCO₃and brine. The organic phase was washed with water

until the solution colorless became colorless; then, the organic

solution was dried over MgSO₄, ﬁltered, and concentrated

under reduced pressure. The resulting residue was puriﬁed by

ﬂash chromatography over silica gel (EtOAc/hexanes, 1:3) to

obtain pure compound S3 (1.215 g, 80%) as a white foam. ¹H

NMR (600 MHz, CDCl₃, 298 K): δ 7.50 (d, J = 8.0 Hz, 2H,

ArH), 7.42−7.28 (m, 23H, ArH), 6.03−5.97 (m, 1H, 

CHCH₂), 5.48 (s, 1H, PhCH), 5.40 (dd, J = 17.5, 1.6 Hz,

1H, CH_aH_b), 5.26 (dd, J = 10.2, 1.0 Hz, 1H, CHaH_b),

5.06 (d, J = 10.7 Hz, 1H, ArCH_aH_b), 4.98 (d, J = 11.5 Hz,

1H, ArCH_aH_b), 4.95 (d, J = 10.8 Hz, 1H, ArCH_aH_b), 4.77

(m, 3H, 3 × ArCH_aH_b), 4.65−4.63 (m, 2H, C1H_β‑man

(overlapped), ArCH_aH_b), 4.51−4.45 (m, 3H, C1H_β‑glc

(overlapped), ArCH_aH_b, OCH_aH_b), 4.21−4.17 (m, 1H,

OCH_aH_b), 4.12 (dd, J = 10.3, 4.7 Hz, 1H, CH_ring), 4.03

(t, J = 9.2 Hz, 1H, CH_ring), 3.75−3.72 (m, 2H, CH_ring, 

CH_aH_b), 3.70−3.67 (m, 2H, CH_ring, CH_aH_b), 3.60 (t, 1H,

J = 9.0 Hz, CH_ring), 3.58−3.50 (m, 3H, CH₂, CH_ring),

3.54−3.41(m, 1H, CH_ring), 3.13−3.09 (m, 1H, CH_ring),

2.38 (d, J = 8.5 Hz, 1H, OH). ¹³C{¹H} NMR (150 MHz,

CDCl₃, 298 K): δ 139.3 (ArC), 138.6 (ArC), 138.3 (Ar

C), 137.8 (ArC), 137.4 (ArC), 134.2 (CH), 129.3

(ArC), 128.8 (ArC), 128.6 (ArC), 128.5 (ArC),

128.4 (ArC), 128.3 (ArC), 128.3 (ArC), 128.2 × 2

(ArC), 128.1 (ArC), 127.8 (ArC), 127.5 (ArC),

126.5 (ArC), 117.5 (=CH₂), 102.8 (C1_β‑glc), 102.2

(C1_β‑man), 101.9 (PhCH), 83.2 (CH_ring), 81.8 (CH_ring),

79.3 (CH_ring), 79.1 (CH_ring), 78.2 (CH_ring), 75.9 (

CH₂), 75.5 (CH₂), 75.2 (CH₂), 74.9 (CH_ring), 73.8 (

CH₂), 71.1 (CH_ring), 70.4 (CH₂), 68.7 (CH₂), 68.7 (

1

as a white foam. H NMR (600 MHz, CDCl₃, 298 K): δ 7.68

(d, J = 6.7 Hz, 2H, ArH), 7.64 (d, J = 6.7 Hz, 2H, ArH),

7.45 (d, J = 6.6 Hz, 2H, ArH), 7.37−7.11 (m, 42H, ArH),

7.08 (d, J = 6.9 Hz, 2H, ArH), 5.97−5.90 (m, 1H, CH),

5.45 (d, J = 3.6 Hz, 1H, C1H_α‑glc), 5.33 (m, 1H, CHaH_b),

5.27 (s, 1H, PhCH), 5.19 (m, 1H, CHaH_b), 4.98 (d, J = 10.9

Hz, 1H, ArCHaH_b), 4.91−4.87 (m, 3H, 3 × ArCH_aH_b),

4.83 (d, J = 11.2 Hz, 1H, ArCH_aH_b), 4.74 (dd, J = 11.2, 2.7

Hz, 2H, ArCH₂), 4.69 (dd, J = 11.2, 2.7 Hz, 2H, ArCH₂),

4.60 (d, J = 10.7 Hz, 1H, ArCH_aH_b), 4.57 (d, J = 11.9 Hz,

1H, ArCH_aH_b), 4.55 (s, 1H, C1H_β‑man), 4.47 (d, J = 12.0

Hz, 1H, ArCH_aH_b), 4.41 (d, J = 7.8 Hz, 1H, C1H_β‑glc),

4.39−4.37 (m, 1H, CH_aH_b), 4.35 (d, J = 12.0 Hz, 1H, Ar

CH_aH_b), 4.31 (d, J = 12.0 Hz, 1H, ArCH_aH_b), 4.15−4.10

(m, 2H, CH_ring, CH_aH_b), 3.99 (t, J = 9.2 Hz, 1H, 

CH_ring), 3.94 (dd, J = 10.4, 4.9 Hz, 1H, CH_aH_b), 3.90−3.87

(m, 2H, CH_ring, CH_aH_b), 3.81−3.79 (m, 2H, CH_ring, 

CH_aH_b), 3.71−3.64 (m, 3H, 3 × CH_ring), 3.59−3.55 (m,

2H, CH_ring, CH_aH_b), 3.48−3.41 (m, 4H, 2 × CH_ring, 

CH₂), 3.34−3.31 (m, 1H, CH_ring), 3.03−2.99 (m, 1H, 

CH_ring), 1.02 (s, 9H, 3 × CH₃). ¹³C{¹H} NMR (150 MHz,

CDCl₃, 298 K): δ 139.4 (ArC), 138.9 (ArC), 138.7 (Ar

C), 138.6 × 3 (ArC), 138.0 (ArC), 137.6 (ArC), 136.0

(ArC), 135.8 (ArC), 134.2 (CH), 133.6 (ArC),

133.4 (ArC), 129.9 (ArC), 129.8 (ArC), 129.4 (Ar

C), 128.6 (ArC), 128.5 × 2 (ArC), 128.4 × 2 (ArC),

128.3 × 3 (ArC), 128.0 (ArC), 127.9 (ArC), 127.8 × 2

(ArC), 127.7 × 2 (ArC), 127.5 (ArC), 127.4 × 2 (Ar

C), 127.3 (ArC), 126.6 (ArC), 117.5 (=CH₂), 102.8

1

(C1_β‑glc, J_C−H= 161.0 Hz), 102.3 (PhCH), 101.3 (C1_β‑man

,

¹J_C−H= 156.7 Hz), 96.6 (C1_α‑glc, J_C−H= 173.3 Hz), 83.2 (

CH_ring), 81.9 (CH_ring), 81.6 (CH_ring), 79.7 (CH_ring),

79.6 (CH_ring), 79.2 (CH_ring), 77.4 × 2 (2 × CH_ring),

75.9 × 2 (2 × CH₂), 75.2 × 2 (2 × CH₂), 75.1 (CH₂), 74.9

(CH_ring), 74.8 (CH_ring), 73.6 (CH₂), 72.5 (CH_ring),

71.1 (CH₂), 70.5 (CH₂), 68.8 (CH₂), 68.7 (CH₂),

1

Y

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Article

67.10 (CH_ring), 63.18 (CH₂), 27.1 (CH₃), 19.5 (tert-

C). HRMS (ESI) m/z: [M + Na]⁺calcd for C₉₃H₁₀₀O₁₆SiNa,

1523.6673; found, 1523.6681.

Allyl Methyl-2,3,4-tri-O-benzyl-α-D-glucopyranosyluro-

nate-(1→3)-2-O-benzyl-4,6-O-benzylidene-β-D-mannopyra-

nosyl-(1→4)-2,3,6-tri-O-benzyl-α-^D-glucopyranoside (S7).

Compound S5 (1.62 g, 1.28 mmol, 1 equiv) was dissolved

in a mixture of CH₂Cl₂/H₂O (30 mL, 1:1 = v/v). The reaction

mixture was cooled to 0 °C; then, BAIB (1.24 g, 3.85 mmol, 3

equiv) and TEMPO (61 mg, 0.385 mmol, 0.3 equiv) were

added. The reaction mixture was allowed to warm to rt and

stirred for 2h. Upon completion, the reaction was quenched by

the addition of satd aq Na₂S₂O₃. The reaction mixture was

diluted with CH₂Cl₂(10 mL), and the organic layer was

separated. The aqueous layer was extracted with CH₂Cl₂(15

mL), and the combined organic layers were dried over MgSO₄,

ﬁltered, and concentrated. The residue was puriﬁed by ﬂash

column chromatography (100% CH₂Cl₂followed by CH₂Cl₂/

MeOH, 98:2) to yield pure compound S6 (1.488 g, 93%) in

cream color foam.

The above puriﬁed compound was dissolved in dry DMF

(20 mL), and MeI (363 μL, 5.83 mmol, 5 equiv) and K₂CO₃

(483 mg, 3.5 mmol, 3 equiv) were added at room temperature

under an argon atmosphere. The reaction mixture was stirred

overnight. Upon completion, the solvent was removed under

high vacuum, diluted with CH₂Cl₂(25 mL) and washed with

water (10 mL). The aqueous layer was extracted with CH₂Cl₂

(10 mL), and the combined organic layers were dried over

MgSO₄, ﬁltered, and concentrated. The obtained residue was

puriﬁed by ﬂash column chromatography (EtOAc/hexanes,

1:3) to yield the pure compound S7 (1.387 g, 92%) as a white

foam. ¹H NMR (600 MHz, CDCl₃, 298 K): δ 7.52 (d, J = 7.2

Hz, 2H, 2 × ArH), 7.38−7.18 (m, 36H, 36 × ArH), 7.03

(d, J = 7.3 Hz, 2H, 2 × ArH), 6.04−5.97 (m, 1H, CH),

5.56 (d, J = 3.5 Hz, 1H, C1H_{α‑glc‑COOMe}), 5.40 (dd, J = 17.2,

1.6 Hz, 1H, CHaH_b), 5.31 (s, 1H, PhCH−), 5.26 (dd, J =

10.7, 1.6 Hz, 1H, CHaH_b), 5.06 (d, J = 10.8 Hz, 1H, Ar

CH_aHb), 4.96 (dd, J = 11.0, 6.5 Hz, 2H, ArCH₂), 4.91 (d, J

= 11.5 Hz, 1H, ArCH_aH_b), 4.86 (d, J = 11.4 Hz, 1H, Ar

CH_aH_b), 4.84 (d, J = 11.0 Hz, 1H, ArCH_aH_b), 4.78 (dd, J =

6.8, 10.6 Hz, 2H, ArCH₂), 4.73 (s, 1H, ArCH_aH_b), 4.71

(s, 1H, ArCH_aH_b), 4.65 (s, 1H, C1H_β‑man), 4.59 (d, J =

11.1 Hz, 1H, ArCH₂), 4.55 (d, J = 12.1 Hz, 1H, Ar

CH₂), 4.50 (s,1H, ArCH₂), 4.49 (s, 1H, C1H_β‑glc),

4.47−4.45 (m, 1H, ArCH₂), 4.33 (d, J = 12.0 Hz, 1H,

ArCH₂), 4.29 (d, J = 9.7 Hz, 1H, CH_ring), 4.21−4.18

(m, 2H, CH_ring, ArCH_aH_b), 4.04−4.01 (m, 2H, 

CH_ring, ArCH_aH_b), 3.99 (t, J = 9.3 Hz, 1H, CH_ring),

3.88−3.85 (m, 2H, 2 × CH_ring), 3.76−3.72 (m, 2H, 

CH_ring, ArCH₂), 3.68 (s, 3H, COOCH₃), 3.67−3.62

(m, 2H, CH_ring, ArCH_aH_b), 3.56−3.49 (m, 3H, 2 × 

CH_ring, ArCH_aH_b), 3.43−3.40 (m, 1H, CH_ring), 3.10−

3.06 (m, 1H, CH_ring). ¹³C{¹H} NMR (150 MHz, CDCl₃,

298 K): δ 170.0 (CO), 139.3 (ArC), 138.6 × 3 (ArC),

137.4 (ArC), 134.2 (CH), 129.5 (ArC), 128.7 (Ar

C), 128.5 × 2 (ArC), 128.4 (ArC), 128.3 × 2 (ArC),

128.2 × 2 (ArC), 128.1 (ArC), 128.0 (ArC), 127.9

(ArC), 127.8 × 2 (ArC), 127.5 (ArC), 127.4 (ArC),

127.3 (ArC), 126.5 (ArC), 117.5 (CH₂), 102.9 (C1β-

glc, ¹J_C−H= 157.6 Hz), 102.3 (Ph−CH), 101.4 (C1_β‑man, ¹J_C−H

Allyl 2,3,4-Tri-O-benzyl-α-D-glucopyranosyl-(1→3)-2-O-

benzyl-4,6-O-benzylidene-β-^D-mannopyranosyl-(1→4)-

2,3,6-tri-O-benzyl-α-^D-glucopyranoside (S5). To a Nalgene

bottle containing silylether compound S4 (2.05 g, 1.37 mmol,

1 equiv) in THF/pyridine (30 mL, 9:1 = v/v), pyridinium

hydorﬂuoride (HF·py) stock solution (22 mL, 15 equiv; stock

solution was prepared from commercially available Aldrich HF·

py (70%) by dissolving 15 mL in 30 mL of pyridine, and 75

mL of THF) was added by plastic syringe over 5 min at 0 °C.

The reaction was allowed to warm to room temperature and

stirred overnight. By then, TLC conﬁrmed all starting material

had disappeared. The reaction mixture was poured into cold

satd aq NaHCO₃(200 mL) and extracted with CH₂Cl₂(2 ×

50 mL). The combined organic extracts were dried over

MgsO₄, ﬁltered and concentrated. The residue was puriﬁed by

ﬂash chromatography over silica gel (EtOAc/hexanes, 2:3) to

1

aﬀord pure compound S5 (1.62 g, 94%) as a white foam. H

NMR (600 MHz, CDCl₃, 298 K): δ 7.43 (d, J = 7.2 Hz, 2H,

ArH), 7.30−7.09 (m, 36H, ArH), 6.95 (d, J = 7.2 Hz, 2H,

ArH), 5.93−5.87 (m, 1H, CH), 5.36 (d, J = 3.6 Hz, 1H,

C1−H_α‑glc), 5.30 (dd, J = 17.3, 1.6 Hz, 1H, CHaH_b), 5.26 (s,

1H, PhCH), 5.16 (dd, J = 10.6, 1.4 Hz, 1H, CHaH_b),

4.98 (d, J = 10.7 Hz, 1H, ArCHaH_b), 4.89−4.81 (m, 4H, 2

× ArCH₂), 4.78 (d, J = 11.6, 1H, Hz, ArCH_aH_b), 4.69−

4.61 (m, 4H, 2 × ArCH₂), 4.59 (s, 1H, C1−H_β‑man), 4.53

(d, J = 11.0 Hz, 1H, ArCH_aH_b), 4.49 (d, J = 12.3 Hz, 1H,

ArCH_aH_b), 4.41 (d, J = 11.7 Hz, 1H, ArCH_aH_b), 4.39 (d,

J = 7.7 Hz, 1H, C1−H_β‑glc), 4.37−4.34 (m, 1H, CH_aH_b),

4.26 (d, J = 12.2 Hz, 1H, ArCH_aH_b), 4.14−4.08 (m, 2H, 

CH_ring, CH_aH_b), 3.97−3.89 (m, 3H, 2 × CH_ring, 

CH_aH_b), 3.78 (dd, J = 9.8, 3.0 Hz, 1H, CH_ring), 3.73 (d, J =

2.6 Hz, 1H,−CH_ring), 3.65−3.61 (m, 3H, CH_ring, 2 × 

CH_aH_b), 3.56−3.51 (m, 3H, CH_ring, 2 × -CH_aH_b), 3.47−

3.35 (m, 4H, 3 × CH_ring, CH_aH_b), 3.31−3.29 (m, 1H, 

CH_ring), 3.09−3.05 (m, 1H, CH_ring). ¹³C{¹H} NMR (150

MHz, CDCl₃, 298 K): δ 139.3 (ArC), 138.8 (ArC), 138.7

(ArC), 138.6 (ArC), 138.4 (ArC), 138.3 (ArC),

138.2 (ArC), 137.5 (ArC), 134.2 (CH), 129.4 (Ar

C), 128.7 (ArC), 128.6 (ArC), 128.5 (ArC), 128.3 × 3

(ArC), 128.2 × 3 (ArC), 128.0 × 2 (ArC), 127.9 (Ar

C), 127.8 × 2 (ArC), 127.7 (ArC), 127.5 (ArC), 127.4

(ArC), 127.3 (ArC), 126.5 (ArC), 117.5 (=CH₂),

1

102.9 (C1_β‑glc, J_C−H= 160.8 Hz), 102.4 (C1_β‑man, J_C−H

=

1

163.4 Hz), 101.5 (PhCH), 96.8 (C1_α‑glc, J_C−H= 171.6 Hz),

83.1 (CH_ring), 81.9 − (CH_ring), 81.4 (CH_ring), 79.4 (

CH_ring), 79.2 (CH_ring), 78.9 (CH_ring), 77.4 (CH_ring),

77.3 (CH_ring), 75.7 × 2 (2 × CH₂), 75.3 × 2 (CH₂, 

CH_ring), 75.2 × 2 (2 × CH₂), 74.9 (CH_ring), 73.7 (

CH₂), 71.7 (CH_ring), 70.9 (CH₂), 70.5 (CH₂), 68.8 (

CH₂), 68.7 (CH₂), 67.30 (CH_ring), 62.30 (CH₂).

HRMS (ESI) m/z: [M + Na]⁺calcd for C₇₇H₈₂O₁₆Na,

1285.5495; found, 1285.5503.

Z

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J. Org. Chem. XXXX, XXX, XXX−XXX

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Article

= 158.7 Hz), 97.4 (C1_{α‑glc‑COOMe}, ¹J_C−H= 175.8 Hz), 83.0 (

CH_ring), 81.9 (CH_ring), 80.7 (CH_ring), 79.3 (CH_ring),

79.2 (CH_ring), 79.1 (CH_ring), 78.6 (CH_ring), 77.8

(−CH_ring), 75.9 (CH₂), 75.8 (CH₂), 75.4 (CH_ring),

75.3 (CH₂), 75.2 × 2 (CH₂), 74.8 (CH_ring), 73.7

(CH₂), 71.2 (CH_ring), 71.0 (CH₂), 70.5 (CH₂), 68.7 ×

2 (CH₂), 67.1 (CH_ring), 52.6 (COOCH₃). HRMS

(ESI) m/z: [M + Na]⁺calcd for C₇₈H₈₂O₁₇Na, 1313.5444;

found, 1313.5463.

(CH₂), 68.9 × 2 (CH₂, CH_ring), 52.6 (COOCH₃).

HRMS (ESI) m/z: [M + Na]⁺calcd for C₇₈H₈₄O₁₇Na,

1315.5601; found, 1315.5615.

Allyl 2-O-Benzoyl-3-O-p-methoxy benzyl-4,6-O-benzyli-

dene-β-D-glucopyranosyl-(1→4)-2,6-di-O-benzyl-[methyl-

2,3,4-tri-O-benzyl-α-D-glucopyranosyluronate-(1→3)]-β-D-

mannopyranosyl-(1→4)-2,3,6-tri-O-benzyl-α-^D-glucopyra-

noside (S9). A mixture of acceptor S8 (692 mg, 0.53 mmol),

donor 9 (480 mg, 0.8 mmol, 1.5 equiv), and dried 4 Å

molecular sieves (1 g) in an anhydrous CH₂Cl₂(30 mL) was

stirred under an argon atmosphere for 30 min. The reaction

mixture was cooled to −30 °C. Then, NIS (216 mg, 0.96

mmol, 1.2 equiv with respect to the donor) and TfOH (0.5 M

in Et₂O, 480 μL, 0.24 mmol, 0.3 equiv with respect to the

donor) were added, and the reaction mixture was stirred at

−20 °C until TLC analysis (EtOAc/toluene, 1:10) indicated

the disappearance of the starting materials (50 min). Upon

completion, the reaction was quenched by the addition of NEt₃

(35 μL). The reaction mixture was slowly warmed to rt. The

mixture was ﬁltered through a pad of Celite, and the ﬁltrate

was concentrated under reduced pressure. The residue was

puriﬁed by ﬂash chromatography over silica gel (EtOAc/

toluene, 1:10) to give pure compound S9 (836 mg, 93%) as a

white foam. ¹H NMR (600 MHz, CDCl₃, 298 K): δ 7.89 (d, J

= 8.0 Hz, 2H, 2 × ArH), 7.48−7.06 (m, 48H, 48 × ArH),

7.01 (d, J = 8.7 Hz, 2H, 2 × ArH), 6.60 (d, J = 8.6 Hz, 2H, 2

× ArH), 5.99−5.92 (m, 1H, CH), 5.35 (dd, J = 17.1, 1.9

Hz, 1H, CH_aH_b), 5.22−5.19 (m, 2H, CH_aH_b, CH_ring),

5.18 (d, J = 3.1 Hz, 1H, C1H_α‑glc), 5.06 (d, J = 11.8 Hz, 1H,

ArCH_aH_b), 5.02 (d, J = 11.2 Hz, 1H, ArCH_aH_b), 4.99 (s,

1H, PhCH), 4.90−4.82 (m, 5H, 2 × ArCH₂, Ar

CH_aH_b), 4.80 (d, J = 11.6 Hz, 1H, ArCH_aH_b), 4.77 (d, J =

12.1 Hz, 1H, ArCH_aH_b), 4.68−4.65 (m, 4H, ArCH₂,

ArCH_aH_b, C1H_β‑glc), 4.61 (d, J = 10.8 Hz, 1H, Ar

CH_aH_b), 4.57 (dd, J = 11.9, 3.7 Hz, 2H, ArCH₂), 4.51 (d, J

= 12.0 Hz, 1H, ArCH_aH_b), 4.48 (d, J = 9.4 Hz, 1H, 

CH_ring), 4.42−4.34 (m, 5H, C1H_β‑man, C1H_{β‑allylglc}, 

CH_ring, ArCH₂), 4.19−4.10 (m, 3H, CH_ring, 2 × 

CH_aH_b), 4.05 (d, J = 11.8 Hz, 1H, CH_aH_b), 3.84−3.80 (m,

3H, 3 × CH_ring), 3.72 (s, 3H, OCH₃), 3.69 (d, J = 10.1 Hz,

1H, CH_aH_b), 3.65 (dd, J = 9.5, 3.0 Hz, 1H, CH_ring), 3.63

(s, 3H, COOCH₃), 3.62 (dd, J = 11.0, 4.1 Hz, 1H, CH_aH_b),

3.57−3.51 (m, 5H, 3 × CH_ring, 2 × CH_aH_b), 3.44 (t, 1H, J

= 8.0 Hz, CH_ring), 3.39 (dd, 1H, J = 9.7, 2.2 Hz, CH_ring),

3.33 (d, J = 11.2 Hz, 1H, CH_aH_b), 3.27 (t, 1H, J = 9.3 Hz,

CH_ring), 3.16 (m, 1H, CH_ring), 2.81 (d, 1H, J = 9.1 Hz, 

CH_ring). ¹³C{¹H} NMR (150 MHz, CDCl₃, 298 K): δ 170.4

(CO), 164.86 (CO), 159.1 (ArC), 139.5 (ArC), 139.2

(ArC), 138.9 (ArC), 138.8 (ArC), 138.6 (ArC),

138.5 (ArC), 138.4 (ArC), 138.1 (ArC), 137.7 (Ar

C), 134.2 (CH), 133.3 (ArC), 130.4 (ArC), 129.9

(ArC), 129.8 (ArC), 129.6 (ArC), 129.0 (ArC),

128.7 (ArC), 128.6 × 2 (ArC), 128.5 × 3 (ArC), 128.4

× 2 (ArC), 128.3 (ArC), 128.2 × 2 (ArC), 128.1 (Ar

C), 128.0 × 2 (ArC), 127.9 × 2 (ArC), 127.8 (ArC),

127.7 (ArC), 127.5 (ArC), 127.3 (ArC), 127.2 (Ar

C), 127.0 (ArC), 126.2 (ArC), 117.4 (CH₂), 113.6

Allyl Methyl-2,3,4-tri-O-benzyl-α-D-glucopyranosyluro-

nate-(1→3)-2,6-di-O-benzyl-β-^D-mannopyranosyl-(1→4)-

2,3,6-tri-O-benzyl-α-^D-glucopyranoside (S8). A mixture of the

starting material S7 (615 mg, 0.476 mmol, 1 equiv) and 4 Å

molecular sieves (3 g) in CH₂Cl₂was stirred under an argon

atmosphere for 1 h. The reaction mixture was cooled to −78

°C, and triethylsilane (230 μL, 1.43 mmol, 3 equiv) and TfOH

(150 μL, 1.67 mmol, 3.5 equiv) were added. The reaction

mixture continued to stir at −78 °C until the complete

disappearance of the starting material (monitored by TLC, 1

h). Then, the reaction was quenched by the addition of satd aq

NaHCO₃. The reaction mixture was warmed to room

temperature and ﬁltered, and the ﬁltrate was washed with

saturated NaHCO₃and brine. The organic phase was dried

over MgSO₄and ﬁltered, and the ﬁltrate was concentrated

under reduced pressure. The residue was puriﬁed by ﬂash

chromatography over silica gel (EtOAc/hexanes, 1:5) to give

pure compound S8 (578 mg, 89%) as a viscous colorless oil.

¹H NMR (600 MHz, CDCl₃, 298 K): δ 7.43 (d, J = 7.1 Hz,

2H, 2 × ArH), 7.35−7.19 (m, 38H, 38 × ArH), 6.02−

5.95 (m, 1H, CH), 5.38 (dd, J = 17.2, 1.5 Hz, 1H, 

CH_aH_b), 5.24 (dd, J = 10.2, 1.5 Hz, 1H, CH_aH_b), 5.10−5.09

(m, 2H, C1H_{α‑glc‑COOMe}(overlapped), ArCH_aH_b), 4.97−

4.91 (m, 3H, ArCH₂, ArCH_aH_b), 4.83−4.72 (m, 7H, 3 ×

ArCH₂, ArCH_aH_b), 4.68 (d, J = 12.1 Hz, 1H, Ar

CH_aH_b), 4.60 (d, J = 11.2 Hz, 1H, ArCH_aH_b), 4.56 (s, 1H,

C1H_β‑man), 4.53 (d, J = 12.1 Hz, 1H, ArCH_aH_b), 4.46 (s,

1H, C1H_β‑glc), 4.45−4.42 (m, 2H, ArCH_aH_b, CH_aH_b),

4.40 (d, J = 10.1 Hz, 1H, CH_ring), 4.37 (d, J = 12.1 Hz, 1H

ArCH_aH_b), 4.17 (dd, J = 12.8, 5.8 Hz, 1H −CH_aH_b), 4.05−

3.97 (m, 3H, 3 × CH_ring), 3.78−3.73 (m, 3H, 2 × CH_ring

,

CH_aH_b), 3.69−3.61 (m, 7H, 2 × CH_ring, CH₂, 

COOCH₃), 3.51−3.46 (m, 3H, 2 × CH_ring, CH_aH_b), 3.31

(dd, 1H, J = 9.3, 2.9 Hz, CH_ring), 3.26 (m, 1H, CH_ring).

¹³C{¹H} NMR (150 MHz, CDCl₃, 298 K): δ 170.2 (CO),

139.5 (ArC), 138.9 (ArC), 138.7 (ArC), 138.6 (Ar

C), 138.5 (ArC), 138.3 (ArC), 138.2 (ArC), 137.4

(ArC), 134.3 (CH), 128.7 (ArC), 128.6 × 2 (ArC),

128.5 × 3 (ArC), 128.4 × 2 (ArC), 128.3 × 2 (ArC),

128.2 (ArC), 128.1 (ArC), 128.0 (ArC), 127.9 × 2

(ArC), 127.8 × 2 (ArC), 127.7 (ArC), 127.6 (ArC),

127.5 (ArC), 127.2 (ArC), 117.5 (=CH₂), 102.8

1

(C1_{β‑allylicglc}, J_C−H= 162.6 Hz), 100.7 (C1_β‑manJ_C−H= 155.8

1

Hz), 100.6 (C1_α‑glc, J_C−H= 171.4 Hz), 83.9 (CH_ring), 82.7

(CH_ring), 82.0 (CH_ring), 81.5 (CH_ring), 79.7 (CH_ring),

79.1 (CH_ring), 78.7 (CH_ring), 77.2 (CH_ring), 75.2 × 2

(CH₂), 75.1 (CH_ring), 74.9 (CH₂), 73.8 (CH₂), 73.7

(CH₂), 73.6 (CH₂), 71.2 (CH_ring), 71.0 (CH₂), 70.5

AA

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(ArC), 102.8 (C1_{β‑allylicglc}

,

¹J_C−H= 162.2 Hz), 101.21

Glycosyl bromide (20 g, 30.3 mmol) and allyl alcohol (2.7

mL, 39.4 mmol, 1.3 equiv) were dissolved in anhydrous

CH₂Cl₂(200 mL) under argon. Freshly activated powdered 4

Å MS (15 g) were added, and the reaction mixture was stirred

for 30 min. Then, it was cool to 0 °C, and AgOTf (10.13 g,

39.4 mmol, 1.3 equiv) was added in portions over 10 min; the

reaction mixture was stirred overnight at room temperature in

darkness. Upon completion, the reaction was quenched by the

addition of NEt₃(4 mL). Then the mixture was stirred for 10

min and ﬁltered through a wet Celite pad. The ﬁltrate was

washed with saturated NaHCO₃(2 × 40 mL) and brine (40

mL), dried over MgSO₄, ﬁltered, and concentrated. The crude

product was subjected to ﬂash chromatography on silica gel

(EtOAC/hexanes, 1:4) to give pure compound S15 (17 g,

88%) as a white foam. ¹H NMR (600 MHz, CDCl₃, 298 K): δ

8.07 (dd, J = 8.6, 1.5 Hz, 2H, ArH), 8.01 (dd, J = 8.2, 1.2

Hz, 2H, ArH), 7.94 (dd, J = 8.2, 1.2 Hz, 2H, ArH), 7.87

(dd, J = 8.2, 1.5 Hz, 2H, ArH), 7.58−7.50 (m 3H, ArH),

7.46−7.35 (m 7H, ArH), 7.31−7.28 (m, 2H, ArH), 5.96

(t, J = 9.6 Hz, 1H, CH_ring), 5.86−5.80 (m, 1H, CH),

5.73 (t, J = 9.6 Hz, 1H, CH_ring), 5.62 (dd, J = 9.6, 7.7 Hz,

1H, CH_ring), 5.27−5.23 (m, 1H, CH_aH_b), 5.16−5.14 (m,

1H, CH_aH_b), 4.95 (d, J = 7.8 Hz, 1H, C1H), 4.69 (dd, J =

12.1, 3.6 Hz, 1H, CH_aH_bOBz), 4.56 (dd, J = 12.1, 5.6 Hz,

1H, CH_aH_bOBz), 4.41−4.38 (m, 1H, CH_ring), 4.22−4.18

(m, 2H, OCH₂CHCH). ¹³C{¹H} NMR (150 MHz,

CDCl₃, 298 K): δ 166.3 (CO), 166.0 (CO), 165.4 (CO), 165.3

(CO), 133.6 (ArC), 133.5 (ArC), 133.4 × 2 (ArC),

133.3 (ArC), 130.0 (ArC), 129.9(ArC), 129.9 (Ar

C), 129.8 (ArC), 129.5, 128.9 (ArC), 128.6 (ArC),

128.5 × 3 (ArC), 118.1 (CH₂), 100.0 (C1_β‑glc), 73.1 (

CH_ring), 72.4 (CH_ring), 72.0 (CH_ring), 70.3 (CH₂), 70.0

(CH_ring), 63.4 (CH₂). HRMS (ESI) m/z: [M + Na]⁺

calcd for C₃₇H₃₂O₁₀Na 659.1888; found, 659.1932.

1

(C1_β‑manJ_C−H= 158.3 Hz), 100.9 (Ph−CH), 100.4 (C1_β‑glc

,

¹J_C−H= 164.2 Hz), 97.6 (C1_α‑glc, J_C−H= 171.3 Hz), 82.9 (

CH_ring), 81.9 (CH_ring), 81.6 (CH_ring), 80.7 (CH_ring),

80.1 (CH_ring), 79.3 (CH_ring), 78.9 (CH_ring), 78.1 (

CH_ring), 77.7 (CH_ring), 77.6 (CH_ring), 75.8 (CH_ring),

75.6 (CH₂), 75.1 (CH₂), 75.0 (CH₂), 74.9 (CH₂), 74.8 (

CH_ring), 74.2 (CH₂), 73.8 (CH_ring), 73.5 × 2 (CH₂), 73.4

(CH₂), 72.7 (CH₂), 72.1 (CH_ring), 71.5 (CH_ring), 70.4

(CH₂), 68.8 (CH₂), 68.6 (CH₂), 68.3 (CH₂), 66.2 (CH_ring),

55.3 (OCH₃), 52.5 (COOCH₃). HRMS (ESI) m/z: [M +

Na]⁺calcd for C₁₀₆H₁₁₀O₂₄Na, 1789.7279; found, 1789.7279.

1

1,2,3,4,6-Penta-O-benzyl-β-D-glucopyranoside (S14). D-

Glucose (15 g, 83.2 mmol) and a catalytic amount of

DMAP (1.02 g, 8.32 mmol, 0.1 equiv) were dissolved in

pyridine (175 mL). After cooling the reaction mixture to 0 °C,

benzoyl chloride (72 mL, 62.44 mmol, 7.5 equiv) was added,

and the mixture was left stirring overnight at rt. Upon

completion, pyridine was removed under reduced pressure,

and the obtained residue was dissolved in CH₂Cl₂(175 mL).

Then, H₂O (75 mL) was added carefully, and the reaction

mixture was stirred vigorously to decompose excess benzyol

chloride. The product was extracted with CH₂Cl₂, and the

organic phase was washed with satd aq NaHCO₃(75 mL) and

brine (50 mL) and dried over MgSO₄and concentrated in

vacuo. The residue was crystallized from CH₂Cl₂/hexane (1:3)

to give the title compound S14 (58 g, 99%, white solid) in an

1

α,β-mixture (α/β, 6:1). H NMR (600 MHz, CDCl₃, 298 K):

(major anomer) δ 8.15 (dd, J = 7.1, 1.3 Hz, 2H, ArH), 8.01

(dd, J = 7.2, 0.9 Hz, 2H, ArH), 7.94 (dd, J = 7.2, 0.9 Hz, 2H,

ArH), 7.87−7.85 (m, 4H, ArH), 7.65−7.63 (m 1H, Ar

H), 7.53−7.27 (m 14H, ArH), 6.84 (d, J = 3.7 Hz, 1H,

C1H), 6.32 (q, J = 9.9 Hz, 1H, CH_ring), 5.86 (t, J = 9.9

Hz, 1H, CH_ring), 5.68 (dd, J = 10.2, 3.6 Hz, 1H, CH_ring),

4.62−4.59 (m, 2H, CH_ring, CH_aH_bOBz), 4.48−4.45 (m,

1H, CH_aH_bOBz). ¹³C{¹H} NMR (150 MHz, CDCl₃, 298

K): δ 166.3 (CO), 166.1 (CO), 165.5 (CO), 165.3 (CO), 164.6

(CO), 134.1 (ArC), 133.7 × 2 (ArC), 133.5 (ArC),

133.3 (ArC), 130.2 (ArC), 130.1 (ArC), 130.0 (Ar

C), 129.9 × 2 (ArC), 129.0 (ArC), 128.6 × 2 (ArC),

128.5 × 2 (ArC), 90.2 (C1_α‑glc), 70.6 × 3 (CH_ring), 69.0

(CH_ring), 62.6 (CH₂).

Allyl β-D-Glucopyranoside (S16). To a stirred solution of

S15 (17 g, 26.7 mmol) in methanol was added NaOMe (30%

in MeOH, 1.4 mL, 8 mmol, 0.3 equiv) under an argon

atmosphere. After overnight stirring, the reaction mixture was

neutralized with Amberlite IR-120 resin. The resin was

removed by ﬁltration, and the ﬁltrate was concentrated. The

residue was puriﬁed by ﬂash chromatography over silica

(MeOH/CH₂Cl₂, 1:6) to aﬀord title compound S16 (5.62 g,

95%) as a white solid. ¹H NMR (600 MHz, CD₃OD, 298 K): δ

5.94−5.88 (m, 1H, CH), 5.29 (dd, J = 17.1, 1.5 Hz, 1H, 

CH_aH_b), 5.11 (d, J = 10.3 Hz, 1H, CH_aH_b), 4.33 (dd, J =

12.7, 5.0 Hz, 1H, OCH_aH_bCHCH₂), 4.25 (d, J = 7.9 Hz,

1H, C1−H), 4.10 (dd, J = 12.7, 6.0 Hz, 1H, OCH_aH_bCH

CH₂), 3.81 (dd, J = 12.0, 2.1 Hz, 1H, H_aH_bOH), 3.62 (dd, J

= 11.9, 5.6 Hz, 1H, H_aH_bOH), 3.31−3.28 (m, 1H, 

CH_ring), 3.25−3.17 (m, 2H, 2 × CH_ring), 3.17−3.12 (t, J =

7.8 Hz, 1H, CH_ring). ¹³C{¹H} NMR (150 MHz, CD₃OD,

298 K): δ 135.8 (CH), 117.6 (CH₂), 103.4 (C1_β‑glc), 78.2

(CH_ring), 78.0 (CH_ring), 75.2 (CH_ring), 71.7 (CH_ring),

71.2 (CH₂), 62.8 (CH₂). HRMS (ESI) m/z: [M + Na]⁺

calcd for C₉H₁₆O₆Na, 243.0839; found, 243.0854.

Allyl 2,3,4,6-Tetra-O-benzyl-β-D-glucopyranoside (S15).

Glucose pentabenzoate S14 (50 g) was dissolved in CH₂Cl₂

(250 mL), and 100 mL of HBr (33% in AcOH, 2 mL/1 g) was

added at 0 °C over 20 min under argon. Then, the reaction

mixture was allowed to react for 1 h at the same temperature

and for another 3 h at room temperature. Upon completion,

the reaction mixture was diluted with 75 mL of CH₂Cl₂and

poured over ice. The organic layer was washed with cold H₂O

(2 × 100 mL), satd aq NaHCO₃(2 × 150 mL), and brine (2 ×

100 mL) and dried over MgSO₄. The sample was ﬁltered and

concentrated, and the solid was crystallized from EtOAC/

hexane (1:3) to give pure glycosyl bromide (38 g, 79%) in

white crystals.

AB

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Allyl 4,6-O-Benzylidene-β-D-glucopyranoside (S17). To a

suspension of the tetraol S16 (8 g, 36.35 mmol) in anhydrous

acetonitrile (120 mL) were added DL-10-camphorsulfonic acid

(CSA) (2.110 g, 9.09 mmol, 0.25 equiv) and benzaldehyde

dimethyl acetal (13.7 mL, 90.09 mmol, 2.5 equiv) dropwise

under an argon atmosphere at room temperature, and the

mixture was allowed to stir vigorously for 15 min. The reaction

was quenched with Et₃N (1 mL). Then, the reaction mixture

was concentrated in vacuo, and the residue was dissolved in

CH₂Cl₂(100 mL) and washed with satd aq NaHCO₃(50 mL)

and water. The combined organic layers were dried over

MgSO₄and concentrated in vacuo. The solid residue was

recrystallized in ethyl acetate and hexane (1:3) to aﬀord pure

compound S17 (10 g, 89%) in a white solid, which was pure

C), 117.8 (CH₂), 103.4 (Ph−CH), 101.3 (C1_β‑glc), 82.3 (

CH_ring), 81.7 (CH_ring), 81.1 (CH_ring), 75.6 (CH₂), 75.3

(CH₂), 70.9 (CH₂), 68.9 (CH₂), 66.2 (CH_ring).

HRMS (ESI) m/z: [M + Na]⁺calcd for C₃₀H₃₂O₆Na,

511.2091; found, 511.2125.

Allyl 2,3,6-Tri-O-benzyl-β-D-glucopyranoside (S1). A mix-

ture of starting material S18 (6 g, 12.29 mmol, 1 equiv) and 4

Å molecular sieves (9 g) in CH₂Cl₂(150 mL) was stirred

under argon for 1 h. The reaction was cooled to −78 °C, and

triethylsilane (5.9 mL, 36.86 mmol, 3 equiv) and TFA (3.805

mL, 43.01 mmol, 3.5 equiv) were added. The reaction mixture

was stirred at −78 °C until TLC analysis indicated the

disappearance of the starting material (1 h); then, the reaction

was quenched by the addition of satd aq NaHCO₃(10 mL)

solution. The reaction mixture was slowly warmed to room

temperature and ﬁltered. The ﬁltrate was washed with

saturated NaHCO₃(50 mL) and brine. The organic phase

was dried over MgSO₄and ﬁltered. The ﬁltrate was

concentrated under reduced pressure. The residue was puriﬁed

by ﬂash chromatography over silica gel (EtOAc/hexanes, 1:4)

1

enough for further reaction. H NMR (600 MHz, CD₃OD,

298 K): δ 7.42−7.41 (m, 2H, ArH), 7.26−7.25 (m, 3H,

ArH), 5.91−5.84 (m, 1H, CH), 5.49 (s, 1H, Ph−CH),

5.27 (dd, J = 17.5, 1.0 Hz, 1H, CH_aH_b), 5.10 (dd, J = 10.5,

1.0 Hz, 1H, CH_aH_b), 4.37 (dd, J = 7.7, 2.1 Hz, C1H),

4.26−4.23 (m, 2H, CH_ring, CH_aH_b), 4.21−4.18 (m, 1H,

CH_aH_b), 3.70 (ddd, J = 10.2, 2.4 Hz, 1H, CH_ring), 3.57

(ddd, J = 9.0, 2.4 Hz, 1H, CH_ring), 3.40 (ddd, J = 9.2, 2.2 Hz,

1H, CH_ring), 3.36 (ddd, J = 4.8, 2.4 Hz, 1H, CH_ring),

3.25−3.22 (m, 3H, 2 × OH). ¹³C{¹H} NMR (150 MHz,

CD₃OD, 298 K): δ 139.3 (ArC), 135.7 (ArC), 130.1

(ArC), 129.2 (ArC), 127.6 (ArC), 117.7 (CH₂),

104.3 (Ph−CH), 103.1 (C1_β‑glc), 82.4 (CH_ring), 76.1 (

CH_ring), 74.8 (CH_ring), 71.6 (CH₂), 69.8 (CH₂), 67.7

(CH_ring). HRMS (ESI) m/z: [M + Na]⁺calcd for

C₁₆H₂₀O₆Na, 331.1152; found, 331.1169.

1

to give pure product S1 (4.88 g, 81%) as a white solid. H

NMR (600 MHz, CDCl₃, 298 K): δ 7.36−7.26 (m, 15H, Ar

H), 5.98−5.92 (m, 1H, CH), 5.34 (dd, J = 17.2, 1.5 Hz, 1H,

CH_aH_b), 5.21 (dd, J = 10.2, 1.5 Hz, 1H, CH_aH_b), 4.96

(dd, J = 16.6, 10.9 Hz, 2H, ArCH₂), 4.73 (dd, J = 11.6, 5.3

Hz, 2H, ArCH₂), 4.60 (d, J = 12.2 Hz, 1H, ArCH_aH_b),

4.57 (d, J = 12.2 Hz, 1H, ArCH_aH_b), 4.46 (dd, J = 7.5, 2.0

Hz, 1H, C1H_β‑glc), 4.43−4.39 (m, 1H, CH_aH_b), 4.15−

4.12 (m, 1H, CH_aH_b), 3.77 (dd, J = 10.5, 3.9 Hz, 1H, 

CH_aH_b), 3.70 (dd, J = 10.5, 5.5 Hz, 1H, CH_aH_b), 3.60−3.56

(m, 1H, CH_ring), 3.46−3.41 (m, 3H, 3 × CH_ring), 2.53 (d,

J = 2.0 Hz, 1H, −OH). ¹³C{¹H} NMR (150 MHz, CDCl₃, 298

K): δ 138.8 (ArC), 138.6 (ArC), 138.1 (ArC), 134.2

(ArC), 128.7 (ArC), 128.6 × 2 (ArC), 128.4 (ArC),

128.2 (ArC), 128.0 (ArC), 127.9 (ArC), 127.9 (Ar

C), 117.5 (CH₂), 102.9 (C1_β‑glc), 84.2 (CH_ring), 81.9 (

CH_ring), 75.5 (CH₂), 75.0 (CH₂), 74.2 (CH_ring), 73.8

(CH₂), 71.7 (CH_ring), 70.5 × 2 (CH₂). HRMS (ESI)

m/z: [M + Na]⁺calcd for C₃₀H₃₄O₆Na, 513.2248; found,

513.2277.

Allyl 2,3-Di-O-benzyl-4,6-O-benzylidene-β-D-glucopyra-

noside (S18). To a suspension of NaH (60%, 2.65 g, 66.2

mmol, 3 equiv) in dry DMF (70 mL) was added diol

compound S17 (6.8 g, 22.06 mmol) dissolved in dry DMF (35

mL) at 0 °C under an argon atmosphere. The reaction mixture

was stirred for 10 min; then, benzyl bromide (10.5 mL, 88.27

mmol, 4 equiv) was added dropwise, and the reaction mixture

was stirred for 5−6 h at room temperature. Upon completion,

the reaction was carefully quenched with MeOH at 0 °C, and

the mixture was concentrated in vacuo. The residue was diluted

with CH₂Cl₂(75 mL) and washed with H₂O. The combined

organic layers were dried over MgSO₄, ﬁltered, and

concentrated in vacuo. The residue was puriﬁed by ﬂash

column chromatography on silica gel (100% hexane and

EtOAC/hexanes, 1:9) to yield compound S18 (10.5 g, 98%) as

Phenyl 4,6-O-Benzylidene-3-p-methoxybenzyl-1-thio-α-D-

mannopyranoside (S19). To a well-stirred suspension of α-

mannose thioglycoside (8.17 g, 30 mmol, 1 equiv) in dry

CH₃CN (200 mL) were added CSA (70 mg, 3 mmol, 10 mol

%) and benzaldehyde dimethyl acetal (BDA) (5.17 mL, 36.0

mmol, 1.2 equiv) at room temperature. The reaction mixture

was stirred under argon until TLC analysis indicated the

disappearance of the starting material (45 min); then, the

reaction was quenched by triethyl amine (600 μL, 2 equiv to

CSA). The solvent was removed under reduced pressure to

give a solid compound, which was dissolved in CH₂Cl₂(125

mL) and washed with satd aq NaHCO₃(2×) and water. The

combined organic layers were dried over MgSO₄, ﬁltered, and

concentrated under reduced pressure. The resulting solid

compound was recrystallized from CH₂Cl₂:hexanes (1:3), and

the crystallized compound was separated and repeatedly

1

a white solid. H NMR (600 MHz, CDCl₃, 298 K): δ 7.48−

7.42 (m, 2H, ArH), 7.38−7.25 (m, 13H, ArH), 5.97−

5.5.91 (m, 1H, CH), 5.56 (s, 1H, Ph−CH), 5.36 (dd, J =

17.3, 1.6 Hz, 1H, CH_aH_b), 5.23 (dd, J = 10.5, 2.8 Hz, 1H, 

CH_aH_b), 4.91 (d, J = 11.1 Hz, 2H, ArCH₂), 4.81 (d, J = 11.4

Hz, 1H, ArCH_aH_b), 4.77 (d, J = 10.9 Hz, 1H, ArCH_aH_b),

4.56 (d, J = 7.6 Hz, 1H, C1H_β‑glc), 4.42−4.38 (m, 1H, 

CH_aH_b), 4.35 (dd, J = 10.5, 5.0 Hz, 1H, CH_aH_b), 4.17−4.14

(m, 1H, CH_aH_b), 3.80 (t, J = 9.8 Hz, 1H, CH_aH_b), 3.76

(t, J = 9.5 Hz, 1H, CH_ring), 3.68 (t, J = 9.5 Hz, 1H, 

CH_ring), 3.50 (dd, J = 8.6, 7.6 Hz, 1H, CH_ring), 3.42−3.38

(m, 1H, CH_ring). ¹³C{¹H} NMR (150 MHz, CDCl₃, 298 K):

δ 138.7 (ArC), 138.5 (ArC), 137.5 (ArC), 133.9 (Ar

C), 129.1(ArC), 128.5 × 2 (ArC), 128.4 × 2 (ArC),

128.2 (ArC), 127.9 (ArC), 127.8 (ArC), 126.2 (Ar

AC

https://dx.doi.org/10.1021/acs.joc.0c01404

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

pubs.acs.org/joc

Article

washed with hexane or diethyl ether to remove excess BDA.

The obtained white solid was almost pure and can be used as is

for further reactions (9.3 g, 86%).

until the solution was neutralized. The reaction mixture was

ﬁltered and concentrated in vacuo to aﬀord tetraol S20 (28 g,

94%) as a white solid. ¹H NMR (600 MHz, CD₃OD, 298 K): δ

7.37 (d, J = 8.0 Hz, 2H, ArH), 7.03 (d, J = 8.0 Hz, 2H, Ar

H), 4.42 (d, J = 9.8 Hz, 1H, C1H_β‑glc), 3.76 (dd, J = 12.1, 1.5

Hz, 1H, CH_aH_b), 3.57 (dd, J = 12.1, 5.1 Hz, 1H, CH_aH_b),

3.28 (t, J = 8.6 Hz, 1H, CH_ring), 3.19−3.15 (m, 2H,

−CH_ring), 3.09 (t, J = 9.3 Hz, 1H, (CH_ring), 2.22 (s, 3H,

PhCH₃). ¹³C{¹H} NMR (150 MHz, CD₃OD, 298 K): δ 138.9

(ArC), 133.6 (ArC), 131.3 (ArC), 130.6 (ArC), 89.8

(C1_β‑glc), 82.1 (CH_ring), 79.8 (CH_ring), 73.8 (CH_ring),

71.5 (CH_ring), 63.0 (CH₂), 21.2 (CH₃). HRMS (ESI)

m/z: [M + Na]⁺calcd for C₁₃H₁₈O₅SNa, 309.0767; found,

309.0786.

A portion of the above dried mannose diol (6.1 g, 17 mmol)

and Bu₂SnO (4.655 g, 18.7 mmol, 1.1 equiv) in toluene (120

mL) were stirred at reﬂux for 3 h with azeotropic removal of

water using a Dean−Stark apparatus (clear solution was

formed during the reﬂuxing). The solvent was removed under

reduced pressure and coevaporated twice with toluene. The

residue was dissolved in DMF (50 mL), and Bu₄NBr (5.77 g,

17.9 mmol, 1.05 equiv, CsF (2.72 g, 17.9 mmol, 1.05 equiv),

and PMBCl (2.5 mL, 17.9 mmol, 1.05 equiv) were added

sequentially under an argon atmosphere. The reaction mixture

was stirred overnight. Upon completion, the solvent was

removed under reduced pressure, and the residue was diluted

with EtOAc (120 mL) and washed with sat aq NaHCO₃

solution (75 mL) and brine. The separated organic layer was

ﬁltered through a pad of Celite to remove the inorganic salts.

The residue was dried over MgSO₄, ﬁltered, and concentrated

under reduced pressure. The obtained solid was recrystallized

from Et₂O/hexane (1:3) to get white crystallized compound

S19, which was pure enough for further reactions (7.8 g, 88%).

¹H NMR (600 MHz, CDCl₃, 298 K): δ 7.51−7.42 (m, 4H,

ArH), 7.39−7.35 (m, 3H, ArH), 7.31−7.25 (m, 5H, Ar

H), 6.88−6.86 (m, 2H, ArH), 5.60 (s, 1H, Ph−CH), 5.57

(s, 1H), 4.81 (d, J = 11.4 Hz, 1H), 4.66 (d, J = 11.4 Hz, 1H),

4.33−4.29 (m, 1H), 4.22 (br d, J = 3.2 Hz, 1H), 4.20 (dd, J =

10.2, 4.8 Hz, 1H), 4.16 (t, J = 9.4 Hz, 1H), 3.9 (dd, J = 9.6, 3.4

Hz, 1H), 3.85 (t, J = 10.3 Hz, 1H), 3.80 (s, 3H, OCH₃),

2.89 (s, 1H, OH). ¹³C{¹H} NMR (150 MHz, CDCl₃, 298

K): δ 159.7 (ArC), 137.6 (ArC), 133.5 (ArC), 131.9

(ArC), 129.9 (ArC), 129.8 (ArC), 129.3 (ArC),

129.2 (ArC), 128.4 (ArC), 127.9 (ArC), 126.3 (Ar

C), 114.1 (ArC), 101.8 (Ph−CH), 87.9 (C1α-man), 79.2

(CH_ring), 75.6 (CH_ring), 73.1 (CH₂), 71.6 (CH_ring),

68.7 (CH₂), 64.8 (CH_ring), 55.5 (OCH₃). HRMS (ESI)

m/z: [M + Na]⁺calcd for C₂₇H₂₈O₆SNa, 503.1499; found,

503.1530.

p-Methylphenyl 4,6-O-Benzylidene-3-p-methoxybenzyl-1-

thio-β-D-glucopyranoside (S21). To a suspension of the

tetraol S20 (7.5 g, 26.2 mmol) in anhydrous acetonitrile (100

mL) were added DL-10-camphorsulfonic acid (CSA) (1.52 g,

6.55 mmol, 0.25 equiv) and benzaldehyde dimethyl acetal (7.9

mL, 52.4 mmol, 2 equiv) dropwise under an argon atmosphere

at room temperature. The mixture was stirred vigorously for 45

min. Then, the reaction was quenched with Et₃N (2 mL), and

the mixture was concentrated in vacuo. The residue was

dissolved in CH₂Cl₂(150 mL) and washed with satd aq

NaHCO₃(50 mL) and water. The combined organic layers

were dried over MgSO₄and concentrated in vacuo. The solid

residue was crystallized from CH₂Cl₂/hexane (1:3) and

separated by ﬁltering. The solid mass was repeatedly washed

with hexane or diethyl ether to remove excess BDA and later

dried under a vacuum. The white solid was almost pure and

could be used as is for further reactions (9.5 g, 88%).

A portion of the above dried thioglycoside (5.5 g, 14.69

mmol) and Bu₂SnO (4.02 g, 16.89 mmol, 1.15 equiv) in

toluene (75 mL) were stirred at reﬂux for 3−4 h with

azeotropic removal of water using a Dean−Stark apparatus

(clear solution was formed during the reﬂuxing). The solvent

was removed under reduced pressure and coevaporated twice

with toluene. The residue was dissolved in DMF (50 mL), and

Bu₄NBr (5.21 g, 16.16 mmol, 1.1 equiv), CsF (2.46 g, 16.16

mmol, 1.1 equiv), and PMBCl (2.3 mL, 16.16 mmol, 1.1

equiv) were added sequentially under an argon atmosphere.

The reaction mixture was stirred overnight at rt. Upon

completion, DMF was removed under reduced pressure, and

the residue was diluted with EtOAc (150 mL) and washed

with sat aq NaHCO₃solution (75 mL) and brine. The

separated organic layer was ﬁltered through a pad of Celite to

remove the inorganic salts, dried over MgSO₄, ﬁltered, and

concentrated under reduced pressure. The solid residue was

puriﬁed by ﬂash chromatography over silica gel (hexanes/

EtOAc/CH₂Cl₂, 2:1:1) to give pure compound S21 as a white

p-Methylphenyl 1-Thio-β-D-glucopyranoside (S20).⁴⁸D-

Glucose penta-acetate (45 g, 11.54 mmol) and p-thiocreosl

(21.5 g, 17.31 mmol, 1.5 equiv) were dissolved in anhydrous

CH₂Cl₂(250 mL) under argon in a ﬂame-dried 1 L ﬂask. The

solution was cooled to 0 °C, and BF₃·Et₂O (43 mL, 14.4

mmol, 1.25 equiv) was added. The mixture was stirred at room

temperature overnight. Upon completion, the reaction mixture

was diluted with CH₂Cl₂(50 mL) and neutralized with satd aq

NaHCO₃solution (until the evaluation of CO₂ceased). The

organic layer was separated, and the aqueous layer was

extracted with CH₂Cl₂(2 × 50 mL). The combined organic

layers were washed with water, dried over MgSO₄, and

concentrated in vacuo. The residue was crystallized from the

EtOAc/hexane system (1:3) by storing in a 4 °C refrigerator

overnight. The crystals were ﬁltered through a sintered funnel

by repeatedly washing with hexane to aﬀord pure tetra-acetyl

β-thioglycoside (47 g, 90%) in a white crystalline solid. The

compound was dissolved in methanol. Sodium methoxide in

methanol (30 wt %, 9.3 mL, 51.8 mmol, 0.5 equiv) was added

to the solution at rt, and the mixture was stirred for 4−5 h.

Then, ion-exchange resin (IR-120) was added portionwise

1

solid (6.4 g, 88%). H NMR (600 MHz, CDCl₃, 298 K): δ

7.46−7.22 (m, 9H, ArH), 7.10 (d, J = 7.9 Hz, 2H, ArH),

6.82 (d, J = 8.5 Hz, 2H, ArH), 5.52 (s, 1H), 4.85 (d, J = 11.2

Hz, 1H), 4.69 (d, J = 11.2 Hz, 1H), 4.53 (d, J = 9.7 Hz, 1H),

4.35 (dd, J = 10.5, 4.9 Hz, 1H), 3.78−3.73 (m, 4H, OCH₃),

3.61 (dt, J = 18.4, 9.0 Hz, 2H), 3.47−3.40 (m, 2H), 2.32 (s,

3H, CH₃). ¹³C{¹H} NMR (150 MHz, CDCl₃, 298 K): δ

159.6 (ArC), 138.9 (ArC), 137.4 (ArC), 134.1 (Ar

C), 130.5 (ArC), 130.0 (ArC), 129.2 (ArC), 128.5

(ArC), 127.4 (ArC), 126.2 (ArC), 114.1 (ArC),

AD

https://dx.doi.org/10.1021/acs.joc.0c01404

J. Org. Chem. XXXX, XXX, XXX−XXX

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