Development of Pseudomonas aeruginosa Lectin LecA Inhibitor by using Bivalent Galactosides Supported on Polyproline Peptide Scaffolds

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Accepted Article

Title: Development of Pseudomonas aeruginosa Lectin LecA Inhibitors

using Bivalent Galactosides Supported on Polyproline Peptide

Scaffolds

Authors: Shao-Feng Huang, Cin-Hao Lin, Yu-Tsung Lai, Chia-Lung

Tsai, Ting-Jen Rachel Cheng, and Sheng-Kai Wang

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To be cited as: Chem. Asian J. 10.1002/asia.201701724

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Development of Pseudomonas aeruginosa Lectin LecA Inhibitors

using Bivalent Galactosides Supported on Polyproline Peptide

Scaffolds

Shao-Feng Huang,^[a]Cin-Hao Lin,^[a]Yu-Tsung Lai,^[a]Chia-Lung Tsai,^[a]Ting-Jen R. Cheng^[b]and

Sheng-Kai Wang*^[a]

Abstract: LecA is

a

galactose-binding tetrameric lectin from

infection in the airway of cystic fibrosis or immunocompromised

patients. The antibiotic resistances in P. aeruginosa infection

involve the formation of biofilm, which is partly contributed by a

bacterial lectin called LecA, previously known as PA-IL.^[9]In

addition, LecA is also reported to bind to galactosyl residue

presented on the lung epithelial cell membrane, leading to lung

injury.^[10]Therefore, inhibition of LecA has been studied by many

research groups as a novel strategy to control P. aeruginosa

infection. From the galactoside derivatives that have been

synthesized, it is known that a -aromatic moiety significantly

improves the affinity toward LecA.^[11–15]This binding advantage

was explained by the aid of structural studies of LecA-galactoside

complex showing that the -aromatic group initiates CH-

interaction with histidine residue at the binding site of LecA.^[16]

Another approach to enhance inhibitor interaction to LecA is

through multivalent binding. Because LecA is a tetrameric lectin

and the two closest binding sites are separated by about 26 Å,^[17]

Pseudomonas aeruginosa involved in infection and biofilm formation.

The emergent antibiotic resistance of P. aeruginosa has made LecA

a promising pharmaceutical target to treat such infections. To develop

LecA inhibitors, we exploit the unique helical structure of polyproline

peptides to create a scaffold that controls the galactoside positions to

fit their binding sites on LecA. With a modular scaffold design, both

the galactoside ligands and the inter-ligand distance can be altered

conveniently. We prepared scaffolds with spacings of 9, 18, 27 and

36 Å for ligand conjugation and found that glycopeptides with

galactosides ligands three helical turns (27 Å ) apart best fit LecA. In

addition, we tested different galactose derivatives on the selected

scaffold (27 Å ) to improve binding avidity to LecA. The results validate

a new multivalent scaffold design and provide useful information for

LecA inhibitor development.

18–24]

various artificial scaffolds including dendrimers,^[11,

Introduction

clusters,^[12-14,

and linear rigid spacers^[33–36]have been

25–32]

exploited to present galactoside ligands to the binding sites to

enhance inhibition activity.^[37]In other cases, the artificial scaffolds

are further functionalized to generate extra interactions with LecA

for stronger binding.^[38,39]

Carbohydrate-protein interactions are essential in cellular

processes including cell recognition^[1]and invasion of

pathogens.^[2]Through these interactions, pathogens and host

cells employ carbohydrates as tools for invasion and defense, and

this relationship likely has evolved with time.^[3]Since the late

1920s, the discovery and introduction of antibiotics, which shifted

the balance between bacteria and humans, seemed to solve the

threat of bacterial infection. However, more recently, antibiotic

resistance has emerged rapidly at a speed that new antibiotic

development cannot match,^[4]and multi-resistant genes have

been found widely distributed in bacteria around the world.^[5]

These observations have indicated the arrival of a “post-antibiotic

age”, so the development of anti-bacterial agents with novel

mechanisms is urgently needed.^[6–7]

In this study, we investigated the design of the scaffold connecting

two galactoside ligands for suitable bivalent presentation, as well

as the aromatic moiety that enhances interaction to LecA binding

sites. Notably, instead of using a high-valency cluster or

dendrimer, we focus on a dimeric scaffold with adjustable but

stable spacing to provide spatial selectivity for LecA. To fulfill the

requirements of a stable, adjustable and biocompatible inhibitor,

we employed peptides with the polyproline structure as the

scaffolds. Polyproline is a unique peptide that adopts a left-

handed polyproline helix II (PPII) structure under aqueous media.

This helix structure has an almost 3-fold symmetry along the axis,

and each of its proline residues gives a rise of about 3 Å (Figure

1).^[40]Therefore, when ligands are conjugated to this peptide

scaffold, the direction and spacing can be controlled.

Pseudomonas aeruginosa, one of the ESKAPE pathogens^[8]that

are the principal cause of nosocomial infection, leads to lethal

[a]

[b]

S.-F. Huang, C.-H. Lin, Y.-T. Lai, C.-L. Tsai, and Prof. S.-K. Wang

Department of Chemistry,

National Tsing Hua University,

Hsinchu, Taiwan R.O.C.

E-mail: skwang@mx.nthu.edu.tw

Dr. T.-J. R. Cheng

Genomics Research Center,

Academia Sinica,

Taipei, Taiwan R.O.C.

Figure 1. Structure of polyproline helix II. Left: side view; Right: axial view.

Produced by Discovery Studio Visualizer with structural data from ref. 40.

Supporting information for this article is given via a link at the end of

the document.

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Having a well-defined PPII structure and conformational stability,

this peptide has been exploited for various applications, including

as a molecular ruler to calibrate distance for Förster resonance

energy transfer (FRET)^[41]and as a scaffold to control the location

of ligands to probe cell surface receptors.^[42]With such a peptide

scaffold, we tested bivalent galactoside with varied spacing to find

an optimal design for LecA. Besides the scaffold design, we

introduced the combination of the aromatic moiety with different

linkers on galactosides to reveal their structure-activity

relationship through surface plasmon resonance (SPR) analysis.

reaction, azide substitution and deacetylation reactions. As a

result, the conjugation of 6b to the scaffold would provide two

triazole rings separated by

a methylene group. Likewise,

cycloaddition of propargyl phenol derivatives to 12 installed both

phenyl and triazole rings on the galactosides. Further hydroxyl

group mesylation, azide substitution and deacetylation gave 6c

and 6d with extended para and meta-phenyl groups, respectively.

For the preparation of 6e, the triazole intermediate obtained with

CuAAC reaction between 12 and 4-nitrophenylacetylene was

reduced to give an amine that was further coupled to 5-

azidovaleric acid and deacetylated to give the desired galactoside

6e. In addition to the galactosides derived from galactosyl azide,

we also synthesized 2-azidoethyl--galactose 6f following a

known route,^[45]in which galactosyl trichloroacetimidate was

prepared to glycosylate with 2-azidoethanol and followed by

deacetylation. Galactoside 6f was selected to move the triazole

ring away from the anomeric carbon for comparison with 6a–6e.

Results and Discussion

In this study, we developed LecA inhibitors in two steps: first,

finding a suitable ligand spacing on scaffold to allow strong

bivalent binding to LecA, and second, using the optimized scaffold

to test the derivatives of galactose as ligands for improved avidity.

To allow convenient preparation of a series of galactosides with

varied spacing on the polyproline scaffolds, we introduced

chemical handles to aid ligand conjugation. Such design shares a

common galactoside ligand for different synthetic peptide

scaffolds (or vice versa), and greatly reduces the effort of peptide

synthesis. To exploit the high conjugation efficiency of Cu

catalyzed Alkyne-Azide Cycloaddition (CuAAC) reaction, we

designed the placement of the azide group on glycan and the

alkynyl groups on the peptide scaffold. In addition, due to the

three-fold symmetry of PPII along its axis, a suitable arrangement

of conjugating proline residues was necessary to ensure that the

galactosides would be displayed on the same face of the

polyproline helix. Based on these requirements, we decided to

install a chemical handle on proline ring to control the relative

directions

of

the

two

ligands.

Therefore,

trans-4-

propargyloxyproline building block 1^[43]was employed in Fmoc

solid phase peptide synthesis (SPPS) to locate the alkyne-

modified prolines at the (2, 5), (2, 8), (2, 11) and (2, 14) positions

on peptide 2–5, respectively (Scheme 1). As a result, these

peptides had propargyl groups facing in the same direction, with

ligand spacings of about 9, 18, 27 and 36 Å .

With the peptide scaffolds available, we prepared the first

collection of galactosides, of which the triazole groups were

employed as the aromatic moiety to enhance LecA binding. The

locations and the number of triazole rings were altered as well as

combined with other phenyl groups (Scheme 1). The first

galactose derivative, -galactosyl azide 6a, was prepared from

bromination of peracetylated galactose, which gave

tetraacetylated -galactosyl bromide,^[11]followed by a phase

[44]

transfer catalyzed (PTC) reaction

with tetrabutylammonium

hydrogen sulfate (TBAHS) and sodium azide to provide

tetraacetyl -galactosyl azide 12 (Scheme 2). Then deacetylation

with sodium methoxide provided the desired -galactosyl azide

6a, which afforded the shortest linkage between the galactoside

and the scaffold. The peracetylated galactosyl azide 12 also

served as an intermediate to provide other galactose derivatives.

For example, galactoside 6b was prepared by CuAAC reaction

between propargyl alcohol and 12, followed by Appel bromination

Scheme 1. Design and preparation of glycopeptides based on polyproline helix.

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Figure 2. The circular dichroism spectra of polyproline peptides. a) Alkynyl

peptides 2–5; b) glycopeptides 7a–10a.

Table 1. LecA binding results of glycopeptides with varying ligand spacing

measured by surface plasmon resonance assay

[a]

[b]

k_a

k_d

K_D

Peptide

Scaffold

Ligand

(10³/s M) (10⁻³/s)

(nM)

—

17.9

11.1

—

7.1

—

6.1

7a

8a

9a

10a

7f

2 (9 Å)

6a

6f

inactive

340

ND^[c]

3 (18 Å)

4 (27 Å)

5 (36 Å)

2 (9 Å)

ND^[c]

Scheme 2. Synthesis of azido-galactose derivatives 6a–6f.

5.9

535

ND^[c]

—

5.7

inactive

808

ND

To investigate the effect of ligand spacing toward LecA, we

employed the simple galactosides 6a and 6f as model ligands to

conjugate to the polyproline scaffolds. These galactoside

derivatives with azido chemical handles were subjected to

CuAAC conjugation with alkynyl peptides 2–5 to give

glycopeptides 7a–10a and 7f–10f in good yields (Scheme 1). As

the PPII structure was the basis for our ligand spacing control, we

measured the circular dichroism spectra of alkynyl peptides 2–5

and glycopeptides 7a–10a in the far UV range (Figure 2). The

results indicated that the conjugation of galactoside ligands did

not affect the PPII conformation of the scaffold; therefore, the

spacing control was valid for our system. In order to evaluate the

1338±226

656±268

ND

8f

3 (18 Å)

4 (27 Å)

5 (36 Å)

6f

7.2

3.2

442

9f

6f

—

10f

6f

inactive

[a] SPR condition 1: with 5% DMSO

[b] SPR condition 2: without DMSO. Complete table can be found in supporting

information

[c] Not determined due to solubility in assay conditions

the 26 Å distance between LecA binding sites. To confirm these

results, another simple ligand 6f was employed to conjugate to

scaffolds 2–5 to give glycopeptides 7f–10f. This series of

conjugates showed a similar trend upon LecA binding in that 9f,

with the 27 Å spacing scaffold, was the best of the tested scaffolds.

This set of compounds is particularly interesting because we have

recently created a fluorous microarray system using polyproline

as the scaffold. That microarray system is set to analyzed protein

spatial specificity in order to rapidly develop corresponding

inhibitors.^[46]With a same ligand 6f as used in the microarray

analysis, the present results have validated our previous

prediction to determine the protein preference for a dimer scaffold.

With the conclusion on suitable ligand spacing, we moved on to

investigate the galactoside structures with the selected

polyproline scaffold 4, which separates the conjugated ligands by

three helical turns (27 Å ) on the left-handed helix of PPII. We first

focused on the galactose derived ligands 6b–6e, which had extra

aromatic rings in addition to the triazole ring formed during ligand

conjugation. When the product glycopeptides 9b–9e were

evaluated by SPR, the results indicate that when triazole was

glycopeptide activity, we established

a surface plasmon

resonance (SPR) assay, which immobilized LecA to SPR sensor

chip and flowed glycopeptides through the protein immobilized

surface. This setting would allow a series of glycopeptide samples

to be tested conveniently on the same sensor chip for more

accurate comparison. Due to the solubility of several

glycopeptides, all samples were tested in assay buffer containing

5% DMSO (condition 1), which would require solvent correction.

For the active glycopeptides that were soluble without DMSO,

normal SPR analyses (condition 2, DMSO-free) were further

performed. The binding analysis of glycopeptide 7a–10a indicated

that 7a and 8a bound to LecA very weakly, so reliable kinetic

constants could not be determined (Table 1 and supporting

information). In contrast, peptides 9a and 10a had dissociation

constant (K_D) measurements in the high nanomolar ranges,

where 9a was stronger than 10a. These results suggested that 9a

was the best inhibitor for LecA among the four different scaffolds

for this ligand, and this finding can be explained by the 27 Å

spacing of scaffold 4 being well-suited to

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directly connected to anomeric carbon, in the presence of another

phenyl group, whether as a conjugated system or not, the

compounds were not favored by LecA (9c–9e, Table 2). However,

when two triazole rings were separated by a methylene group in

9b, the dissociation constant was comparable to those of 9a and

9f.

derivative 14i from benzyl 3-hydroxybenzoate and then

transformed it into 6i. An extension of the phenyl group with an

indole moiety was also accomplished by substitution of 5-

nitroindole to 15, which was obtained from PTC glycosylation of

4-iodophenol to tetraacetyl galactosyl bromide. After reduction of

its nitro group to amine, intermediate 16 further coupled to the 6-

azidohexanoic acid linker and gave azido galactoside 6k after

deacetylation.

Table 2. LecA binding results of glycopeptide 9a–9k measured by by surface

plasmon resonance

SPR condition 1^[a]

SPR condition 2^[b]

k_d

.

Compd

k_a

k_d

K_D

(nM)

k_a

K_D

(nM)

(10³/s M) (10⁻³/s)

17.9

3.6

6.1

1.3

340

363

ND^[c]

858±371

ND

9a

9b

9c

9d

9e

9f

—

18.1±3.7 16.1±9.3

inactive

442

—

ND

—

7.2

3.2

31.5±3.0 20.8±9.5

656±268

71±14

ND

45.3

6.1

136

62.3±3.8

4.4±0.7

9g

9h

9i

inactive

174

—

32.4

5.6

34.2±2.8

7.6±1.1

224±48

106±8

ND^[c]

30.3

43.0

8.4

7.7

278

75.2±62.1 8.0±6.7

9j

180

9k

—

Scheme 3. Synthesis of azido-galactose derivatives 6g–6k.

[a] SPR assay performed with 5% DMSO

[b] SPR assay performed without DMSO

[c] Not determined due to solubility in assay conditions

The prepared 6g–6k^[47]azido galactosides were conjugated to

scaffold 4 to give glycoconjugate 9g–9k to test the effect of the

aromatic moiety in LecA binding (Scheme 1). The SPR assays on

9g–9k indicated that this series of glycopeptide were generally

better than the 9a–9f, the exception of 9h, which did not initiate

significant binding to LecA (Table 2 and supporting information).

Among this set of glycopeptides, the enhancement of the aromatic

moiety of p-phenyl and 2-naphthyl (correspond to peptide 9g and

9j, respectively) have been observed by Reymond et al.,^[11]Roy

et al.,^[12]Chevolot and Morvan et al.^[13]These selected ligands with

aromatic aglycon indeed performed better than the triazole

galactosides of 9a–9e. Notably, m-phenyl derivative 9i gave

bivalent binding comparable to that of 9g, while 1-naphthyl

modification on 9h was significantly weaker than the 2-naphthyl

moiety on 9j. Compared to p-phenyl aglycon, the 1-naphthyl

moiety had an extra fused ring extruding sideways that may play

a role in this discrimination in LecA. However, it is interesting that

an indole moiety connected to p-phenyl on peptide 9k was

tolerated by LecA with a comparable K_Dto 9g. To understand how

the ligand may interact with LecA, we performed molecular

modeling analysis of truncated 6k with the LecA complex

structure from a protein data base (3ZYB).^[19]The docking results

We further investigated the aromatic derivatives that have been

reported to initiate strong interactions to LecA, such as para-

phenyl and naphthyl galactosides, which have been

demonstrated by the Reymond group,^[11,16]Roy and Imberty

groups^[12], and Morvan and Chevolot groups^[13]to be good

inhibitors. We prepared these moieties as well as their derivatives

with the synthetic routes shown in Scheme 3. The synthesis of

galactoside 6g was adapted from a reported strategy;^[13]PTC

glycosylation of tetraacetyl galactosyl bromide with 4-nitrophenol,

followed by reduction of the nitro group through catalytic

hydrogenation to provide intermediate 13g. Then the coupling of

the linker molecule 6-azidohexanoic acid to 13g and subsequent

deacetylation gave 6g. With a similar approach, 5-amino-1-

naphthol was glycosylated with tetraacetyl galactosyl bromide to

give 13h and then transformed to 6h. The preparation of

intermediate 14j was carried out according to the reported

methods^[13]with PTC glycosylation of benzyl 6-hydroxy-2-

naphthoate and hydrogenolysis. Then coupling of 14j to 2-(2-

Azidoethoxy)ethanamine and deacetylation afforded galactoside

6j. Similar procedures were exploited to prepare the meta-phenyl

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(Figure 3) indicated that the way of truncated ligand 6k binding to

LecA was similar to the interaction reported in the -

phenylgalactopyranoside–LecA complex structure,^[16]where the

hydroxyl groups (3′ and 4′ OHs) directly interact with calcium ion

and the p-phenyl ring initiates T-shape CH– interaction with the

His50 residue (Figure 3). The distance from the His50 C(1)–H to

the centroid of the phenyl ring^[16]is 2.4 Å , which is shorter than the

expected distance of van der Waals interaction at 2.9 Å ,^[48]

suggesting a contribution to the binding interactions. In addition,

the indole substituent slightly rotates from the attached phenyl

ring and may initiate interaction with the Pro51 residue to benefit

binding to LecA.^[13,49]

It has been reported that -phenyl moiety may account for more

than 5 fold affinity enhancement to LecA in terms of dissociation

constant.^[11,16]In our case when two -phenyl galactosides

combined in a bivalent binding to LecA, we would expect the 9g

to have much better avidity than that of 9f. It is possible that our

current linker design did not allow both -phenyl galactosides of

9g to perfectly orient in LecA binding sites to fully initiate CH-

interaction. Therefore, there are opportunities for modifications to

further improve the activity of our bivalent LecA inhibitors, such as

slightly increase the length of the linker.

dimer, 82 nM, ITC),^[38]and Chevolot, Morvan et al. (pyranose

cluster, <20 nM, fluorescence).^[13]However, it is difficult to draw a

direct comparison between the activities since different methods

were used to measure the dissociation constants. Nonetheless,

our strategy provides a convenient method to generate a potent

lead glycoconjugate that can be further refined by modifying the

scaffold and ligands. Due to the relatively rigid PPII structure, the

activity also reflects the arrangement of the binding sites on

protein to aid future inhibitor developments. In addition, as the

polyproline scaffolds with different ligand spacing used in this

study were not yet further customized for a specific protein, this

system can be applied to selectively target other multivalent

proteins simply by switching the conjugated ligand. Therefore, we

expect that lead multivalent inhibitors for other proteins can be

developed conveniently with this scaffold system. Moreover, as

polyproline peptides have been demonstrated to have high

proteolytic stability in serum,^[50]our scaffolds have potential for

pharmaceutical applications.

Conclusions

In conclusion, we introduce a new scaffold based on the well-

defined polyproline helix II structure to prepare bivalent inhibitors

for LecA. By adjusting the ligand spacing, a preferred scaffold to

match the binding site arrangement on LecA was identified as the

one having ligands separated by 3 helical turns at 27 Å . Such

design enhances the avidity for LecA and also benefits the spatial

selectivity due to scaffold rigidity. The bivalent inhibitor activity

was further improved through convenient ligand changes,

effected by the use of chemical handles on the scaffold. From the

tested galactosides, we found that indole extension from phenyl

galactoside provides opportunities for further modifications.

These results demonstrate the capability of using polyproline

scaffolds to develop multivalent inhibitors against LecA, and this

strategy can be extended to other protein targets in the

development of multivalent inhibitors.

Experimental Section

Materials: Lectin LecA was purchased from Sigma-Aldrich; SPR sensor

chip CM5 was purchased from GE Healthcare.

General chemical synthesis: All chemicals were purchased as reagent

grade without further purification. Ethyl acetate (EtOAc) and hexanes were

distilled before column chromatography. Dichloromethane (CH₂Cl₂) and

acetonitrile (CH₃CN) were distilled over calcium hydride. Tetrahydrofuran

(THF) was extra-dry grade stored with molecular sieves purchase from

Acros. Anhydrous N,N-dimethylformamide (DMF) was purchase from JT

Baker. Molecular sieves for glycosylation were AW-300 (Aldrich) and

activated by flame and microwave under vacuum. Reactions were

monitored with analytical thin layer chromatography (TLC) on E. Merck

silica gel 60G F₂₅₄plate and visualized under UV (254 nm) and staining

with p-anisaldehyde, ceric ammonium molybdate, ninhydrin or potassium

permanganate. Column chromatography was carried out on Silica Gel 60

(E. Merck). High resolution ESI mass spectra were measured on a Bruker–

Impact HD, EVOQ system. Matrix-assisted laser desorption ionization-

time of flight (MALDI-TOF) mass spectra were measured on a Bruker

Figure 3. Interaction model of truncated 6k with LecA by molecular docking. 6k

is shown in green and calcium ion in magneta. a) 6k binding to surface of LecA;

b) Interaction 6k with LecA residues His50 (center), Pro51 (right) in cyan.

The activities of our glycoconjugates are comparable to the

glycodendrimers reported by Roy et al. (230 nM, SPR).^[21]Others

have also developed very potent LecA inhibitors through

structural modifications and refinements, such as Reymond et al.

(glycopeptide dendrimer, 2.5 nM, ITC),^[22]Pieters et al. (rigid dimer,

28 nM, ITC),^[34]Imberty, Römer, Winssinger et al. (glycopeptide

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Daltonics Autoflex III smartbeam LRF200-CID spectrometer. Reverse-

phase HPLC was performed on an Agilent technologies 1260 Infinity

system. Peptides were purified with a Vydac C18 (25 cm x 10 mm) column

operating at a flow rate of 3 mL min^-1using a mobile phase of 0.1% TFA

in H₂O and acetonitrile. All reactions were carried out in oven-dried

glassware under argon atmosphere. NMR spectra were recorded on a

Mercury 400 or Bruker AV-400 spectrometers and the chemical shift δ is

expressed in parts per million (ppm) units. For ¹H NMR spectra, chemical

shifts are expressed in ppm from CDCl₃(7.24 ppm) or CD₃OD (3.31 ppm)

or D₂O (4.80 ppm) or (CD₃)₂CO (2.04 ppm) or (CD₃)₂SO (2.50 ppm).

Coupling constants (J) are reported in hertz (Hz) and the multiplicity

assigned as s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet. ¹H NMR

spectra were reported in this order: chemical shifts (multiplicity, coupling

constant(s), number of proton).

General procedure for the Cu-catalyzed alkyne azide cycloaddition

reaction (protocol E): To a solution of the azido glycoside (1 equiv.) and

desired propargyl aglycon (1.1 equiv.) in acetone was added aqueous

CuSO₄solution (1 equiv.) and then sodium ascorbate aqueous solution (2

equiv.). The reaction mixture was stirred vigorously for 2 h. After TLC

indicated the completion of reaction, water (30 mL) was added and the

aqueous layer was extracted twice with CH₂Cl₂. The combined organic

phase was washed with brine, dried over Na₂SO₄, filtered, and evaporated

for further purification.

2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl azide (12)

According to protocol A, penta-O-acetyl D-galactopyranoside 11 (1.0 g,

2.56 mmol) underwent bromination to provide glycosyl bromide. The

solution of the bromide intermediate in CH₂Cl₂(14mL) was added TBAHS

(0.9 g, 2.65 mmol), NaN₃(0.8 g, 12.3 mmol) and 14 mL of NaHCO_3(sat.)(aq),

and stirred vigorously. After phase transfer catalyzed (PTC) glycosylation

completed, the reaction mixture was diluted with EtOAc and washed

sequentially with NaHCO_3(sat.)(aq)(× 3), water, brine. The organic phase

was dried over Na₂SO₄, concentrated and purified using silica gel column

chromatography (EtOAc/hexanes = 1:2) to give product 12 (740 mg, 77%).

¹H NMR (400 MHz, CDCl₃) δ 5.39 (dd, J = 3.4, 1.1 Hz, 1H, H-4), 5.13 (dd,

J = 10.4, 8.7 Hz, 1H, H-2), 5.00 (dd, J = 10.4, 3.4 Hz, 1H, H-3), 4.57 (d, J

= 8.7 Hz, 1H, H-1), 4.18–4.09 (m, 2H, H-6), 3.98 (td, J = 6.4, 1.0 Hz, 1H,

H-5), 2.14, 2.06, 2.03, 1.95 (s, 3H × 4, Ac). The results show good

accordance with the reported data.^[51]

Galactose derivative synthesis

General procedure for the glycosyl bromide synthesis and PTC

(phase transfer catalyzed) glycosylation (protocol A): To the solution

of acetylated glycoside (compound 11) in CH₂Cl₂stirred at 0 °C under an

iced bath was slowly added 33% HBr in acetic acid and stirred for 0.5 h.

Then the ice bath was removed to allow the reaction mixture gradually

return to room temperature and continued stirring for 1.5 h. Upon

completion of reaction, the reaction mixture were quenched by iced water

and diluted with EtOAc. The resultant organic phase was washed with

saturated aqueous NaHCO₃(× 3), brine, dried over Na₂SO₄and

evaporated to dryness. The resulted glycosyl bromide was kept under

vacuum for

1

h

and dissolved in CH₂Cl₂with 1.1 equiv. of

equiv. of

O-(4-Aminophenyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside

(13g).

tetrabutylammonium hydrogen sulfate (TBAHS) and

2

corresponding aglycons, then 1 M aqueous NaOH was added to the

reaction mixture and stirred vigorously for few hours. Upon completion, the

reaction mixture was diluted with EtOAc and the organic phase was

sequentially washed with 1 M NaOH(aq) (× 3), water, brine, then dried over

Na₂SO₄. After solvent evaporation, the residue was purified using silica gel

chromatography to give the desired product.

According protocol A, penta-O-acetyl galactopyranoside 11 (2.00 g, 5.12

mmol) was dissolved in 4 mL 33% HBr in acetic acid for bromination. After

bromination procedures, to the glycosyl bromide dissolved in 25 mL

CH₂Cl₂was added TBAHS (1.74 g, 5.12 mmol), 4-nitrophenol (0.93 g, 6.66

mmol), and then 8 mL 1 M NaOH(aq). After PTC glycosylation procedure

and extraction in protocol A, the crude was purified by silica gel

chromatography (EtOAc/hexanes/CH₂Cl₂= 1:5:0.6 to 1:2:0.3) to provide O-

(4-Nitrophenyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (1.17 g,

General procedure for hydrogenation or hydrogenolysis (protocol B):

To a solution of the starting material dissolved or suspended in methanol

with 10% v/v formic acid was added 10% Pd/C. The air in the reaction

container was removed under vacuum for few seconds and back-filled with

hydrogen gas for 3−5 times. Then the reaction mixture was stirred under

an atmosphere of hydrogen for few hours. After the completion of the

reaction, the reaction mixture was diluted with methanol and filtered

[ ]³⁰

= −7.9 (c 1.0, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 8.20–8.17

D

49%).

α

(m, 2H), 7.07−7.05 (m, 2H), 5.50 (dd, J = 10.4, 7.9 Hz, 1H, H-2), 5.45 (app.

d, J = 3.3 Hz, 1H, H-4), 5.15 (d, J = 7.9 Hz, 1H, H-1), 5.12 (dd, J = 10.4,

3.3 Hz, 1H, H-3), 4.22−4.09 (m, 3H, H-6a, H-6b, H-5), 2.17, 2.05, 2.00 (s,

3H × 4, Ac); HRMS (ESI): calc. for C₂₀H₂₃NNaO₁₂[M + Na]⁺: 492.1112,

found: 492.1112. The results show good accordance with the reported

data.^[52]

through

a pad of Celite to remove the catalyst. The filtrate was

concentrated and purified with flash column chromatography.

General procedure for deacetylation (protocol C): To a solution of

acetylated starting material dissolved in methanol under ice bath was

added 30% NaOMe in methanol. Upon completion, the reaction mixture

was diluted with methanol and neutralized with Dowex 50 WX2 resins. The

resins were removed by filtration and further rinsed with additional

methanol. The combined filtrate was evaporated under reduced pressure

to give the deacetylated product.

According to protocol B, the previous intermediate (1.17 g, 2.50 mmol)

was dissolved in 20 mL methanol, then 100 mg 10% Pd/C was added for

hydrogenation. After the hydrogenation procedure in protocol B had been

performed, the filtrate was concentrated and purified by silica gel

chromatography (EtOAc/hexanes = 1:3 to 1:1) to provide compound 13g

[ ]³⁰

1

(0.96 g, 87%).

α

= +5.7 (c 1.0, CHCl₃); H NMR (400 MHz, CDCl₃) δ

D

6.81−6.77 (m, 2H), 6.57−6.54 (m, 2H), 5.41−5.36 (m, 2H, H-2, H-4), 5.03

(dd, J = 10.4, 3.4 Hz, 1H, H-3), 4.82 (d, J = 8.0 Hz, 1H, H-1), 4.20−4.04

(m, 2H, H-6a, H-6b), 3.94 (app. t, J = 6.7 Hz, 1H, H-5), 3.54 (br, 2H, NH₂),

2.12, 2.04, 2.00, 1.96 (s, 3H × 4, Ac); HRMS (ESI): calc. for C₂₀H₂₅NNaO₁₀

[M + Na]⁺: 462.1371, found: 462.1371. The results show good accordance

with the reported data.^[53]

General procedure for the linker coupling to glycan (protocol D): To

a solution of galactoside derivatives with amino or carboxylic acid groups

(13, 14 and 16) and corresponding azido linker dissolved in CH₂Cl₂stirred

at 0 °C, was added N,N-Diisopropylethylamine (DIEA) and HATU. The

reaction mixture was then allowed to return to room temperature and

stirred overnight. Upon completion of reaction, the mixture was diluted with

CH₂Cl₂and washed sequentially with saturated aqueous NaHCO₃, water,

and brine. After the organic phase was dried over Na₂SO₄, filtered, and

concentrated, the residue was purified by silica gel chromatography to give

the coupled product.

O-(5-Aminonaphthyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside

(13h).

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According to protocol A, penta-O-acetyl galactopyranoside 11 (2.0 g, 5.13

mmol) was dissolved in 4 mL of 33% HBr in acetic acid for bromination.

After performing bromination procedures, the resultant glycosyl bromide

was dissolved in CH₂Cl₂(25 mL) and added TBAHS (1.25 g, 3.67 mmol),

5-amino-1-naphthol (1.23 g, 7.70 mmol), and 1M NaOH(aq) (8 mL) with

vigorous stir. After the PTC glycosylation and extraction procedures in

protocol A, the resulted crude product was purified by silica gel

chromatography (EtOAc/hexanes/CH₂Cl₂= 1:4:0.5 to 1:1:0) to give

(1.30 g, 4.69 mmol), and 1 M NaOH(aq) (6 mL) with vigorous stir. After

performing PTC glycosylation and extraction procedures described in

protocol A, the crude intermediate was purified by silica gel

chromatography (EtOAc/hexanes = 1:4 to 1:2) to provide Benzyl 4’-

(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyloxy)-2-naphthoate (0.85 g,

42.0%).¹H NMR (400 MHz, CDCl₃) δ 8.56 (s, 1H), 8.06 (dd, J = 8.6, 1.5

Hz, 1H), 7,85 (d, J = 9.0 Hz, 1H), 7.73 (d, J = 8.6 Hz, 1H), 7.46 (d, J = 7.0

Hz, 2H), 7.39−7.31 (m, 4H), 7.22 (dd, J = 9.0, 2.4 Hz, 1H), 5.55 (dd, J =

10.4, 7.9 Hz, 1H, H-2), 5.48 (d, J = 3.3 Hz, 1H, H-4), 5.38 (s, 2H), 5.23 (d,

J = 7.9 Hz, 1H, H-1), 5.15 (dd, J = 10.4, 3.3 Hz, 1H, H-3), 4.24−4.14 (m,

3H, H-5, H-6a, H-6b), 2.17, 2.05, 2.04, 2.00 (s, 3H × 4, Ac); ¹³C NMR (100

MHz, CDCl₃) δ 170.2, 170.1, 170.0, 169.3, 166.4, 156.3, 136.5, 136.0,

131.2, 130.9, 128,9, 128.5, 128.2, 127.1, 126.2, 126.1, 119.4, 110.8, 99.1,

71.2, 70.7, 68.5, 66.8, 66.7, 61.5, 20.62, 20.56, 20.54, 20.48; HRMS (ESI):

calc. for C₃₂H₃₂NaO₁₂[M + Na]⁺: 631.1786, found: 631.1789.

[ ]²⁵

= −41.5 (c 1.0, CHCl₃); ¹H NMR (400

D

compound 13h (0.77 g, 31%).

α

MHz, CDCl₃) δ 7.51 (app. t, J = 8.8 Hz, 2H, Ar), 7.29 (t, J = 8.1 Hz, 1H, Ar),

7.24 (t, J = 7.9 Hz, 1H, Ar), 7.02 (d, J = 7.6 Hz, 1H, Ar), 6.75 (d, J = 7.2 Hz,

1H, Ar), 5.66 (dd, J = 10.4, 7.9 Hz, 1H, H-2), 5.47 (d, J = 3.1 Hz, 1H, H-4),

5.14 (dd, J = 10.4, 3.4 Hz, 1H, H-3), 5.12 (d, J = 7.8 Hz, 1H, H-1),

4.26−4.06 (m, 3H, H-6a, H-6b, H-5), 2.15, 2.04, 2.01, 2.00 (s, 3H × 4, Ac);

¹³C NMR (100 MHz, CDCl₃) δ 170.24, 170.19, 170.0, 169.6, 153.2, 141.9,

126.5, 126.3, 124.5, 124.2, 115.8, 112.1, 110.3, 109.0, 99.9, 70.9, 70.6,

68.4, 66.8, 61.3, 20.7, 20.5(3C); HRMS (ESI): calc. for C₂₄H₂₈NO₁₀[M +

H]⁺: 490.1708, found: 490.1711.

According to protocol B, the intermediate (313 mg, 0.515 mmol) was

dissolved in 5 mL CH₂Cl₂with 10% v/v (0.5 mL) formic acid and added

10% Pd/C (30mg) for hydrogenolysis. After performing hydrogenolysis

procedures in protocol B, the crude product was purified by silica gel

chromatography (EtOAc/hexanes = 1:1) to give product 14j (217 mg, 81%).

¹H NMR (400MHz, CDCl₃) δ 8.63 (s,1H), 8.08 (d, J = 8.5 Hz, 1H), 7.89 (d,

J = 9.0 Hz, 1H), 7.77 (d, J = 8.7 Hz, 1H), 7.36 (d, J = 1.7 Hz, 1H), 7.24 (dd,

J = 8.8, 2.1 Hz, 1H), 5.55 (dd, J = 10.4, 8.0 Hz, 1H, H-2), 5.49 (d, J = 3.2

Hz, 1H, H-4), 5.24 (d, J = 7.9 Hz, 1H, H-1), 5.16 (dd, J = 10.4, 3.3 Hz, 1H,

H-3), 4.28−4.14 (m, 3H, H-5, H-6), 2.18, 2.06, 2.05, 2.01 (s, 3H × 4, Ac);

¹³C NMR (100 MHz, CDCl₃) δ 171.6, 170.4, 170.2, 170.1, 169.4, 156.7,

137.0, 131.9, 131.4, 129.0, 127.4, 126.3, 125.5, 119.6, 110.9, 99.1, 71.3,

70.8, 68.6, 66.9, 61.5, 20.72, 20.66 (2C), 20.6; HRMS (ESI): calc. for

C₂₅H₂₆NaO₁₂[M + Na]⁺: 541.1316, found: 541.1309. The results show

good accordance with the reported data.^[13]

3-(Tetra-O-acetyl-β-D-galactopyranosyloxy)benzoic acid (14i).

According to protocol A, compound 11 (1.72 g, 4.41 mmol) was dissolved

in 4 mL of 33% HBr in acetic acid for bromination. After bromination

procedures, the resultant glycosyl bromide was dissolved in CH₂Cl₂(20

mL), and added TBAHS (2.5 g, 4.41 mmol), 3-benzyloxycarbonylphenol

(1.36 g, 5.96 mmol) and 7 mL 1M NaOH(aq) with vigorous stir. After PTC

glycosylation and extraction procedures, the crude intermediate was

purified by silica gel chromatography (EtOAc/hexanes/CH₂Cl₂= 1:4:0.5 to

1:2:0.3) to give the intermediate O-(3-Benzyloxycarbonylphenyl)-2,3,4,6-

[ ]³¹

tetra-O-acetyl-β-D-galactopyranoside (0.94 g, 38%).

α

= −45.0 (c 1.0,

D

CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 7.78−7.76 (m, 1H), 7.68−7.67 (m,

1H), 7.43−7.30 (m, 6H), 7.18 (ddd, J = 8.2, 2.6, 0.9 Hz, 1H), 5.48 (dd, J =

10.2, 7.9 Hz, 1H, H-2), 5.44 (app. d, J = 3.4 Hz, 1H, H-4), 5.33 (d, J = 1.2

Hz, 2H, CH₂Ar), 5.10 (dd, J = 10.2, 3.4 Hz, 1H, H-3), 5.08 (d, J = 7.9 Hz,

1H, H-1), 4.15 (d, J = 6.5 Hz, 2H, H-6), 4.06 (app. t, J = 6.5 Hz, 1H, H-5),

2.16, 2.04, 2.02, 1.99 (s, 3H × 4, Ac); ¹³C NMR (100 MHz, CDCl₃) δ 170.4,

170.2, 170.0, 169.3, 165.7, 156.7, 135.8, 131.7, 129.6, 128.6, 128.3, 128.1,

124.5, 122.0, 117.6, 99.4, 77.2, 71.2, 70.8, 68.5, 66.9, 61.4, 20.7, 20.6,

20.5 (2C); HRMS (ESI): calc. for C₂₈H₃₀NaO₁₂[M + Na]⁺: 581.1629, found:

581.1638.

O-(4-Iodophenyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranose (15).

According to protocol A, compound 11 (1.00 g, 2.56 mmol) was dissolved

in 2 mL of 33% HBr in acetic acid for bromination. After bromination

procedures performed, the glycosyl bromide dissolved in 12 mL CH₂Cl₂

was added TBAHS (0.87 g, 2.56 mmol), 4-iodophenol (0.85 g, 3.84 mmol),

and 4.5 mL 1 M NaOH(aq), with vigorous stir. After performing PTC

glycosylation and extraction procedures as in protocol A, the crude product

was purified by silica gel chromatography (EtOAc/hexanes/CH₂Cl₂=

[ ]²⁶

According to protocol B, the intermediate from the previous step (491 mg,

0.879 mmol) was dissolved in 5 mL CH₂Cl₂with 10% v/v formic acid, and

added 10% Pd/C (50 mg) for hydrogenolysis. After performing

hydrogenolysis procedures in protocol B, the crude product was purified

1:4:0.5 to 1:2:0.3) to give compound 15 (0.70 g, 50%). α = +7.4 (c 1.0,

D

CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 7.58−7.55 (m, 2H), 6.77−6.74 (m,

2H), 5.44 (dd, J = 10.4, 7.9 Hz, 1H, H-2), 5.42 (d, J = 3.0 Hz, 1H, H-4),

5.07 (dd, J = 10.4, 3.4 Hz, 1H, H-3), 4.97 (d, J = 7.9 Hz, 1H, H-1),

4.21−4.06 (m, 2H, H-6a, H-6b), 4.03 (app. t, J = 6.6 Hz, 1H, H-5), 2.15,

2.04, 1.98 (s, 3H × 4, Ac); ¹³C NMR (100 MHz, CDCl₃) δ 170.3, 170.2,

170.1, 169.3, 156.7, 138.4, 119.1, 99.4, 86.1, 71.0, 70.7, 68.4, 66.7, 61.3,

20.7, 20.65, 20.64, 20.56; HRMS (ESI): calc. for C₂₀H₂₃INaO₁₀[M + Na]⁺:

573.0228, found: 573.0232. The results show good accordance with the

reported data.^[54]

by silica gel chromatography (EtOAc/hexanes = 1:2 to 1:1) to give

[ ]³¹

1

compound 14i (356 mg, 86%). α = −2.5 (c 0.5, CHCl₃); H NMR (400

D

MHz, CDCl₃) δ 7.81 (d, J = 7.7 Hz, 1H, Ar), 7.71 (s, 1H, Ar), 7.38 (app. t, J

= 7.9 Hz, 1H, Ar), 7.23 (d, J = 7.8 Hz, 1H, Ar), 5.50 (dd, J = 10.4, 7.9 Hz,

1H, H-2), 5.45 (d, J = 3.4 Hz, 1H, H-4), 5.12 (dd, J = 10.4, 3.4 Hz, 1H, H-

3), 5.11 (d, J = 7.9 Hz, 1H, H-1), 4.18 (d, J = 6.2 Hz, 2H, H-6), 4.11 (app.

t, J = 6.2 Hz, 1H, H-5), 2.17, 2.06, 2.05, 2.00 (s, 3H×4, Ac); ¹³C NMR (100

MHz, CDCl₃) δ 170.9, 170.5, 170.2, 170.1, 169.4, 156.7, 130.8, 129.6,

124.9, 122.7, 117.5, 99.1, 71.2, 70.7, 68.5, 66.9, 61.6, 20.6, 20.53, 20.46

(2C); HRMS (ESI): calc. for C₂₁H₂₄NaO₁₂[M + Na]⁺: 491.1160, found:

491.1151.

O-(4-(5-Amino-1H-indol-1-yl)phenyl)-2,3,4,6-tetra-O-acetyl-β-D-

galactopyranoside (16).

Compound 15 (440 mg, 0.80 mmol), 5-nitroindole (156 mg, 0.96 mmol),

CuI (46 mg, 0.24 mmol), K₂CO₃(276 mg, 2.00 mmol) and L-proline (55 mg,

0.48 mmol) were premixed and maintained under vacuum for 1 h. Then

degassed DMSO (4 mL) was added to the mixture and stirred at 100 °C

under argon atmosphere for 8 h. After returned to room temperature, the

reaction mixture was diluted with EtOAc (20 mL), and filtered through a

layer of Celite. The in vacuo-concentrated filtrate was re-dissolved in

pyridine (3 mL) and added acetic anhydride (2 mL) with catalytic amount

DMAP (4-Dimethylaminopyridine) to stir for 2 h. The reaction was

6-(2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyloxy)-2-naphthoic acid

(14j).

According to protocol A, compound 11 (1.30 g, 3.34 mmol) was dissolved

in 3 mL of 33% HBr in acetic acid for bromination. After performing

bromination procedure, the glycosyl bromide dissolved in CH₂Cl₂(17 mL)

was added TBAHS (1.25 g, 3.67 mmol), benzyl-6-hydroxy-2-naphthoate

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quenched by MeOH under ice bath, and concentrated under reduced

pressure. The resultant residue was purified by silica gel chromatography

(EtOAc/hexanes = 1:4 to 1:1) to give intermediate O-(4-(5-Nitro-1H-indol-

1-yl)phenyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (331 mg, 71%).

NaHCO₃(aq) and brine. The organic layer was dried over Na₂SO₄and

evaporated in vacuo. The crude residue dissolved in dry DMF (2 mL) was

added NaN₃(80 mg, 5 equiv.) and stirred at 60 °C for 24 h. After

completion of reaction, CH₂Cl₂was added to dilute the reaction solution

and washed with distilled water and brine. The organic layer was dried

over Na₂SO₄, filtered, and evaporated to afford the crude product as an

off-white solid. Recrystallize from CH₂Cl₂/hexanes provided 1-(4-

azidomethyl-[1,2,3]-triazol-1-yl)-2,3,4,6-Tetra-O-acetyl-1-deoxy-β-D-

galactopyranoside as an white needle shaped crystalline intermediate

(110 mg, 97%). ¹H NMR (400 MHz, CDCl₃) δ 7.84 (s, 1H, triazole), 5.83 (d,

J = 9.3 Hz, 1H, H-1), 5.52 (app. t, J = 9.8 Hz, 1H, H-2) 5.53–5.52 (m, 1H,

H-4), 5.23 (dd, J = 10.3, 3.3 Hz, 1H, H-3), 4.47 (s, 2H, –CH₂N₃), 4.23–4.07

(m, 3H, H-5 and H-6), 2.19, 2.01, 1.98, 1.86 (s, 3H × 4, Ac). The results

show good accordance with the reported data.^[57]

[ ]²⁸

α

= +4.7 (c 1.0, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 8.61 (d, J = 2.2

D

Hz, 1H, Ar), 8.08 (dd, J = 9.1, 2.2 Hz, 1H, Ar), 7.42−7.37 (m, 4H, Ar),

7.19−7.15 (m, 2H, Ar), 6.82 (dd, J = 3.4, 0.5 Hz, 1H, Ar), 5.52 (dd, J = 10.4,

7.9 Hz, 1H, H-2), 5.47 (dd, J = 3.4, 0.6 Hz, 1H, H-4), 5.13 (dd, J = 10.4,

3.4 Hz, 1H, H-3), 5.11 (d, J = 7.9 Hz, 1H, H-1), 4.27−4.23 (m, 1H, H-6a),

4.18−4.14 (m, 1H, H-6b), 4.11−4.08 (m, 1H, H-5), 2.19, 2.09, 2.05, 2.01 (s,

3H × 4, Ac); ¹³C NMR (100 MHz, CDCl₃) δ 170.2, 170.1, 170.0, 169.3,

155.9, 141.9, 138.7, 133.6, 131.3, 128.1, 126.0, 118.1, 118.0, 117.7, 110.1,

105.3, 99.3, 71.0, 70.6, 68.4, 66.7, 61.2, 20.6, 20.5 (2C), 20.4; HRMS

(ESI): calc. for C₂₈H₂₈N₂NaO₁₂[M + Na]⁺: 607.1534, found: 607.1535.

According to protocol B, the intermediate from the previous step (235 mg,

0.402 mmol) dissolved in methanol (4 mL) was added 10% Pd/C (23 mg)

for hydrogenation. After performing hydrogenation procedure in protocol B,

the crude product was purified by silica gel chromatography

(EtOAc/hexanes = 1:2 to 1:1 with 1% Et₃N) to give compound 16 (170 mg,

According to protocol C, the intermediate (110 mg, 0.24 mmol) from the

previous step was dissolved in 1 mL MeOH at 0 °C and added 130 μL of

30% NaOMe in methanol. After deacetylation procedures performed, the

residue was evaporated in vacuo without further purification to give

compound 6b (42 mg, 68%). ¹H NMR (400 MHz, D₂O) δ 8.32 (s, 1H,

triazole), 5.72 (d, J = 9.2 Hz, 1H, H-1), 4.58 (s, 2H, -CH₂N₃), 4.23 (t, J =

9.5 Hz, 1H, H-2), 4.09 (d, J = 3.2 Hz, 1H, H-4), 4.01 (t, J = 6.2 Hz, 1H, H-

5), 3.90 (dd, J = 9.6, 3.2 Hz, 1H, H-3), 3.79 (d, J = 6.2 Hz, 2H, H-6); HRMS

(ESI): calc. for C₉H₁₄N₆NaO₅[M + Na]⁺: 309.0918, found: 309.0911. The

results show good accordance with the reported data.^[57]

[ ]²⁵

= +3.1 (c 1.0, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 7.38−7.36

D

76%).

α

(m, 2H, Ar), 7.27 (d, J = 8.8 Hz, 1H, Ar), 7.17 (d, J = 3.2 Hz, 1H, Ar),

7.12−7.09 (m, 2H, Ar), 6.95 (d, J = 2.2 Hz, 1H, Ar), 6.65 (dd, J = 8.7, 2.2

Hz, 1H, Ar), 6.46 (d, J = 3.2 Hz, 1H, Ar), 5.50 (dd, J = 10.4, 7.9 Hz, 1H, H-

2), 5.46 (d, J = 2.9 Hz, 1H, H-4), 5.12 (dd, J = 10.4, 3.4 Hz, 1H, H-3), 5.06

(d, J = 7.9 Hz, 1H, H-1), 4.26−4.22 (m, 1H, H-6a), 4.18−4.13 (m, 1H, H-

6b), 4.08−4.02 (m, 1H, H-5), 3.55 (br, 2H, NH₂), 2.18, 2.09, 2.04, 2.01 (s,

3H × 4, Ac); ¹³C NMR (100 MHz, CDCl₃) δ 170.3, 170.2, 170.1, 169.3,

154.9, 140.1, 135.5, 130.9, 130.1, 128.2, 125.2, 117.9, 113.0, 110.8, 105.7,

102.3, 99.8, 71.1, 70.8, 68.6, 66.8, 61.3, 20.7, 20.6 (2C), 20.5; HRMS

(ESI): calc. for C₂₈H₃₁N₂O₁₀[M + H]⁺: 555.1973, found: 555.1980.

1-(4-(4-(2-azidoethoxy)-phenoxymethyl)-[1,2,3]-triazol-1-yl)-1-deoxy-

β-D- galactopyranoside (6c)

Compound

12

(120

mg,

0.32

mmol)

and

2-(4-

propargyloxyphenoxy)ethanol (76 mg, 0.38 mmol) were dissolved in

acetone (3 mL) to react according to protocol E procedures. The

purification by silica gel chromatography (EtOAc/hexanes = 1:2 to 2:1)

afforded the triazole intermediate (140 mg, 77%). ¹H NMR (400 MHz,

CDCl₃) δ 7.87 (s, 1H), 6.90−6.81 (m, 4H), 5.82 (d, J = 9.3 Hz, 1H, H-1),

5.53 (app. t, J = 10.0 Hz, 1H, H-2), 5.53–5.50 (m, 1H, H-4), 5.22 (dd, J =

10.3, 3.3 Hz, 1H, H-3), 5.12 (s, 2H, triazole–CH₂), 4.22–4.07 (m, 3H, H-5

and H-6), 4.00 (t, J = 4.5 Hz, 2H), 3.93−3.87 (m, 2H), 2.18, 2.01, 1.97, 1.83

(s, 3H × 4, Ac); ¹³C NMR (100 MHz, CDCl₃) δ 170.3, 169.9, 169.7, 169.0,

153.2, 152.6, 145.1, 121.1, 115.9, 115.5, 86.2, 74.0, 70.7, 69.8, 67.8, 66.8,

62.6, 61.4, 61.2, 20.6 (2C), 20.4, 20.2.

β-D-galactopyranosyl azide (6a)

According to protocol C, compound 12 (84 mg, 0.22 mmol) dissolved in 1

mL MeOH at 0 °C was added 121 μL of 30% NaOMe in methanol solution.

After performing deacetylation procedures, the filtrate was evaporated in

vacuo without further purification to give product 6a (33 mg, 71%). ¹H NMR

(400 MHz, D₂O) δ 4.68 (d, J = 8.4 Hz, 1H, H-1), 3.98 (s, 1H, H-4), 3.80–

3.77 (m, 3H, H-5, H-6), 3.70 (d, J = 9.6 Hz, 1H, H-3), 3.53 (t, J = 9.6 Hz,

1H, H-2); HRMS (ESI): calc. for C₆H₁₁N₃NaO₅[M + Na]⁺: 228.0591, found

228.0592. The results show good accordance with the reported data.^[55]

To a solution of the triazole intermediate (120 mg, 0.21 mmol) from the

previous step in dry CH₂Cl₂(2 mL) stir under nitrogen at 0 °C was added

Et₃N (42 L, 0.3 mmol) and methanesulfonyl chloride (18 L, 0.24 mmol).

After the disappearance of starting material based on TLC, the reaction

mixture was diluted with CH₂Cl₂and sequentially washed with 1N HCl(aq),

saturated NaHCO₃(aq) and brine. The organic layer was dried over

Na₂SO₄, filtered, and evaporated to afford the mesylate intermediate as

pale yellow oil, which was dissolved in dry DMF (10 mL), added NaN₃(69

mg, 5 equiv.) and stirred at 50 °C for 24 h. After the completion of reaction,

the DMF in the reaction mixture was removed under reduced pressure.

The resulting residue was dissolved in CH₂Cl₂and sequentially washed

with distilled water and brine. The resulted organic layer was dried over

Na₂SO₄, filtered, and evaporated to afford the crude product as an off-

white solid. Further recrystallize from EtOAc/hexanes gave pure azido

intermediate (104 mg, 84%). ¹H NMR (400 MHz, CDCl₃) δ 7.86 (s, 1H,

triazole), 6.89−6.79 (m, 4H, Ph), 5.83 (d, J = 9.3 Hz, 1H, H-1), 5.51 (dd, J

= 10.3, 9.6 Hz, 1H, H-2) 5.51–5.48 (m, 1H, H-4), 5.22 (dd, J = 10.3, 3.4 Hz,

1H, H-3), 5.10 (s, 2H, triazole–CH₂), 4.23–4.06 (m, 3H, H-5, H-6), 4.05 (t,

J = 5.0 Hz, 2H), 3.51 (t, J = 5.0 Hz, 2H), 2.16, 1.98, 1.95, 1.81 (s, 3H × 4,

Ac); ¹³C NMR (100 MHz, CDCl₃) δ 170.2, 169.8, 169.6, 168.9, 152.72,

152.66, 144.9, 121.1, 115.9, 115.5, 86.1, 73.9, 70.6, 67.7, 67.5, 66.8, 62.4,

1-(4-azidomethyl-[1,2,3]-triazol-1-yl)-1-deoxy-β-D-galactopyranoside

(6b)

Compound 12 (110 mg, 0.3 mmol) and propargyl alcohol (0.1 mL, 1.73

mmol) were dissolved in acetone (3 mL). Then protocol E was performed

and the resulted crude product was purified by silica gel chromatography

(EtOAc/hexanes = 1:2 to 2:1) to give 1-(4-hydroxymethyl-[1,2,3]-triazol-1-

yl)-2,3,4,6-Tetra-O-acetyl-1-deoxy-β-D-galactopyranoside

as

an

intermediate (110 mg, 80%). ¹H NMR (400 MHz, CDCl₃) δ 7.80 (s, 1H,

triazole), 5.82 (d, J = 9.2 Hz, 1H, H-1), 5.48 (app. t, J = 10.4 Hz, 1H, H-2),

5.50−5.45 (m, 1H, H-4), 5.22 (dd, J = 10.5, 3.4 Hz, 1H, H-3), 4.70 (s, 2H,

triazole−CH₂), 4.21 (t, J = 6.4 Hz, 1H, H-5), 4.13−4.02 (m, 2H, H-6), 2.14,

1.95, 1.93, 1.80 (s, 3H × 4, Ac). The results show good accordance with

the reported data.^[56]

To a solution of the intermediate (110 mg ,0.25 mmol) from the previous

step in 2 mL dry THF was added CBr₄(170 mg, 0.5 mmol) and PPh₃(135

mg, 0.5 mmol) to stir under nitrogen at 0 °C. After the disappearance of

starting material based on TLC, CH₂Cl₂was added to dilute the reaction

mixture, which was then sequentially washed with 1 N HCl(aq), saturated

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61.1, 50.1, 20.5 (2C), 20.3, 20.0; HRMS (ESI): calc. for C₂₅H₃₀N₆NaO₁₁[M

+ Na]⁺: 613.1865, found: 613.1879.

off-white solid. Recrystallization from EtOAc/hexanes to give the azido

intermediate (146 mg, 89%). ¹H NMR (400 MHz, CDCl₃) δ 7.87 (s, 1H,

triazole), 7.13 (t, J = 8.1 Hz, 1H, Ph), 6.57−6.47 (m, 3H, Ph), 5.83 (d, J =

9.2 Hz, 1H, H-1), 5.51 (app. t, J = 9.9 Hz, 1H, H-2), 5.50–5.47 (m, 1H, H-

4), 5.22 (dd, J = 10.3, 3.2 Hz, 1H, H-3), 5.13 (s, 2H, triazole−CH₂), 4.21

(app. t, J = 6.4 Hz, 1H, H-5), 4.16–4.04 (m, 4H), 3.51 (t, J = 4.8 Hz, 2H),

2.15, 1.97, 1.94, 1.79 (s, 3H × 4, Ac); ¹³C NMR (100 MHz, CDCl₃) δ 170.1,

169.8, 169.6, 168.8, 159.2 (2C), 144.5, 129.9, 121.1, 107.43, 107.36,

101.8, 86.0, 73.8, 70.6, 67.7, 66.8 (2C), 61.7, 61.1, 49.9, 20.4 (2C), 20.3,

20.0; HRMS (ESI): calc. for C₂₅H₃₀N₆NaO₁₁[M + Na]⁺: 613.1865, found:

613.1874.

According to protocol C, the azido intermediate (104 mg, 0.17 mmol) from

the previous step dissolved in 1 mL MeOH and stirred at 0 °C, was added

95 μL 30% NaOMe in methanol. After deacetylation procedures performed,

the residual solution was evaporated in vacuo without further purification

[ ]³²

= +7.0 (c 0.78, MeOH); ¹H NMR

퐷

to give compound 6c (66 mg, 89%).

α

(400 MHz, (CD₃)₂SO) δ 8.35 (s, 1H, triazole), 7.01−6.89 (m, 4H, Ph), 5.50

(d, J = 9.2 Hz, 1H, H-1), 5.09 (s, 2H, triazole−CH₂), 4.11 (t, J = 4.4 Hz, 2H),

4.04 (app. t, J = 9.2 Hz, 1H, H-2), 3.76 (br, 1H, H-4), 3.71 (t, J = 6.0 Hz,

1H, H-5), 3.61 (t, J = 4.4 Hz, 2H), 3.55−3.48 (m, 3H, H-3, H-6); ¹³C NMR

(100 MHz, (CD₃)₂SO) δ 152.5, 152.2, 142.9, 123.4, 115.6, 115.5, 88.1,

78.4, 73.6, 69.3, 68.4, 67.3, 61.4, 60.4, 49.6; HRMS (ESI): calc. for

C₁₇H₂₂N₆NaO₇[M + Na]⁺: 445.1442, found 445.1437.

According to protocol C, the azido intermediate (146 mg, 0.25 mmol)

stirred in 1 mL MeOH at 0 °C, was added 134 μL 30% NaOMe in methanol.

After deacetylation procedures, the solvents were evaporated in vacuo

[ ]³³

=

퐷

without further purification to furnish compound 6d (70 mg, 67%).

α

+5.4 (c 0.38, MeOH); ¹H NMR (400 MHz, (CD₃)₂SO) δ 8.37 (s, 1H, triazole),

7.21 (t, J = 8.2 Hz, 1H, Ph), 6.68–6.55 (m, 3H, Ph), 5.51 (d, J = 9.2 Hz, 1H,

H-1), 5.14 (s, 2H, triazole−CH₂), 4.16 (t, J = 4.5 Hz, 2H), 4.04 (t, J = 9.2

Hz, 1H, H-2), 3.76 (d, J = 3.1 Hz, 1H, H-4), 3.72 (t, J = 6.0 Hz, 1H, H-5),

3.64 (t, J = 4.5 Hz, 2H), 3.56–3.45 (m, 3H, H-3 and H-6); ¹³C NMR (100

MHz, (CD₃)₂SO) δ 159.3, 159.2, 142.6, 130.1, 123.5, 107.3 (2C), 101.4,

88.1, 78.4, 73.5, 69.2, 68.4, 66.7, 61.0, 60.4, 49.5; HRMS (ESI): calc. for

C₁₇H₂₂N₆NaO₇[M + Na]⁺: 445.1442, found: 445.1445.

Preparation of 2-(4-propargyloxyphenoxy)ethanol:

To a solution of 4-propargyloxyphenol^[58](1.6 g, 10.8 mmol) in dry DMF

(40 mL) with excess anhydrous K₂CO₃(9 g) at 90 °C was dropwise added

2-bromoethanol (1.6 mL, 21 mmol) over 2 h. The reaction mixture was

heated for additional 30 h, then cooled and filtered. The brown oily residue

from evaporated filtrate was dissolved in CH₂Cl₂(250 mL) and washed

with distilled water (100 mL × 2) and brine solution (100 mL). The organic

layer was dried over anhydrous Na₂SO₄and concentrated to allow

recrystallization of the residue from EtOAc/hexanes to give 2-(4-

propargyloxyphenoxy)ethanol (610 mg, 30%). ¹H NMR (400 MHz, CDCl₃)

δ 6.92−6.84 (m, 4H), 4.62 (d, J = 2.4 Hz, 2H), 4.03−4.01 (m, 2H),

Preparation of 2-(3-propargyloxyphenoxy)ethanol:

To a solution of 3-Propargyloxyphenol^[58](1.4 g, 9.5 mmol) dissolved in dry

DMF (40 mL) with excess of anhydrous K₂CO₃(8 g) at 90 °C was dropwise

added 2-bromoethanol (1.6 mL, 21 mmol) with stir over 2 h. The reaction

was heated for an additional 30 h and then cooled and filtered. The brown

oily residue from evaporated filtrate was dissolved in CH₂Cl₂(250 mL) and

washed with water (100 mL × 2) and brine (100 mL). The organic layer

was dried over anhydrous Na₂SO₄and concentrated to give the crude

intermediate that were further purified by recrystallization from

EtOAc/hexanes to give 2-(3-propargyloxyphenoxy)ethanol (542 mg, 29%).

¹H NMR (400 MHz, CDCl₃) δ 7.18 (t, J = 8.6 Hz, 1H), 6.59−6.54 (m, 3H),

4.66 (d, J = 2.4 Hz, 2H), 4.06−4.04 (m, 2H), 3.95−3.93 (m, 2H), 2.51 (t, J

= 2.4 Hz, 1H), 2.02 (t, J = 6.4 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 159.7,

158.6, 129.9, 107.6, 107.3, 102.0, 78.4, 75.5, 69.1, 61.2, 55.7; HRMS

(ESI): calc. for C₁₁H₁₂NaO₃[M + Na]⁺: 215.0679, found: 215.0681.

3.94−3.90 (m, 2H), 2.48 (t, J = 2.4 Hz, 1H), 1.99 (t, J = 6.4 Hz, -OH); ¹³

C

NMR (100 MHz, CDCl₃) δ 153.3, 151.9, 116.1, 115.4, 78.7, 75.4, 69.7,

61.3, 56.5; HRMS (ESI): calc. for C₁₁H₁₂NaO₃[M + Na]⁺: 215.0679, found:

215.0678.

1-(4-(3-(2-azidoethoxy)-phenoxymethyl)-[1,2,3]-triazol-1-yl)-1-deoxy-

β-D- galactopyranoside (6d)

Compound

12

(135

mg,

0.32

mmol)

and

2-(3-

propargyloxyphenoxy)ethanol (76 mg, 0.38 mmol) were dissolved in

acetone (3 mL) to perform protocol E procedures. Purification of the crude

product by silica gel chromatography (EtOAc/hexanes = 1:2 to 2:1)

afforded the desired triazole intermediate (165 mg, 87%). ¹H NMR (400

MHz, CDCl₃) δ 7.88 (s, 1H, triazole), 7.15 (t, J = 8.1 Hz, 1H, Ph), 6.58−6.49

(m, 3H, Ph), 5.83 (d, J = 9.3 Hz, 1H, H-1), 5.53 (app. t, J = 10.0 Hz, 1H, H-

2), 5.53–5.49 (m, 1H, H-4), 5.22 (dd, J = 10.3, 3.1 Hz, 1H, H-3), 5.16 (s,

2H, triazole–CH₂O−), 4.24–4.06 (m, 3H, H-5 and H-6), 4.02 (t, J = 4.3 Hz,

2H), 3.91 (t, J = 4.3 Hz, 2H), 2.18, 2.00, 1.97, 1.82 (s, 3H × 4, Ac); ¹³C

NMR (100 MHz, CDCl₃) δ 170.3, 169.9, 169.7, 169.1, 159.8, 159.3, 144.8,

130.0, 121.2, 107.5, 107.4, 101.9, 86.2, 74.0, 70.7, 69.2, 67.8, 66.8, 61.8,

61.3, 61.2, 20.6 (2C), 20.4, 20.1.

1-(4-(4-(5-Azidopentanamido)phenyl)-[1,2,3]-triazol-1-yl)-1-deoxy-β-

D-galactopyranoside (6e)

Compound 12 (110 mg, 0.3 mmol) and 1-Ethynyl-4-nitrobenzene (44 mg,

0.295 mmol) were dissolved in 3 mL 50% (v/v) aqueous acetone solution.

Then protocol E was performed with sonication at 40 °C for 2 h and the

resulted crude residue was purified by silica gel chromatography to give 1-

(4-(4-nitrophenyl)-[1,2,3]-triazol-1-yl)-2,3,4,6-tetra-O-acetyl-1-deoxy-β-D-

1

galactopyranoside (45 mg, 32%) as an intermediate. H NMR (400 MHz,

To a solution of the triazole intermediate (164 mg, 0.29 mmol) in dry

CH₂Cl₂(1.5 mL) at 0 °C stirred under nitrogen was added triethylamine (60

L, 0.45 mmol) and methanesulfonyl chloride (26 L, 0.32 mmol). After the

disappearance of starting material based on TLC, the reaction solution was

diluted with CH₂Cl₂and washed sequentially washed with 1N HCl(aq),

saturated NaHCO₃(aq) and brine. The organic layer was dried over

Na₂SO₄, filtered, and evaporated to afford the mesylate intermediate as

pale yellow oil, which was dissolved in dry DMF (10 mL) and added NaN₃

(69 mg, 5 equiv.) and stirred for 24 h at 50 °C. After the completion of

reaction, the DMF in the reaction mixture was removed under reduced

pressure. The resulting residue was dissolved in CH₂Cl₂and sequentially

washed with distilled water and brine. The resulted organic layer was dried

over Na₂SO₄, filtered, and evaporated to afford the crude product as an

CDCl₃) δ 8.28 (d, J = 8.8 Hz, 2H), 8.18 (s, 1H, triazole), 8.01 (d, J = 8.8 Hz,

2H), 5.90 (d, J = 9.3 Hz, 1H, H-1), 5.57 (dd, J = 10.1, 9.4 Hz, 1H, H-2),

5.56 (d, J = 2.9 Hz, 1H, H-4), 5.27 (dd, J = 10.3, 3.3 Hz, 1H, H-3), 4.26 (t,

J = 6.2 Hz, 1H, H-5), 4.22−4.14 (m, 2H, H-6), 2.22, 2.03, 2.00, 1.89 (s, 3H

× 4, Ac); ¹³C NMR (100 MHz, CDCl₃) δ 170.3, 169.8, 169.7, 169.3, 147.6,

146.2, 136.2, 126.4, 124.3, 119.5, 86.5, 74.3, 70.6, 67.9, 66.8, 61.1, 20.6

(2C), 20.4, 20.2; HRMS (ESI): calc. for C₂₂H₂₅N₄O₁₁[M+H]⁺: 521.1514,

found: 521.1514.

To a solution of the previous intermediate (45 mg, 0.086 mmol) in methanol

(0.5 mL) was added acetic acid (0.35 mL) and zinc powder (20 mg, 0.301

mmol) to stir under argon for 30 min. Then the reaction mixture was diluted

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with methanol and filtered through a pad of Celite. The residue from the

concentrated filtrate was purified using flash column (MeOH/CHCl₃= 1:10)

to give 1-(4-(4-aminophenyl)-[1,2,3]-triazol-1-yl)-2,3,4,6-tetra-O-acetyl-1-

deoxy-β-D-galactopyranoside (38.7 mg, 91%) as an intermediate.

8.0 Hz, 1H, H-1), 4.17 (dd, J = 11.3, 6.6 Hz, 1H, H-6a), 4.10 (dd, J = 11.3,

6.7 Hz, 1H, H-6b), 4.03 (ddd, J = 10.6, 4.7, 3.5 Hz, 1H), 3.90 (td, J = 6.7,

1.0 Hz, 1H, H-5), 3.67 (ddd, J = 10.7, 8.5, 3.3 Hz, 1H), 3.49 (ddd, J = 13.4,

8.5, 3.5 Hz, 1H), 3.28 (ddd, J = 13.4, 4.6, 3.3 Hz, 1H), 2.14, 2.05, 2.03,

1.96 (s, 3H × 4, Ac). The results show good accordance with the reported

data.^[60]

A solution of intermediate from last step (33.4 mg, 0.068 mmol), 5-

azidovaleric acid (8 mg, 0.056 mmol), HATU (39 mg, 0.1 mmol) and HOBt

(7.5 mg, 0.056 mmol) in DMF (0.5 mL) were stirred at room temperature

and added DIEA (23 L, 0.130 mmol) to react for overnight. Upon

completion of reaction, the reaction mixture was diluted with CH₂Cl₂and

sequentially washed with saturated aqueous NaHCO₃, water, and brine.

After dried over Na₂SO₄, the concentrated organic phase was then purified

by silica gel chromatography (EtOAc/hexanes = 1:3 to 1:1) to give 1-(4-(4-

(5-Azidopentanamido)phenyl)-[1,2,3]-triazol-1-yl)-2,3,4,6-tetra-O-acetyl 1-

deoxy-β-D-galactopyranoside (36 mg, 84%) as an intermediate.

According to protocol C, the azido intermediate (380 mg, 0.48 mmol) from

the previous step dissolved in 1 mL MeOH and stirred at 0 °C was added

260 μL of 30% NaOMe in methanol. After deacetylation procedures, the

residual solution was evaporated in vacuo without further purification to

give compound 6f as product (225 mg, 99%). ¹H NMR (400 MHz, D₂O) δ

4.46 (dd, J = 7.8, 1.1 Hz, 1H, H-1), 4.10−4.06 (m, 1H), 3.95 (d, J = 3.4 Hz,

1H, H-4) 3.89−3.70 (m, 4H), 3.68 (ddd, J = 9.9, 3.4, 1.2 Hz, 1H, H-3),

3.60−3.54 (m, 3H); HRMS (ESI): calc. for C₈H₁₅N₃NaO₆[M + Na]⁺:

272.0853, found: 272.0848. The results show good accordance with the

reported data.^[61]

According to protocol C, the previous intermediate (36 mg, 0.057 mmol)

dissolved in 0.5 mL MeOH and stirred at 0 °C was added 1.3 μL 30%

NaOMe in methanol solution. After deacetylation procedures, the residue

was evaporated in vacuum without further purification to give compound

O-(4-(6-Azidohexanamido)phenyl)-β-D-galactopyranoside (6g).

[ ]³²

= −12.4 (c 1.0, MeOH); ¹H NMR (400 MHz,

퐷

6e (14.1 mg, 55%).

α

According to protocol D, compound 13g (150 mg, 0.341 mmol) and 6-

azidohexanoic acid (73 μL, 0.512 mmol) were dissolved in 1.5 mL DMF at

0 °C, to which DIEA (92 μL, 0.512 mmol), HATU (195 mg, 0.512 mmol)

was added sequentially. After coupling procedure and extraction in

protocol D, the residual solution was concentrated and purified by silica

gel chromatography (EtOAc/hexanes = 1:3 to 1:1) to give O-(4-(6-

Azidohexanamido)phenyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside

CD₃OD) δ 8.54 (s, 1H, triazole), 7.79 (d, J = 8.6 Hz, 2H), 7.66 (d, J = 8.6

Hz, 2H), 5.61 (d, J = 9.2 Hz, 1H, H-1), 4.20 (app. t, J = 9.2 Hz, 1H, H-2),

4.01 (d, J = 3.0 Hz, 1H, H-4), 3.87 (t, J = 5.9 Hz, 1H, H-5), 3.83−3.76 (m,

2H, H-6), 3.73 (dd, J = 9.5, 3.2 Hz, 1H, H-3), 3.36 (t, J = 6.7 Hz, 2H,

N₃CH₂-), 2.43 (t, J = 7.3 Hz, 2H, NHCO-CH₂-), 1.83−1.75 (m, 2H, CH₂),

1.71−1.64 (m, 2H, CH₂); ¹³C NMR (100.6 MHz, MeOD) δ 174.1, 148.7,

140.1, 127.3, 127.2, 121.4, 120.6, 90.4, 80.0, 75.3, 71.5, 70.4, 62.5, 52.2,

37.3, 29.4, 24.0; HRMS (ESI): calc. for C₁₉H₂₅N₇NaO₆[M+Na]⁺: 470.1759,

found: 470.1759.

[ ]³¹

1

(150 mg, 76 %) as an intermediate.

α

= +4.0 (c 1.0, CHCl₃); H NMR

D

(400 MHz, CDCl₃) δ 7.42−7.38 (m, 2H), 6.96−6.92 (m, 2H), 5.44 (dd, J =

10.4, 8.0 Hz, 1H, H-2), 5.42 (d, J = 3.4 Hz, 1H, H-4), 5.07 (dd, J = 10.4,

3.4 Hz, 1H, H-3), 4.95 (d, J = 8.0 Hz, 1H, H-1), 4.22−4.11 (m, 2H, H-6a, H-

6b), 4.01 (app. t, J = 6.7 Hz, 1H, H-5), 3.26 (t, J = 6.8 Hz, 2H), 2.33 (t, J =

7.4 Hz, 2H), 2.15, 2.05, 2.03, 1.99 (s, 3H × 4, Ac), 1.77−1.70 (m, 2H),

1.65−1.58 (m, 2H), 1.47−1.40 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 170.9,

170.3, 170.2, 170.0, 169.4, 153.4, 133.4, 121.3, 117.5, 100.1, 71.0, 70.7,

68.6, 66.8, 61.3, 51.1, 37.1, 28.5, 26.3, 24.9, 20.7, 20.6 (2C), 20.5; HRMS

(ESI): calc. for C₂₆H₃₄N₄NaO₁₁[M + Na]⁺: 601.2116, found: 601.2129.

1-(2-Azidoethyl)-β-D-galactopyranoside (6f)

To a stirred solution of Penta-O-acetyl-D-galactopyranose 11 (1.0 g, 2.54

mmol) in DMF (12 mL) under argon was added hydrazine acetate (0.29 g,

3 mmol). After TLC (EtOAc/hexanes = 1:1) showed the disappearance of

starting material, the reaction mixture was diluted with EtOAc, washed with

5% NaCl(aq) and distilled water, and dried over Na₂SO₄. After evaporation

of solvents under reduced pressure, the crude product was purified by

According to protocol C, the solution of previous intermediate (34 mg,

0.059 mmol) dissolved in 0.5 mL MeOH and stirred at 0 °C, was added 6

μL of 30% NaOMe in methanol solution. After performing deacetylation

procedures, the residual solution was evaporated in vacuum without

[ ]³¹

= −32.0 (c

D

silica

gel

chromatography

to

give

2,3,4,6-tetra-O-acetyl-D-

1

galactopyranose (710 mg, 80%) as an intermediate. H NMR (400 MHz,

CDCl₃) δ 5.44 (app. t, J = 3.0 Hz, 1H, H-1), 5.41 (d, J = 3.1 Hz, 1H, H-4),

5.35 (dd, J = 10.7, 3.3 Hz, 1H, H-3), 5.08 (dd, J = 10.8, 3.5 Hz, 1H, H-2),

4.42 (t, J = 6.6 Hz, 1H, H-5), 4.13−4.03 (m, 2H, H-6a, H-6b), 2.09, 2.04,

2.00, 1.94 (s, 3H × 4, Ac).The results show good accordance with the

reported data.^[59]

further purification to give compound 6g (23 mg, 94%).

α

0.5, MeOH); ¹H NMR (400 MHz, CD₃OD) δ 7.47−7.43 (m, 2H), 7.09−7.05

(m, 2H), 4.81 (d, J = 7.7 Hz, 1H, H-1), 3.90 (app. d, J = 3.3 Hz, 1H, H-4),

3.81−3.72 (m, 3H, H-6a, H-6b, H-2), 3.67 (app. t, J = 6.1 Hz, 1H, H-5), 3.57

(dd, J = 9.7, 3.4 Hz, 1H, H-3), 3.33−3.29 (m, 2H), 2.36 (t, J = 7.5 Hz, 2H),

1.76−1.68 (m, 2H), 1.68−1.60 (m, 2H), 1.50−1.42 (m, 2H); ¹³C NMR (100

MHz, CD₃OD) δ 174.2, 155.8, 134.4, 122.6, 118.1, 103.3, 77.0, 74.9, 72.3,

70.2, 62.4, 52.3, 37.6, 29.7, 27.4, 26.4; HRMS (ESI): calc. for

C₁₈H₂₆N₄NaO₇[M + Na]⁺: 433.1694, found: 433.1684.

To a solution of the intermediate (1.0 g, 2.87 mmol) from the previous step

in 15 mL CH₂Cl₂under argon was added trichloroacetonitrile (2.9 mL, 10

equiv.) and DBU (0.1 mL, 0.67 mmol). When TLC showed the conversion

completed, the solution was concentrated and purified by flash

chromatography to give the trichloroacetimidate intermediate, which was

dissolved in dry CH₂Cl₂(14 mL) with 2-azidoethanol (1.5 mL, 17.2 mmol)

and activated molecular sieves (4 Å, 1.0 g) to stir at room temperature for

1 h. After the reaction mixture was cooled to – 40 °C, TMSOTf (0.1 mL,

0.55 mmol) was added and the reaction continued to stir at −40 °C for 4 h.

After the completion of the transformation as monitored by TLC, the

reaction was quenched with Et₃N (1.0 mL, 7.2 mmol) at −40 °C. The

reaction mixture was diluted with CH₂Cl₂and washed with NaHCO₃(aq)

and brine. The organic phase was dried over Na₂SO₄, filtered, and

concentrated for column chromatography to give 1-(2-Azidoethyl)-2,3,4,6-

tetra-O-acetyl-β-D-galactopyranoside as an intermediate (0.64 g, 53%). ¹H

NMR (400 MHz, CDCl₃) δ 5.38 (dd, J = 3.4, 1.0 Hz, 1H, H-4), 5.22 (dd, J

= 10.4, 8.0 Hz, 1H, H-2), 5.00 (dd, J = 10.4, 3.4 Hz, 1H, H-3), 4.54 (d, J =

O-(5-(6-Azidohexanamido)naphthalenyl)-β-D-galactopyranoside (6h).

According to protocol D, compound 13h (150 mg, 0.306 mmol) and 6-

azidohexanoic acid (65 μL, 0.460 mmol) was dissolved in 1.5 mL DMF at

0 °C, and DIEA (82 μL, 0.460 mmol), HATU (175 mg, 0.460 mmol) was

added sequentially to the stirred solution. After the coupling and extraction

procedures in protocol D were performed, the residue was concentrated

and purified by silica gel chromatography (EtOAc/hexanes = 1:3 to 1:1) to

give O-(5-(6-Azidohexanamido)naphthalenyl)-2,3,4,6-tetra-O-acetyl-β-D-

[ ]²⁶

galactopyranoside (141 mg, 73%) as an intermediate.

α

= −48.0 (c 1.0,

D

CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 7.98 (d, J = 8.4 Hz, 1H), 7,87 (d, J =

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7.4 Hz, 1H), 7.53–7.38 (m, 4H), 7.07 (d, J = 7.6 Hz, 1H), 5.69 (dd, J = 10.5,

8.0 Hz, 1H, H-2), 5.49 (d, J = 3.2 Hz, 1H, H-4), 5.17 (d, J = 8.0 Hz, 1H, H-

1), 5.15 (dd, J = 10.1, 3.2 Hz, 1H, H-3), 4.29–4.24 (m, 1H, H-6a), 4.19–

4.12 (m, 2H, H-6b, H-5), 3.29 (t, J = 6.4 Hz, 2H), 2.51 (t, J = 7.2 Hz, 2H),

2.19, 2.06, 2.02 (s, 3H × 4, Ac), 1.86–1.79 (m, 2H), 1.68–1.63 (m, 2H),

1.55–1.51 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 171.5, 170.3, 170.2,

170.1, 169.7, 153.3, 132.0, 128.6, 126.4, 125.9, 125.8, 122.3, 119.6, 115.8,

108.9, 100.0, 71.1, 70.7, 68.5, 66.8, 61.3, 51.2, 37.1, 28.6, 26.4, 25.2, 20.8,

20.63 (2C), 20.56; HRMS (ESI): calc. for C₃₀H₃₆N₄NaO₁₁[M + Na]⁺:

651.2273, found: 651.2295.

According to protocol D, the stirred solution of compound 14j (157 mg,

0.304 mmol) and 2-(2-azidoethoxy)ethanamine (0.15 mL, 0.75 mmol) in

1.5 mL DMF at 0 °C was added DIEA (81 μL, 0.455 mmol), HATU (173

mg, 0.455 mmol) sequentially. After performing coupling and extraction

procedures in protocol D, the residual solution was concentrated and

purified by silica gel chromatography (EtOAc/hexanes = 1:3 to 1:1) to give

O-(6-((2-(2-Azidoethoxy)ethyl)carbamoyl)naphthalen-2-yl)-2,3,4,6-tetra-

O-acetyl-β-D-galactopyranoside (179 mg, 94%) as an intermediate.

[ ]³¹

α

= +11.8 (c 1.0, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 8.26 (s, 1H),

D

7.85 (dd, J = 8.6, 1.8 Hz, 1H), 7.83 (d, J = 9.0 Hz, 1H), 7.75 (d, J = 8.6 Hz,

1H), 7.34 (d, J = 2.4 Hz, 1H), 7.22 (dd, J = 8.9, 2.4 Hz, 1H), 5.54 (dd, J =

10.4, 7.9 Hz, 1H, H-2), 5.48 (d, J = 3.4 Hz, 1H, H-4), 5.21 (d, J = 8.0 Hz,

1H, H-1), 5.14 (dd, J = 10.5, 3.4 Hz, 1H, H-3), 4.27−4.22 (m, 1H, H-6a),

4.19−4.12 (m, 2H, H-5, H-6b), 3.75−3.68 (m, 6H, CH₂× 3), 3.41−3.39 (m,

2H, CH₂), 2.18, 2.06, 2.05, 2.01 (4s, 3H × 4, Ac); ¹³C NMR (100 MHz,

CDCl₃) δ 170.3, 170.2, 170.1, 169.4, 167.3, 155.9, 135.7, 130.8, 130.4,

129.1, 127.4, 127.3, 124.5, 119.6, 110.9, 99.2, 71.2, 70.8, 70.2, 69.9, 68.6,

66.9, 61.5, 50.6, 39.7, 20.7, 20.6 (2C), 20.5; HRMS (ESI): calc. for

C₂₉H₃₄N₄NaO₁₂[M + Na]⁺: 653.2065, found: 653.2088.

According protocol C, the intermediate (28 mg, 0.045 mmol) from the

previous step dissolved in 0.5 mL MeOH at 0 °C was added 5 μL of 30%

NaOMe in methanol. After performing deacetylation procedures, the

residual solution was evaporated in vacuo without further purification to

[ ]³¹

= −22.2 (c 1.0, MeOH); ¹H NMR

D

give compound 6h (16 mg, 76%).

α

(400 MHz, CD₃OD) δ 8.36 (d, J = 8.4 Hz, 1H), 7.64 (d, J = 8.5 Hz, 1H),

7.58 (d, J = 7.3 Hz, 1H), 7.49−7.43 (m, 2H), 7.28 (d, J = 7.6 Hz, 2H), 5.08

(d, J = 7.8 Hz, 1H, H-1), 3.99 (dd, J = 9.7, 7.8 Hz, 1H, H-2), 3.95 (d, J =

3.4 Hz, 1H, H-4), 3.83−3.73 (m, 3H, H-6a, H-6b, H-5), 3.64 (dd, J = 9.7,

3.4 Hz, 1H, H-3), 3.35 (t, J = 6.8 Hz, 2H), 2.56 (t, J = 7.4 Hz, 2H), 1.83 (m,

2H), 1.73−1.66 (m, 2H), 1.60−1.52 (m, 2H); ¹³C NMR (100 MHz, CD₃OD)

δ 175.5, 155.0, 133.8, 131.3, 128.1, 127.3, 125.8, 124.8, 122.1, 117.3,

110.6, 103.2, 77.1, 75.0, 72.4, 70.3, 62.4, 52.4, 37.2, 29.7, 27.5, 26.6;

HRMS (ESI): calc. for C₂₂H₂₈N₄NaO₇[M + Na]⁺: 483.1850, found:

483.1838.

According protocol C, a stirred solution of the previous intermediate (53

mg, 0.084 mmol) in 0.5 mL MeOH at 0 °C was added 8 μL of 30% NaOMe

in methanol solution. After performing deacetylation procedures, the

residual solution was evaporated in vacuo without further purification to

[ ]³¹

= –47.4 (c 1.0, MeOH); ¹H NMR

D

give compound 6j (37 mg, 95%).

α

(400 MHz, CD₃OD) δ 8.32 (s, 1H), 7.90 (dd, J = 9.1, 2.1 Hz, 1H), 7.87−7.82

(m, 2H), 7.55 (s, 1H), 7.39−7.36 (m, 1H), 5.06 (d, J = 7.7 Hz, 1H, H-1),

3.94 (d, J = 3.4 Hz, 1H, H-4), 3.88 (dd, J = 9.7, 7.8 Hz, 1H, H-2), 3.85−3.76

(m, 3H, H-6a, H-6b, H-5), 3.73−3.69 (m, 4H), 3.65−3.62 (m, 3H, H-3, CH₂),

3.41−3.39 (m, 2H); ¹³C NMR (100 MHz, CD₃OD) δ 170.4, 158.4, 137.5,

131.5, 131.1, 130.1, 128.6, 128.5, 125.3, 121.0, 111.7, 102.7, 77.2, 74.8,

72.3, 71.0, 70.5, 70.3, 62.5, 51.8, 41.0; HRMS (ESI): calc. for

C₂₁H₂₆N₄NaO₈[M + Na]⁺: 485.1643, found: 485.1636.

O-(3-((2-(2-Azidoethoxy)ethyl)carbamoyl)phenyl)-β-D-

galactopyranoside (6i).

According to protocol D, the stirred solution of compound 14i (227 mg,

0.485 mmol) and 2-(2-azidoethoxy)ethanamine (0.2 mL, 0.970 mmol) in

2.5 mL CH₂Cl₂at 0 °C, was sequentially added DIEA (130 μL, 0.730 mmol),

HATU (277 mg, 0.730 mmol). After the coupling reaction and extraction

were performed as describe in protocol D, the residual solution was

concentrated and purified by silica gel chromatography (EtOAc/hexanes =

1:3 to 1:1) to give O-(3-((2-(2-Azidoethoxy)ethyl)carbamoyl)phenyl)-

2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (205 mg, 73%) as an

O-(4-(5-(6-Azidohexanamido)-1H-indol-1-yl)phenyl)-β-D-

galactopyranoside (6k).

According to protocol D, to a stirred solution of compound 16 (87 mg,

0.157 mmol) and 6-azidohexanoic acid (34 μL, 0.236 mmol) in 1 mL DMF

at 0 °C was added DIEA (42 μL, 0.236 mmol) and HATU (90 mg, 0.236

mmol) sequentially. After performing the coupling and extraction

procedures in protocol D, the residual solution was concentrated and

purified by silica gel chromatography (EtOAc/hexanes = 1:3 to 1:1) to give

[ ]³¹

= −3.2 (c 1.0, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ

D

intermediate.

α

7.46 (t, J = 2.0 Hz, 1H), 7.42 (dt, J = 7.9, 1.2 Hz, 1H), 7.32 (app. t, J = 7.9

Hz, 1H), 7.10 (ddd, J = 8.3, 2.6, 1.0 Hz, 1H), 5.47 (dd, J = 10.5, 7.9 Hz,

1H, H-2), 5.43 (dd, J = 3.4, 0.8 Hz, 1H, H-4), 5.09 (d, J = 8.0 Hz, 1H, H-1),

5.08 (dd, J = 10.4, 3.4 Hz, 1H, H-3), 4.20−4.04 (m, 3H, H-6a, H-6b, H-5),

3.68 (app. t, J = 4.8 Hz, 2H), 3.67−3.72 (m, 4H), 3.39−3.35 (m, 2H), 2.15,

2.04, 2.03, 1.98 (s, 3H × 4, Ac); ¹³C NMR (100 MHz, CDCl₃) δ 170.2, 170.0,

169.8, 169.2, 166.5, 156.7, 135.9, 129.4, 121.2, 120.0, 115.4, 99.0, 70.9,

70.5, 69.9, 69.5, 68.3, 66.7, 61.2, 50.3, 39.5, 20.5, 20.4, 20.3 (2C); HRMS

(ESI): calc. for C₂₅H₃₂N₄NaO₁₂[M + Na]⁺: 603.1909, found: 603.1922.

O-(4-(5-(6-

Azidohexanamido)-1H-indol-1-yl)phenyl)-2,3,4,6-tetra-O-

[ ]²⁶

=

D

acetyl-β-D-galactopyranoside (75 mg, 69%) as an intermediate.

α

+6.1 (c 1.0, CHCl₃); ¹H NMR (400 MHz,CDCl₃) δ 7.87 (d, J = 1.8 Hz, 1H),

7.38−7.35 (m, 3H), 7.30 (br, 1H), 7.24 (d, J = 3.0 Hz, 1H), 7.20 (dd, J = 8.9,

2.0 Hz, 1H), 7.13−7.09 (m, 2H), 6.59 (d, J = 3.1 Hz, 1H), 5.50 (dd, J = 10.4,

8.0 Hz, 1H, H-2), 5.46 (d, J = 3.0 Hz, 1H, H-4), 5.12 (dd, J = 10.4, 3.4 Hz,

1H, H-3), 5.07 (d, J = 8.0 Hz, 1H, H-1), 4.26−4.21 (m, 1H, H-6a), 4.17−4.11

(m, 1H, H-6b), 4.06 (app. t, J = 6.6 Hz, 1H, H-5), 3.26 (t, J = 6.9 Hz, 2H),

2.37 (t, J = 7.4 Hz, 2H), 2.18, 2.08, 2.04, 2.01 (s, 3H × 4, Ac), 1.80−1.73

(m, 2H), 1.66−1.57 (m, 2H), 1.50−1.40 (m, 2H); ¹³C NMR (100 MHz,

CDCl₃) δ 171.1, 170.3, 170.1, 170.0, 169.3, 155.1, 134.9, 133.3, 130.9,

129.1, 128.7, 125.4, 117.9, 116.5, 112.9, 110.3, 103.4, 99.6, 71.0, 70.7,

68.5, 66.8, 61.3, 51.1, 37.2, 28.5, 26.3, 25.1, 20.7, 20.55 (2C), 20.49;

HRMS (ESI): calc. for C₃₄H₃₉N₅NaO₁₁[M + Na]⁺: 716.2538, found:

716.2544.

According to protocol C, the stirred solution of the previous intermediate

(41 mg, 0.072 mmol) in 0.5 mL MeOH at 0 °C was added 7 μL of 30%

NaOMe in methanol. After performing deacetylation procedures, the

residual solution was evaporated in vacuo without further purification to

[ ]³¹

= −35.3 (c 1.0, MeOH);

D

give compound 6i (22 mg, 74%) as product.

α

¹H NMR (400 MHz, CD₃OD) δ 7.55 (s, 1H), 7.47 (dd, J = 7.6, 0.5 Hz, 1H),

7.37 (app. t, J = 7.9 Hz, 1H), 7.29−7.25 (m, 1H), 4.92 (d, J = 7.8 Hz, 1H,

H-1), 3.91 (d, J = 3.2 Hz, 1H, H-4), 3.84−3.66 (m, 8H, H-6a, H-6b, H-2, H-

5, CH₂× 2), 3.61−3.56 (m, 3H, CH₂, H-3), 3.40 (app. t, J = 4.0 Hz, 2H); ¹³

C

NMR (100 MHz, CD₃OD) δ 170.0, 159.2, 137.0, 130.6, 122.0, 121.0, 116.6,

102.8, 77.0, 74.8, 72.2, 70.9, 70.3, 70.2, 62.4, 51.7, 40.9; HRMS (ESI):

calc. for C₁₇H₂₄N₄NaO₈[M +Na]⁺: 435.1486, found: 435.1479.

According to protocol C, the intermediate (45 mg, 0.065 mmol) from the

previous step dissolved in 0.5 mL MeOH at 0 °C was added 6 μL of 30%

NaOMe in methanol solution. After deacetylation procedure, the residual

solution was evaporated in vacuum without further purification to give

O-(6-((2-(2-Azidoethoxy)ethyl)carbamoyl)naphthalen-2-yl)-β-D-

galactopyranoside (6j).

28

compound 6k (37 mg, quant).

α

= −25.6 (c 1.0, MeOH); ¹H NMR (400

[

]

D

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MHz, CD₃OD) δ 7.86 (d, J = 1.9 Hz, 1H), 7.43−7.36 (m, 4H), 7.28 (d, J =

8.6 Hz, 2H), 7.23 (dd, J = 8.8, 1.5 Hz, 1H), 6.59 (d, J = 3.0 Hz, 1H), 4.93

(d, J = 7.7 Hz, 1H, H-1), 3.93 (d, J = 3.3 Hz, 1H, H-4), 3.84 (dd, J = 9.6,

7.9 Hz, 1H, H-2), 3.82−3.71 (m, 3H, H-6a, H-6b, H-5), 3.61 (dd, J = 9.6,

3.4 Hz, 1H, H-3), 3.33−3.31 (m, 2H), 2.40 (t, J = 7.4 Hz, 2H), 1.79−1.72

(m, 2H), 1.69−1.62 (m, 2H), 1.52−1.47 (m, 2H); ¹³C NMR (100 MHz,

CD₃OD) δ 174.3, 157.7, 135.5, 134.8, 132.5, 130.6, 130.0, 126.4 (2C),

118.9 (2C), 117.8, 114.1, 111.2, 104.1, 103.1, 77.1, 74.8, 72.3, 70.2, 62.5,

52.3, 37.7, 29.7, 27.5, 26.6; HRMS (ESI): calc. for C₂₆H₃₁N₅NaO₇[M +

Na]⁺: 548.2116, found: 548.2110.

Data for 9a: MALDI-TOF: calc. for C₈₀H₁₁₅N₁₉NaO₂₅[M + Na]⁺1764.82,

found 1765.84 , HPLC gradient 1, R_t= 21.5 min, 83% yield

Data for 10a: MALDI-TOF: calc. for C₉₅H₁₃₆N₂₂NaO₂₈[M + Na]⁺2055.98,

found 2058.38 , HPLC gradient 1, R_t= 24.4 min, Quant.

Data for 9b: MALDI-TOF: calc. for C₈₆H₁₂₁N₂₅NaO₂₅[M + Na]⁺1926.89,

found 1927.65 , HPLC gradient 1, R_t= 22.9 min, Quant.

Data for 9c: MALDI-TOF: calc. for C₁₀₂H₁₃₇N₂₅NaO₂₉[M + Na]⁺2198.99,

found 2199.31 , HPLC gradient 1, R_t= 35.3 min, 51% yield

General procedures for Solid Phase Peptide Synthesis

Data for 9d: MALDI-TOF: calc. for C₁₀₂H₁₃₇N₂₅NaO₂₉[M + Na]⁺2198.99,

found 2199.43 , HPLC gradient 1, R_t= 37.4 min, 57% yield

Rink amide resins (100 mg, 0.55−0.60 mmol/g determined by loading test)

swollen in CH₂Cl₂for 30 min were washed with CH₂Cl₂(3 × 3 mL) and

DMF (3 × 3 mL). The resins were treated with piperidine (20% v/v in DMF,

2 × 3 mL) for 15 min then washed with DMF (3 × 3 mL), CH₂Cl₂(3 × 3 mL)

and DMF (3 × 3 mL). A pre-activated solution of Fmoc-protected amino

acids (4 equiv.), HOBT (4 equiv.), HBTU (4 equiv.) and (i-Pr)₂EtN (4 equiv.)

in DMF (amino acid final concentration 0.2 M) was added to the resin and

the reaction vessel was shaken at 40 °C for 1 h. The resins were washed

with DMF (3 × 3 mL), CH₂Cl₂(3 × 3 mL) and DMF (3 × 3 mL), then treated

with Ac₂O in pyridine (10% v/v, 3 mL) for 10 min. The resins were washed

with DMF (3 × 3 mL), CH₂Cl₂(3 × 3 mL) and DMF (3 × 3 mL). This

procedure was repeated following the designed sequence until all amino

acids were coupled to the resin.

Data for 9e: MALDI-TOF: calc. for C₁₀₆H₁₄₃N₂₇NaO₂₇[M + Na]⁺2249.05,

found 2249.66 , HPLC gradient 3, R_t= 20.8 min, 49% yield

Data for 7f: MALDI-TOF: calc. for C₅₄H₈₁N₁₃NaO₂₁[M + Na]⁺1270.56,

found 1270.88 , HPLC gradient 1, R_t= 14.9 min, 84% yield

Data for 8f: MALDI-TOF: calc. for C₆₉H₁₀₂N₁₆O₂₄[M + Na]⁺1561.72, found

1562.39 , HPLC gradient 1, R_t= 18.9 min, Quant.

Data for 9f: MALDI-TOF: calc. for C₈₄H₁₂₃N₁₉NaO₂₇[M + Na]⁺1852.87,

found 1853.37 , HPLC gradient 1, R_t= 22.6 min, 71% yield.

Cleavage of peptide from Resin and Purification.

Data for 10f: MALDI-TOF: calc. for C₉₉H₁₄₄N₂₂NaO₃₀[M + Na]⁺2144.03,

The resin was treated with mixture of TFA, triisopropylsilane and water

(95:2.5:2.5 v/v/v, 2 × 2 mL), the reaction mixture was shaken at room

temperature for 1 h and then filtered. The resin was washed with CH₂Cl₂

(3 × 3 mL), and the washes were combined with the filtrate and

concentrated. The residue was dissolved in acetonitrile and purified by

reverse phase HPLC using a linear gradient of a solvent system consisting

of solvent A (acetonitrile) and solvent B (water/0.1% TFA). All peptides

were characterized by MALDI-TOF mass spectrometry.

found 2145.34, HPLC gradient 1, R_t= 25.8 min, Quant.

Data for 9g: MALDI-TOF: calc. for C₁₀₄H₁₄₅N₂₁NaO₂₉[M + Na]⁺2175.04,

found 2175.04; HPLC gradient 2, R_t= 15.3 min, 82% yield.

Data for 9h: MALDI-TOF: calc. for C₁₁₂H₁₅₀N₂₁O₂₉[M + H]⁺2253.09, found

2253.96;HPLC gradient 2, R_t= 16.6 min, 71% yield.

Data for 9i: MALDI-TOF: calc. for C₁₀₂H₁₄₁N₂₁NaO₃₁[M + Na]⁺2179.00,

found 2179.79; HPLC gradient 2, R_t= 14.1 min, 90% yield.

General

procedures

for

copper(I)-catalyzed

alkyne-azide

cycloaddition of glycopeptide synthesis:

Data for 9j: MALDI-TOF: calc. for C₁₁₀H₁₄₅N₂₁NaO₃₁[M + Na]⁺2279.03,

found 2279.08; HPLC gradient 2, R_t= 15.5 min, 96% yield.

Glycopeptide 7−10. A solution of azido glycoside (8 equiv., 160 mM) in

DMSO and dialkynyl peptide 2–5 (1 equiv, 10 mM) in H₂O was mixed; then

CuSO₄, sodium ascorbate and tris(triazolylmethyl)amine ligand^[62]were

added to reach 1 mM of Cu(I) and 1 mM of dialkynyl peptide as the final

concentration. The reaction mixture was shaked at room temperature for

1 h, and then purified by reverse phase HPLC with a solvent system

consisting of solvent A (acetonitrile) and solvent B (water/0.1% TFA). The

product was characterized by MALDI-TOF mass spectrometry.

Data for 9k: MALDI-TOF: calc. for C₁₂₀H₁₅₆N₂₃O₂₉[M + H]⁺2383.14, found

2384.14; HPLC gradient 2, R_t= 20.1 min, 64% yield.

Surface Plasmon Resonance Assay

Surface plasmon resonance experiments were performed on a Biacore

T200 system at 25 °C using a functionalized CM5 sensor chip. Protein

immobilization was performed according to the built-in wizard template of

the instrument. The CM5 sensor chip was activated with a solution

containing N-ethyl-N’-(3-diethyl-aminopropyl)-carbodiimide (EDC) (0.2 M)

and N-hydroxysuccinimide (NHS) (0.05 M). LecA (158 μg/mL) in sodium

acetate buffer (10mM, pH 4) was injected over the activated flow cell at a

flow rate of 10 μL/min for 2100 s. Then 1 M ethanolamine at pH 8.5 was

injected to block the remaining activated groups. Binding assays were

performed with HBS-P+ (condition 1: 10 mM HEPES, 150 mM NaCl, 100

M CaCl₂, 0.05% Tween 20, 5% DMSO, pH 7.5; condition 2: 10 mM

HEPES, 150 mM NaCl, 100 M CaCl₂, 0.05% Tween 20, pH 7.5) as

running buffer. Glycopeptides were diluted with the running buffer at

several concentrations ranging from 500nM to 8000nM and injected onto

HPLC analysis gradient 1: A:B = 5:95 to 10:90 for 0−5 min, to 22:78 for

5−25 min, to 42:58 for 25−45 min.

HPLC analysis gradient 2: A:B = 10:90 to 95:5 for 0−45 min

HPLC analysis gradient 3: A:B = 5:95 to 95:5 for 0−60 min

Data for 7a: MALDI-TOF: calc. for C₅₀H₇₃N₁₃NaO₁₉[M + Na]⁺1182.50,

found 1184.25 , HPLC gradient 1, R_t= 13.6 min, Quant.

Data for 8a: MALDI-TOF: calc. for C₆₅H₉₄N₁₆NaO₂₂[M + Na]⁺1473.66,

found 1475.15 , HPLC gradient 1, R_t= 17.6 min, Quant.

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the sensor chip surface at the rate of 30 μL/min. The surface was

regenerated by 30 s injection of 100 mM methyl -D-galactopyranoside

after each binding event. The sensorgrams were solvent corrected (only

condition 1, for 5% DMSO), reference subtracted, quality controlled and

analyzed by Biacore T200 Evaluation Software. The kinetic parameters

were obtained by fitting curves to a 1:1 Langmuir model.

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Molecular docking experiments were performed using AutoDock ver. 4.2

with AutoDockTools ver. 1.5.6 program.^[63]A co-crystallized LecA complex

structure retrieved from Protein Data Bank (PDB ID: 3ZYB) was used for

docking. Water molecules and original ligand were removed and the

hydrogens were added in the pdb file prior to docking. The docking was

performed with protein kept rigid, while the ligand was left flexible in the

grid box containing the binding site with the calcium ion. The Lamarckian

Genetic Algorithm was used, setting 100 independent Genetic Algorithm

runs for ligand; the other parameters were kept at default values.

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Acknowledgements

We gratefully acknowledge the financial supported from the

Ministry of Science and Technology, R.O.C. (MOST 106−2113-

M-007−009).

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Keywords: LecA inhibitor • polyproline • glycoconjugates •

molecular scaffold • glycosides

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Distance matters. Controlled ligand

spacing provides enhanced avidity

and spatial selectivity to proteins with

matching binding site arrangement.

Using structurally well-defined

polyproline helix II peptide as

scaffolds, we develop inhibitor for P.

aeruginosa lectin LecA by controlled

ligand spacing and altering ligand

structures.

Shao-Feng Huang, Cin-Hao Lin, Yu-

Tsung Lai, Chia-Lung Tsai, Ting-Jen R.

Cheng and Sheng-Kai Wang*

Page No. – Page No.

Development of Pseudomonas

aeruginosa Lectin LecA Inhibitors

using Bivalent Galactosides

Supported on Polyproline Peptide

Scaffolds

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Article Doi

Development of Pseudomonas aeruginosa Lectin LecA Inhibitor by using Bivalent Galactosides Supported on Polyproline Peptide Scaffolds

DOI: 10.1002/asia.201701724

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