Synthesis of novel types of polyester glycodendrimers as potential inhibitors of urinary tract infections

DOI: 10.1039/C4NJ00992D

Synthesis of Novel Types of Polyester Glycodendrimers as Potential Inhibitors of

Urinary Tract Infections

a,b,*

a

a,*

Jean-d'Amour K. Twibanire, Nawal K. Paul, and T. Bruce Grindley

a

Department of Chemistry, Dalhousie University, 6274 Coburg Road, P.O. Box 15000, Halifax, N.S. B3H

4

N2, Canada

b

CanAm Bioresearch Inc., Winnipeg, MB R3T 0P4, Canada

*

Corresponding Author

Email(s): twibanire@dal.ca (J.K.T.), bruce.grindley@dal.ca (T.B.G.)

New Journal of Chemistry

Page 2 of 30

DOI: 10.1039/C4NJ00992D

Abstract

Syntheses of highly mannosylated polyester dendrimers with 2, 4, 8, and 16 α-D-mannopyranose

residues on their peripheries connected by different linker arms are presented. Attractive features of

these systems as potential inhibitors of uropathogenic Escherichia coli (UPEC) include the use of non-

toxic and biocompatible polyester dendrimer backbones, aglycones whose lengths are designed to fit in

the tyrosine gate, and multiple copies of α-linked D-mannopyranose residues, capable of effectively

binding to the exposed mannose-sensitive type 1 pili on UPEC’s outer surface.

Key words: polyester glycodendrimers, urinary tract infections, click chemistry, mannosylated systems

2

Page 3 of 30

New Journal of Chemistry

DOI: 10.1039/C4NJ00992D

INTRODUCTION

One of the medically most significant carbohydrate-protein interactions involves the initial

attachment of pathogenic bacteria to target tissues and it has been suggested that this interaction will

, 2

provide a source of alternative antibiotic targets, the anti-adhesion strategy.

For Gram negative

bacteria, these interactions often are between carbohydrate-binding proteins (lectins) called adhesions on

fimbriae protruding from the bacterial surface and carbohydrates on the tissue surface. Urinary tract

4

infections (UTIs), which are mainly caused by strains of gram negative uropathogenic Escherichia coli,

-7

are among the most frequently occurring bacterial diseases in humans. The UPEC bind to the urinary

tract endothelial surface through the 200 to 500 fimbriae which extend from the surface of each bacterial

cell. At the tip of each fimbria there is a lectin, FimH, that recognizes terminal -D-mannopyranoside

8

residues of trimannose-terminated oligosaccharides present on one of the proteins, uroplakin Ia (UPIa)

9

on the endothelial surface. The mannose-binding pocket of FimH is adjacent to a hydrophobic region

0

known as the tyrosine gate that contains two tyrosines and an isoleucine, that accommodates the non-

1, 12

polar faces of the non-terminal mannopyranoses of the UPIa oligosaccharides.

The combination of a

mannose-binding pocket with an adjacent extended hydrophobic region on FimH has resulted in

compounds that are designed to bind to this site being poorly recognized by the many other mannose-

binding receptors in humans. Binding occurs by a catch-bond mechanism that is critical for attachment

4, 15

in the high-sheer environment of the urinary tract.

Infection commences with FimH binding to UPIa

that triggers a conformational change in the cell surfaces, allowing the bacteria to enter and establish

sub-surface colonies that are difficult to eradicate.

A number of compounds have been designed to evaluate the utility of anti-adhesion strategies

against E. coli. Early on Firon et al. found that phenyl α-D-mannopyranosides, particularly the 2-chloro-

4

-nitro derivative, bound effectively, and recently Hultgren and coworkers have shown that α-linked

8

biphenyl derivatives are even more effective. Other phenyl glycosides with planar substituents on the

3

New Journal of Chemistry

Page 4 of 30

DOI: 10.1039/C4NJ00992D

9-21

phenyl ring have also proved to be quite effective,

and these compounds remain attached to FimH

surface long enough to be clinically relevant. Heptyl α-D-mannopyranoside was found to bind to FimH

0

with strengths comparable to the better phenyl glycosides.

Multivalent presentation of binding molecules to proteins has attracted considerable attention,

8, 23-25

and many of these involve binding to FimH.

Dendritic species are becoming increasingly

6-35

important and are now used in a variety of applications.

The ability of dendrimers to present multiple

copies of biologically active residues in a defined fashion has attracted attention because of the

possibility to achieve reproducible results. In particular, polyester dendrimers are attractive as drug

6, 27, 36-46

delivery carriers and numerous reports on their biomedical applications have appeared.

Herein we report the syntheses of two higher generation polyester dendrimers bearing 8 and 16

mannose residues on their surfaces attached by different linker arms. As far as we are aware, the use of

polyester dendrimers based on the 2,2-bis(hydroxymethyl) propanoic acid (bis-HMPA) monomer, in the

preparation of mannosylated systems as potential inhibitors of urinary tract infections has not been

explored. One concept that guided this work was that glycodendrimers based on a non-toxic core could

combine effectiveness with the absence of side effects. Polyester dendrimers constitute an attractive

class of polymers because they are biodegradable, biocompatible, and they have been found to have low

7, 36, 38, 47-49

toxicity in every reported study.

Consequently, the polyester dendrimer scaffold is attractive

for the preparation of biocompatible glycodendrimers.

RESULTS AND DISCUSSION

We are interested in the functionalization of polyester dendrimers to create highly mannosylated

systems as potential therapeutics against urinary tract infections. Based on knowledge of the FimH

binding site outlined in the introduction, two mannose residues (7 and 10) were prepared as shown in

Scheme 1, in order to attach them to dendrimer surfaces using click chemistry. These particular

4

Page 5 of 30

New Journal of Chemistry

DOI: 10.1039/C4NJ00992D

derivatives were selected for the following reasons. As shown in Figure 1, the original p-nitrophenyl

0, 17, 25

10, 25

glycoside

and the heptyl glycoside

both had nM binding constants to FimH. The hexameric

compound shown in Figure 2 had an even better K of 3 nM (that is a per mannose binding constant of

d

8 nM). In view of the fact that the binding constant of the heptyl glycoside is considerably better than

the butyl derivative, it was considered that the closeness of the branching point to the glycosidic centre

of the derivative in Figure 2 considerably hindered its binding. This led to the first set of compounds

(using 7) which have a longer chain after the triazole ring. The second set of compounds (using 10) was

0

derived to more closely mimic heptyl α-D-mannopyranoside.

After this work was underway,

Bouckaert et al. described how compounds having seven heptyl α-D-mannopyranosides grafted either

onto the primary face of β-cyclodextrin or to short amylose chains bound FimH from E. coli well, and

4, 51

also reduced infection by the UTI89 strain in a mouse model.

We hope that the less spatially defined

nature of the current molecules will allow stronger binding because the complexes formed will be

unhindered by adjacent strands and their higher valency will allow binding to greater numbers of

bacterial cells.

0, 25

Figure 1 Binding constants of two monomeric mannosides with FimH

5

New Journal of Chemistry

Page 6 of 30

DOI: 10.1039/C4NJ00992D

Figure 2 A hexameric compound with a K per mannose residue of 18 nM

d

Scheme 1 Synthesis of mannoside residues 7 and 10

60, 65

52, 53

Compounds 7

and 10

were made by literature methods as outlined in Scheme 1. We

found it more efficient to make the 6-azido glycoside via the 6-chlorohexyl glycoside, rather than

52

make 6-azidohexanol first. In other reactions, 6-azidohexanoic acid was prepared for attachment to

click coupling partners of 7, which must be azide-terminated. Accordingly, 6-chlorohexanol was

oxidized to the corresponding acid using Jones reagent followed by an S 2 displacement with sodium

N

azide (Scheme 2).

6

Page 7 of 30

New Journal of Chemistry

DOI: 10.1039/C4NJ00992D

Scheme 2 Preparation of 6-azidohexanoic acid

Scheme 3 Preparation of divalent azide compounds 17 and 20

The TBTU-promoted esterification using 6-azidohexanoic acid for the preparation of azide-

terminated species worked well in DMF. In the case of diols, reactions were high yielding with short

54-56

reaction times as expected based on our previous studies.

As illustrated in Scheme 3, diol 16 reacted

for 4 hours to afford the corresponding diazide 17 in 85% yield, while 19 yielded 20 in 94% yield after 2

hours. To obtain a highly mannosylated system, a third generation polyester dendrimer was divergently

constructed. We have reported the preparation of second generation dendrimer 21. This octaol reacted

7

New Journal of Chemistry

Page 8 of 30

DOI: 10.1039/C4NJ00992D

with a divalent anhydride (22) under standard conditions to give protected third generation dendrimer

23), which gave crystalline dendrimer 24 after hydrogenolysis (see Scheme 4). Third generation

(

dendrimer 24 then afforded 25 in 79% yield after reacting with 6-azidohexanoic acid for 12 hours as

shown in Scheme 5.

Scheme 4 Divergent synthesis of a third generation polyester dendrimer (24)

Scheme 5 Preparation of an azide-terminated third generation polyester dendrimer (25)

8

Page 9 of 30

New Journal of Chemistry

DOI: 10.1039/C4NJ00992D

Having successfully prepared both the alkyne functionalized mannoside 7 and the azide

terminated species; the next step was to connect them using a click reaction. Modified versions of the

click reaction have been developed that do not require toxic copper (I) so that these reactions can even

8-60

be used in living cells.

The reaction has also been used for the synthesis of monomers, dendrons and

1-64

dendrimer skeletons.

Here, using the click reaction, 17 and 20 were reacted with mannoside 7 to give

the corresponding divalent mannoside clusters 26 and 27, respectively (Scheme 6). Most often, this

reaction is carried out using a mixture of water and tetrahydrofuran and this system worked well for the

preparation of divalent clusters. However, this solvent system did not work for the azide-terminated

third generation polyester dendrimer 25, which precipitated whenever water was introduced into the

system. After a few trials, it was found that the reaction worked well in DMF. As shown in Scheme 7,

dendrimer 25 reacted with mannoside 7 to give mannosylated system 28 with 16 mannose residues on its

periphery.

Scheme 6 Synthesis of divalent mannoside clusters 26 and 27

9

New Journal of Chemistry

Page 10 of 30

DOI: 10.1039/C4NJ00992D

Scheme 7 Synthesis of a highly mannosylated system (28)

1

0

Page 11 of 30

New Journal of Chemistry

DOI: 10.1039/C4NJ00992D

Mannoside 10 was used for the synthesis of glycodendrimers with linker arms more closely

resembling heptyl α-D-mannopyranoside. Divalent glycodendrimer 30 was prepared by forming the bis

ester of core molecule 14 with propynoic acid to give 29 that gave divalent glycodendrimer 30 after the

click reaction with 10 in 89% yield (Scheme 8).

Scheme 8 Synthesis of divalent mannoside cluster 30

A partially convergent approach was used to prepare tetramannoside 34 as shown in Scheme 9.

The bispropargyl ether of HMPA (31) was made by the method of Wu et al. Characterization data for

1 are corrected in the experimental. This compound was converted to the anhydride (32), which was

3

then reacted with core molecule 14 to form the tetrapropargyl ether. The copper-catalyzed click reaction

with azide-terminated mannoside 10 gave the tetramannoside 34 in good yield.

1

New Journal of Chemistry

Page 12 of 30

DOI: 10.1039/C4NJ00992D

Scheme 9 Synthesis of tetravalent mannoside cluster 34

It was necessary to adopt a more convergent approach to prepare the third generation polyester

glycodendrimer octamannoside 40 (Scheme 10). Adronov and coworkers prepared the 2-

toluenesulfonylethyl ester of HMPA (35) and demonstrated that it could be used to prepare high

generation HMPA dendrons. We reacted compound 35 with the anhydride of propargylated HMPA to

give the protected second generation dendron 36. Removal of the carboxyl protecting group with DBU

gave the second generation dendron 37. Formation of its anhydride 38 with DCC gave a reactive

intermediate that was converted into the propargylated third generation dendrimer 39 by reaction with

core molecule 14. The copper-catalysed click reaction with the azido-terminated mannoside 10 yielded

the third generation octamannoside 40 in 65% yield.

1

2

Page 13 of 30

New Journal of Chemistry

DOI: 10.1039/C4NJ00992D

1

3

New Journal of Chemistry

Page 14 of 30

DOI: 10.1039/C4NJ00992D

Scheme 10 Synthesis of octavalent mannoside cluster 40

CONCLUSIONS AND SUMMARY

A number of polyester glycodendrimers including compound 28 with 16 terminal mannose

residues, and compound 40, with eight terminal mannose residues, were efficiently prepared and

characterized. Glycodendrimers 28 and 40 are highly mannosylated systems with non-toxic polyester

backbones based on the bis-HMPA monomer. The lengths of the two aglycones were designed to

present alternative approaches for fitting in the tyrosine gate. These polyester glycodendrimers will be

evaluated against urinary tract infections in work to be presented later.

1

4

Page 15 of 30

New Journal of Chemistry

DOI: 10.1039/C4NJ00992D

EXPERIMENTAL SECTION

General Methods

1

13

H and C NMR spectra were recorded on Bruker Avance 500 or Bruker Avance 300 NMR

spectrometers operating at 500.13 and 125.7 MHz or 300.15 and 75.5 MHz respectively using the

solvent resonances as secondary chemical shift references. The carbon and hydrogen atoms were

1

13

assigned following analysis of their one dimensional ( H, C) and two dimensional (COSY, HSQC,

HMBC, and TOCSY) NMR spectral data. Coupling constant (J) values are reported in Hertz. Proton and

carbon assignments shown with location numbers indicate their positions in 1,3-dioxane or mannose.

High-resolution mass spectra were recorded on a Bruker Micro-TOF mass spectrometer using

electrospray ionization. Melting points were determined on a Fisher-John's melting point apparatus and

are uncorrected. Acetone was refluxed over K CO₃and distilled over molecular sieves.

2

Dichloromethane was refluxed over calcium hydride and distilled onto molecular sieves.

Tetrahydrofuran was refluxed over LiAlH and distilled over molecular sieves. Unless otherwise noted,

4

non-aqueous reactions were carried out under a nitrogen atmosphere. Jones reagent (0.56 M) was

prepared by dissolving sodium dichromate dihydrate (Na Cr O .2H O, 300 g, 1.01 mol) in 1.5 L of

2

7

2

o

water followed by slowly adding conc. sulfuric acid (300 mL) to the cooled solution (0 C). Compounds

were visualized/ located by spraying the TLC plate with a solution of 2 % ceric ammonium sulfate in 0.5

M H SO followed by heating on a hot plate until color developed. Solid compounds were purified on

2

4

silica gel using flash column chromatography and specified eluents, or by crystallization. Liquids and

oils were purified using flash column chromatography. Water soluble compounds were purified using

size exclusion chromatography on a Sephadex LH-20 gel column with water as the eluent.

General procedures

Formation of dendritic esters (anhydride coupling): To an oven-dried round-bottomed flask equipped

with a magnetic stir bar under nitrogen atmosphere, the anhydride, the hydroxyl-terminated dendrimer or

1

5

New Journal of Chemistry

Page 16 of 30

DOI: 10.1039/C4NJ00992D

core, and N,N-dimethyl-4-aminopyridine (DMAP) were dissolved in a 3:1 mixture of CH Cl : pyridine

2

(v/v). The reaction mixture was stirred at rt for 4 to 12 h and diluted with water (3 mL) in pyridine (3

mL). Stirring was continued overnight to quench the excess anhydride. The mixture was diluted with

CH Cl (150 mL) and washed using NaHCO (1 M, 30 mL × 3), 10% aq. Na CO (30 mL × 3), brine

2

3

2

3

(

30 mL × 2), and water (30 mL), then dried (MgSO ), filtered, and concentrated. The crude product was

4

then purified using precipitation out of hexanes/ EtOAc or column chromatography to give the desired

product. The NaHCO layers were combined, acidified (pH = 5 – 6), and the carboxylic acid by-product

3

was recovered.

Deprotection using hydrogenolysis: To an oven-dried round-bottomed flask equipped with a magnetic

stir bar, the benzylidene or benzyl protected dendrimer was dissolved in a 1:2:1 mixture of CH Cl :

2

MeOH : THF (v/v/v) and a catalytic amount of Pd/C was added. The flask was evacuated and back-

filled with hydrogen three times. After stirring the mixture overnight under a H atmosphere, the catalyst

2

was filtered off using celite and this celite was washed with MeOH. The filtrate was concentrated to

dryness to afford the product as a colorless solid.

Esterification procedure using TBTU: In an oven-dried round-bottomed flask equipped with a

magnetic stir bar, an acid (1.20 mmol), TBTU (0.387 g, 1.20 mmol), and 1,8-diazabicyclo[5.4.0]undec-

7

-ene (0.37 mL, 2.40 mmol) were dissolved in anhydrous DMF (3 mL) and the resulting mixture was

stirred at rt for 30 min under a nitrogen atmosphere. An alcohol (1.00 mmol of hydroxyl groups) in

DMF (1 mL) was then injected into the reaction mixture via syringe, and stirring was continued at rt

until TLC confirmed the completion of the reaction (1 – 12 h). The reaction mixture was diluted with

CH Cl (15 mL) and the resulting mixture was washed with 5% HCl (2 x 3 mL), 1M NaHCO (3 x 3

2

3

mL) and water (2 x 3 mL). The organic layer was collected, dried (MgSO ), filtered and concentrated to

4

give a crude ester product, which was purified using column chromatography and specified eluents.

1

6

Page 17 of 30

New Journal of Chemistry

DOI: 10.1039/C4NJ00992D

Bis(2-(3-benzyloxy-2,2-bis-(methoxymethyl)propanoyloxy)ethoxy)benzene (15). Compound 15 was

synthesized as described above in the general procedure for formation of dendritic esters. The core diol

1

4 (0.500 g, 2.52 mmol), dry pyridine (5 mL), CH Cl (15 mL), DMAP (0.185 g, 1.51 mmol) and

2 2

anhydride 13 (3.14 g, 6.05 mmol) were stirred at rt for 12 h under nitrogen. After work up and

purification using column chromatography (hexanes/ EtOAc; 2:1; R 0.26), the product was obtained as

F

1

colorless syrup (1.66 g, 94 % yield): H NMR (500.13 MHz, CDCl ) δ 3.31 (s, 12H, 4OCH ), 3.62 (s,

3

8

4

H, 4CH O), 3.69 (s, 4H, 2CH O), 4.09 (t, J = 5 Hz, 4H, ArOCH ), 4.47 (t, J = 5 Hz, 4H, 2CH OC=O),

2 2 2 2

1

3

.52 (s, 4H, 2CH benzylic), 6.82 (s, 4H, PhH), 7.27 – 7.35 (m, 10H, PhH); C NMR (125.7 MHz,

2

CDCl ) δ 172.4 (C=O), 153.0, 138.4, 128.2, 127.4, 127.2, 115.6 (PhC), 73.1 (2CH benzylic), 70.2

3

2

(

4CH OMe), 67.8 (2CH OBn), 66.5 (2PhOC), 62.9 (2COC=O), 59.2 (4OCH ), 53.5 (2C_q_u_a_t). HR ESI

2

3

MS: m/z calcd for C H NaO 721.3194, found 721.3170.

3

8

50

12

Bis(2-(3-hydroxy-2,2-bis-(methoxymethyl)propanoyloxy)ethoxy)benzene (16). Using the general

procedure for hydrogenolysis described above, compound 15 (1.45 g, 2.08 mmol), dissolved in dry

CH Cl (10 mL), dry MeOH (30 mL), and dry THF (15 mL) afforded 16 as a colorless syrup (1.05 g,

2

1

9

3

8% yield): H NMR (500.13 MHz, acetone-d ) δ 3.24 (s, 12H, 4OCH ), 3.51 – 3.55 (m, 8H, 4CH O),

6 3 2

.73 (d, J = 6 Hz, 4H, 2CH O), 3.80 (t, J = 6 Hz, 2H, OH), 4.14 (t, J = 5 Hz, 4H, ArOCH ), 4.39 (t, J = 5

2

1

3

Hz, 4H, 2CH OC=O), 6.90 (s, 4H, PhH); C NMR (125.7 MHz, acetone-d ) δ 173.0 (2C=O), 153.9,

2

6

1

16.4 (PhC), 71.0 (4CH OMe), 67.3 (2PhOC), 63.4 (2COC=O), 61.0 (2CH OH), 59.3 (4OCH ), 54.9

2 2 3

(

2C_q_u_a_t). HR ESI MS: m/z calcd for C H NaO 541.2255, found 541.2254.

24 38 12

Bis(2-(3-(6-azidohexanoyloxy)-2,2-bis-(methoxymethyl)propanoyloxy)ethoxy)benzene

(17).

Compound 17 was synthesized using the general esterification procedure using TBTU. 6-Azidohexanoic

acid 12 (0.190 g, 1.20 mmol) and diol 16 (0.260 g, 1.00 mmol (OH)) were reacted for 4 h to give 17

(0.34 g, 85% yield) as a colorless syrup after purification using column chromatography (hexanes/

1

EtOAc; 3:2; R 0.36): H NMR (300.15 MHz, CDCl ) δ 1.28 – 1.35, 1.47 – 1.59 (2 m, 12H, 3 CH ),

F

3

2

1

7

New Journal of Chemistry

Page 18 of 30

DOI: 10.1039/C4NJ00992D

2

.21 (t, J = 7 Hz, 4H, O=CCH ), 3.19 (t, J = 7 Hz, 4H, CH N), 3.24 (s, 12H, 4OCH ), 3.50 (s, 8H, 2

2 2 3

C_q_u_a_t2CH O), 4.05 (t, J = 4.5 Hz, 4H, ArOCH ), 4.23 (s, 4H, C CH OC=O), 4.39 (t, J = 4.5 Hz, 4H,

2

quat

2

1

3

CH OC=O), 6.77 (s, 4H, PhH); C NMR (75.5 MHz, CDCl ) δ 172.6, 171.5 (C=O), 153.0, 115.7

2

3

(PhC), 70.2 (4CH OMe), 66.5 (ArOCH ), 63.1 (OCH CH O), 62.0 (CH OC=O), 59.3 (4OCH ), 52.4

2 2 2 2 2 3

(

2C_q_u_a_t), 51.1 (CH N ), 33.8 (CH C=O), 28.4, 26.1, 24.3 (CH ). HR ESI MS: m/z calcd for

2

3

2

C H N NaO 819.3747, found 819.3743.

36

56

6

14

Bis((3-benzyloxypropanoyloxy)ethoxy)benzene (18). Compound 18 was synthesized using the

general esterification procedure using TBTU. 3-Benzyloxypropanoic acid (0.216 g, 1.20 mmol) and diol

4 (0.100 g, 1.00 mmol (OH)) were reacted for 1.5 h to give 18 (0.23 g, 89% yield) as a colorless solid

1

after purification using column chromatography (hexanes/ EtOAc; 2:1; R 0.29): mp 105 – 107 °C; H

F

NMR (500.13 MHz, CDCl ) δ 2.69 (t, J = 6.5 Hz, 4H, CH C=O), 3.78 (t, J = 6.5 Hz, 4H, CH OBn),

3

2

4

1

3

.12 (t, J = 4.5 Hz, 4H, PhOCH ), 4.44 (t, J = 4.5 Hz, 4H, CH OC=O), 4.54 (s, 4H, benzylic), 6.85 (s,

2 2

13

H, PhH), 7.29 – 7.35 (m, 10H, PhH); C NMR (125.7 MHz, CDCl ) δ 171.5 (C=O), 153.0, 138.0,

3

28.4, 127.6, 115.6 (PhC), 73.0 (CH , benzylic), 66.5 (PhOCH ), 65.5 (CH OBn), 62.9 (CH OC=O),

2

5.0 (CH C=O). HR ESI MS: m/z calculated for C H NaO 545.2146, found 545.2139.

2

30 34

8

Bis((3-hydroxypropanoyloxy)ethoxy)benzene (19). Using the general procedure for hydrogenolysis

described above, compound 19 (0.750 g, 1.44 mmol), dissolved in dry CH Cl (5 mL), dry MeOH (10

2

mL), and dry THF (5 mL) afforded 19 as a colorless crystalline solid (0.48 g, 98% yield): mp 118 – 120

1

°

C H NMR (500.13 MHz, methanol-d ) δ 2.56 (t, J = 6.5 Hz, 4H, CH C=O), 3.82 (t, J = 6.5 Hz, 4H,

4

2

13

CH OH), 4.13 (t, J = 4.5 Hz, 4H, PhOCH ), 4.40 (t, J = 4.5 Hz, 4H, CH C=O), 6.87 (s, 4H, PhH); C

2

NMR (125.7 MHz, methanol-d ) δ 173.5 (C=O), 154.5, 116.7 (PhC), 67.7 (PhOCH ), 64.2 (CH OC=O),

4

2

5

8.7 (CH OH), 38.4 (CH C=O). HR ESI MS: m/z calcd for C H NaO 365.1207, found 365.1205.

2 2 16 22 8

Bis((3-(6-azidohexanoyloxy)-propanoyloxy)ethoxy)benzene (20). Compound 20 was synthesized

using the general esterification procedure using TBTU. 6-Azidohexanoic acid 12 (0.190 g, 1.20 mmol)

1

8

Page 19 of 30

New Journal of Chemistry

DOI: 10.1039/C4NJ00992D

and diol 19 (0.170 g, 1.00 mmol (OH)) reacted for 2 h to give 20 (0.29 g, 94% yield) as a colorless solid

after purification using precipitation from diethyl ether. Purification was also achieved using column

1

chromatography (hexanes/ EtOAc; 1:1; R 0.53): mp 102 – 105 °C; H NMR (300.15 MHz, CDCl ) δ

F

3

1

.36 – 1.40, 1.51 – 1.65 (2 m, 12H, 2 x 3 CH ), 2.27 (t, J = 7.4 Hz, 4H, O=CCH CH C), 2.69 (t, J = 6.2

2 2 2

Hz, 4H, O=CCH CH O) 3.23 (t, J = 6.8 Hz, 4H, CH N), 4.10 (t, J = 4.5 Hz, 4H, PhOCH ), 4.33 (t, J =

2

1

3

6

.2 Hz, 4H, O=CCH CH O), 4.42 (t, J = 4.5 Hz, 4H, PhOCH CH ), 6.82 (s, 4H, PhH); C NMR (75.5

2 2 2 2

MHz, CDCl ) δ 173.1, 170.7 (C=O), 153.1, 115.8 (PhC), 66.6 (PhOCH ), 63.2 (PhOCH CH ), 59.7

3

2

(

O=COCH ), 51.2 (CH N ), 33.9 (2 CH C=O), 28.6, 26.2, 24.4 (CCH2C). HR ESI MS: m/z calcd for

2 2 3 2

C H N NaO 643.2696, found 643.2701.

28

40

6

10

Benzylidene-protected hydroquinone-cored third-generation dendrimer (23). Compound 23 was

57

synthesized as described above in the general procedure for dendritic ester synthesis. Compound 21

(

1.01 g, 1.13 mmol), dry pyridine (5 mL), CH Cl (15 mL), DMAP (0.330 g, 2.71 mmol) and anhydride

2 2

2 (4.81 g, 11.3 mmol) were stirred at rt for 10 h under nitrogen. After work up and purification as

1

described above, the product was obtained as a colorless solid (2.71 g, 95% yield): mp 112 – 114 °C; H

NMR (500.13 MHz, CDCl ) δ 0.92 (s, 24H, 8CH ), 1.04 (s, 6H, 2CH ), 1.19 (s, 12H, 4CH ), 3.57 (d, J =

3

1

8

5

1

4

1.5 Hz, 16H, H-4 , H-6 ), 3.97 (t, J = 5 Hz, 4H, ArOCH ), 4.07 (AB q,  = 11 Hz, J_A_B= 11 Hz,

ax

2

AB

H, 4CH OC=O), 4.31 – 4.37 (m, 20H, 8CH OC=O, 2CH OC=O), 4.55 – 4.57 (m, 16H, H-4 , H-6 ),

2

eq

1

3

.39 (s, 8H, H-2), 6.75 (s, 4H, PhH), 7.27 – 7.40 (m, 40H, PhH); C NMR (125.7 MHz, CDCl ) δ

3

73.3, 172.3, 172.0 (C=O), 153.0, 137.9, 129.0, 128.3, 126.3, 115.8 (PhC), 101.8 (C-2), 73.63, 73.56 (C-

, C-6), 66.3 (ArOCH ), 66.0 (4CH OC=O), 65.3 (8CH OC=O), 63.8 (OCH CH O), 47.0 (4C_q_u_a_t), 46.6

2

(

2C_q_u_a_t), 42.7 (C-5), 17.82 (8CH ), 17.78 (4CH ), 17.4 (2CH ). HR ESI MS: m/z calcd for

3

C H Na O /2 1286.4904, found 1286.4887.

136 158

2

46

Third generation hydroquinone-cored dendrimer (24).

Using the general procedure for

hydrogenolysis described above, protected third generation dendrimer 23 (1.55 g, 0.613 mmol)

1

9

New Journal of Chemistry

Page 20 of 30

DOI: 10.1039/C4NJ00992D

dissolved in dry CH Cl (15 mL), dry methanol (30 mL), and dry THF (15 mL) afforded 24 as a

2

1

colorless solid (1.10 g, 98% yield): mp 109 – 111 °C; H NMR (500.13 MHz, DMSO-d ) δ 1.00 (s, 24H,

6

8

CH ), 1.14 (s, 12H, 4CH ), 1.20 (s, 6H, CH ), 3.39 – 3.46 (m, 32H, 16CH OH), 4.08 – 4.23 (m, 28H,

3 3 3 2

1

3

CH O, 4CH O, 2ArOCH ), 4.36 (br, 4H, OCH CH O), 4.66 (br, 16H, OH), 4.86 (s, PhH); C NMR

2

(

125.7 MHz, DMSO-d ) δ 174.1 (6C=O), 172.1 (2C=O), 171.9 (4C=O), 152.5, 115.6 (PhC), 66.0

6

(

OCH CH O), 65.8, 64.5 (4, 8 CH OC=O), 63.7 (CH OH, PhOCH O), 50.3 (8C_q_u_a_t), 46.4 (4C_q_u_a_t), 46.2

2

(

2C_q_u_a_t), 17.2 (4CH ), 17.0 (2CH ), 16.8 (8CH ). HR ESI MS: m/z calcd for C H Na O /2 934.3652,

3 3 3 80 126 2 46

found 934.3654.

Azide-functionalized hydroquinone-cored third-generation dendrimer (25). Compound 25 was

synthesized using the general esterification procedure using TBTU. 6-Azidohexanoic acid 12 (0.190 g,

1

.20 mmol) and dendrimer 24 (0.114 g, 1.00 mmol (OH)) were reacted for 12 h to give 25 (0.20 g, 79%

yield) as a colorless syrup after purification using column chromatography (hexanes/ EtOAc; 3:2; R_F

1

0

1

3

4

6

3

.16): H NMR (500.13 MHz, CDCl ) δ 1.20 (s, 24H, 8CH ), 1.21 (s, 12H, 4CH ), 1.28 (s, 6H, 2CH ),

3

.33 - 1.40, 1.55 - 1.63 (2 m, 96 H, 16 x 3 CH ), 2.30 (t, J = 7.5 Hz, 32H, CH C=O), 3.25 (t, J = 6.9 Hz,

2

2H, CH N), 4.11 - 4.28 (m, 60H, 30 x CH OC=O), 4.42 (t, J = 4.5 Hz, 4H, 2 x PhOCH CH ), 6.81 (s,

2

13

H, PhH); C NMR (125.7 MHz, CDCl ) δ 172.8, 172.03, 172.01, 171.5 (C=O), 153.0, 115.7 (PhC),

3

6.3 (PhOCH ), 66.1, 65.2, 64.9 (CH OC=O), 63.9 (PhOCH CH ), 51.2 (CH N ), 46.7, 46.6, 46.4 (C ),

2

3

q

3.8 (CH C=O), 28.6, 26.2, 24.4 (CCH C), 17.8, 17.6, 17.5 (CH ). HR ESI MS: m/z calcd for

2

3

C H Na N O /3 1372.6356, found 1372.6389.

176 270

3

48 62

Divalent -D-mannopyranoside-terminated dendrimer (26). The azide-functionalized divalent

2, 68

dendrimer 17 (0.440 g, 0.552 mmol) and known propargyl α-D-mannopyranoside 7

mmol) were dissolved in THF (15 mL). To the clear solution was added sodium ascorbate (0.060 g,

.303 mmol) and a solution of copper(II) sulfate pentahydrate (0.030 g, 0.120 mmol) in water (15 mL).

The mixture was then vigorously stirred overnight and concentrated under reduced pressure. Purification

(0.280 g, 1.28

0

2

0

Page 21 of 30

New Journal of Chemistry

DOI: 10.1039/C4NJ00992D

using size exclusion chromatography on Sephadex LH-20 column gave the product as a thick colorless

1

syrup (0.55 g, 81% yield). H NMR (500.13 MHz, D O) δ 1.14 (br, 4H, 2 CH ), 1.44 (br, 4H, 2 CH ),

2

1

.60 – 1.80 (br, m, 4H, 2 CH ), 2.18 (br, 4H, CH C=O), 3.17 – 4.92 (complex m, 54H), 6.88 (s, 4H,

2 2

PhH), 8.05 (br, 2H, Triaz CH). HR ESI MS: m/z calcd for C H N Na O /2 639.2610, found

5

4

84

6

2

26

6

39.2628.

Extended -D-mannopyranoside-terminated dendrimer (27). The azide functionalized divalent

dendrimer 20 (0.700 g, 1.13 mmol) and propargyl α-D-mannopyranoside 7 (0.570 g, 2.61 mmol) were

dissolved in THF (20 ml). To the clear solution was added sodium ascorbate (0.120 g, 0.606 mmol) and

a solution of copper(II) sulfate pentahydrate (0.060 g, 0.240 mmol) in water (15 mL). The mixture was

then vigorously stirred overnight and concentrated under reduced pressure. Purification using size

exclusion chromatography on Sephadex LH-20 column gave the product (27) as a thick colorless syrup

1

(

1.03 g, 86% yield): H NMR (500.13 MHz, methanol-d ) δ 1.25 (pentet (p), J = 7.5 Hz, 4H, CH ), 1.58

4

2

(

p, J = 7.5 Hz, 4H, O=CCH CH ), 1.86 (p, J = 7.5 Hz, 4H, NCH CH ), 2.25 (t, J = 7.5 Hz, 4H,

2

O=CCH CH C), 2.69 (t, J = 6 Hz, 4H, O=CCH CH O), 3.55 – 3.89 (m, 12H, H-2, H-3, H-4, H-5, H-6),

2

4

.23 (t, J = 4.5 Hz, 4H, PhOCH ), 4.30 (t, J = 6 Hz, 4H, CH OC=O), 4.37 (t, J = 7 Hz, 4H, CH N), 4.41

2 2 2

(

t, J = 4.5 Hz, 4H, PhOCH CH ), 4.64 (d, J = 12.5 Hz, 2H, =CCHOC-1), 4.79 (d, J = 12.5 Hz, 2H,

2 2

1

3

=

CCH’OC-1), 4.86 (br, 2H, H-1), 6.86 (s, 4H, PhH), 8.00 (s, 2H, triazCH, triaz); C NMR (125.7 MHz,

methanol-d ) δ 174.8, 172.5 (C=O), 154.4 (PhCq), 145.2 (triaz Cq), 125.3 (triaz CH), 116.6 (PhC),

4

1

00.7 (C-1), 74.9 (C-5), 72.5 (C-3), 71.9 (C-2), 68.5 (C-4), 67.8 (PhOCH ), 64.5 (C-6), 62.9

2

(

PhOCH CH ), 61.1 (OCH Triaz), 60.7 (CH OC=O), 51.1 (CH N), 34.6, 34.5 (CH C=O), 30.8, 26.8,

2 2 2 2 2 2

2

5.2 (CCH C). HR ESI MS: m/z calcd for C H N NaO 1079.4279, found 1079.4246.

2 46 68 6 22

Third generation dendrimer bearing 16 mannose residues (28). The azide functionalized dendrimer

5 (1.40 g, 0.346 mmol) and propargyl α-D-mannopyranoside 7 (1.51 g, 6.92 mmol) were dissolved in

DMF (35 ml). To the clear solution was added sodium ascorbate (0.290 g, 1.46 mmol) and a solution of

2

1

New Journal of Chemistry

Page 22 of 30

DOI: 10.1039/C4NJ00992D

copper(II) sulfate pentahydrate (0.145 g, 0.581 mmol) in water (3 mL). The mixture was then vigorously

stirred overnight and concentrated under reduced pressure. Purification using size exclusion

chromatography on Sephadex LH-20 column gave the product as a thick colorless syrup (2.16 g, 83%

1

yield): H NMR (500.13 MHz, D O) δ 1.09 – 1.30 (br, 74H, 14CH , 16CH ), 1.50 (br, 32H, 16CH ),

2

3

2

1

.79 (br, 32H, 16CH ), 2.25 (br, 32H, 16CH ), 3.57 – 4.89 ((br, m, 240H (32H, 16CH , CH N), (32H,

2 2 2 2

6CH , CH OC-1), (112H, 16sugar x 7H), (8H, OCH CH O), (8H, 2C 2CH O), (16H, 4C 2CH O),

2

quat

2

quat

2

1

3

(

32H, 8C_q_u_a_t2CH O)), 6.82 (br, 4H, PhH), 8.04 (br, 16H, H ); C NMR (125.7 MHz, D O) δ 174.0,

2

g

2

1

5

4

72.7, 172.0 (C=O), 152.8 (PhC), 144.1 (triaz Cq), 125.1 (triaz CH), 115.9 (PhC), 99.4 (C-1), 73.0 (C-

), 70.6 (C-3), 70.0 (C-2), 66.6 (C-4), 65.3 (OCH CH O), 60.8 (C-6), 59.7 (Triaz CH O), 50.1 (CH N),

2

6.5, 46.3 (C_q_u_a_t), 33.3 (CH C=O), 29.3, 25.4, 23.8 (CCH C), 17.3, 17.2, 17.1 (Me). HR ESI MS: m/z

2

calcd for C H N Na O /6 1279.6901, found 1279.6411.

3

20 494 48

6

158

Bis(2-(2-propynyloxy)ethoxy)benzene (29). Compound 29 was synthesized using the general

esterification procedure using TBTU. Propynoic acid (0.084 g, 1.20 mmol) and diol 14 (0.099 g, 1.00

mmol (OH)) were reacted for 1 h to give the title compound (0.12 g, 78% yield) as a colorless solid after

1

purification using column chromatography (hexanes/ EtOAc; 3:1; R 0.18): mp 99 – 102 °C; H NMR

F

(

500.13 MHz, CDCl ) δ 2.92 (s, 2H, CCH), 4.16 (t, J = 4.5 Hz, 4H, PhOCH ), 4.52 (t, J = 4.5 Hz, 4H,

3

2

1

3

CH OC=O), 6.85 (s, 4H, PhH); C NMR (125.7 MHz, CDCl ) δ 153.1 (C=O), 152.7, 115.9 (PhC), 75.5

2

3

(

acetylene CH), 74.5 (acetylene O=CC), 66.2 (PhOCH ), 64.6 (CH OC=O). HR ESI MS: m/z calcd for

2 2

C H NaO 325.0683, found 325.0678.

16

14

6

Divalent -D-mannopyranoside-terminated dendrimer with a hexyl linker (30). Dialkyne 29 (0.260

52

g, 0.860 mmol) and 6-azidohexyl α-D-mannopyranoside 10 (0.550 g, 1.80) were dissolved in THF (15

ml). To the clear solution was added sodium ascorbate (0.090 g, 0.454) and a solution of copper(II)

sulfate pentahydrate (0.045 g, 0.180 mmol) in water (15 mL). The mixture was then vigorously stirred

overnight and concentrated under reduced pressure. Purification using size exclusion chromatography on

2

Page 23 of 30

New Journal of Chemistry

DOI: 10.1039/C4NJ00992D

1

Sephadex LH-20 column gave the product as a colorless solid (0.72 g, 89% yield): mp 132 – 134 °C H

NMR (500.13 MHz, DMSO-d / methanol-d ) δ 1.11 – 1.40 (m, 8H, 4 CH ), 1.41 – 1.50 (m, 4H,

6

4

2

CH CH OC-1), 1.82 (p, J = 7 Hz, 4H, NCH CH ), 3.26 – 3.67 (m, 16H, CH OC-1, H-2, H-3, H-4, H-5,

2

H-6), 4.18 (br, 4H, PhOCH ), 4.36 (t, J = 7 Hz, CH N), 4.54 (br, 4H, PhOCH CH ), 4.58 (br, 2H, H-1),

2

1

3

6

1

.85 (s, 4H, PhH), 8.58 (s, 2H, H ); C NMR (125.7 MHz, DMSO-d / methanol-d ) δ 161.9 (C=O),

c 6 4

54.2 (PhC), 140.2 (triaz Cq), 130.2 (triaz CH), 116.9 (PhC), 101.4 (C-1), 75.0 (C-5), 72.5 (C-3), 72.0

(

C-2), 68.5 (C-4), 67.9 (CH OC-1), 67.7 (PhOCH ), 64.7 (PhOCH CH ), 62.8 (C-6), 51.4 (CH N), 31.0,

2 2 2 2 2

3

2

0.3, 27.1, 26.7 (CH ). HR ESI MS: m/z calcd for C H N NaO 935.3856, found 935.3848.

2 40 60 6 18

,2’-Bis-(2-propynyloxymethy)propanoic acid (31). Compound was synthesized by the method of

65

1

Wu et al. in 48 % yield: H NMR (500.13 MHz, CDCl ) δ 1.25 (s, 3H, CH ), 2.42 (t, J = 2.1 Hz, 2H,

3

acetylenic H), 3.65 (AB q, Δν 0.179 ppm, J = 8.9 Hz, 4H, OCH C), 4.14 (AB part of ABX pattern, J =

2

AX

13

J_B_X= 2.3 Hz, J_A_B= 16.3 Hz, 2H, CH CCH); C NMR (125.7 MHz, CDCl ) δ 180.3 (CO), 79.6 (CCH),

2

3

7

4.7 (HCC), 71.7 (OCH CCH), 58.9 (CH O), 48.0 (C ), 17.9 (CH ). HR ESI MS: m/z calcd for

2 2 q 3

C H NaO 233.0784, found 233.0794.

11

14

4

2

,2’-Bis-(2-propynyloxymethyl)propanoyl anhydride (32). Acid 31 (1.00 g, 4.80 mmol) was added

to a stirred solution of DCC (0.530 g, 2.60 mmol) at rt and reaction mixture was stirred for overnight.

The mixture was filtered to remove N,N’-dicyclohexylurea and the filtrate was concentrated to give 32

1

as a light reddish liquid (1.8 g, 94 % yield): H NMR (500.13 MHz, CDCl ) δ 1.28 (s, 3H, CH ), 2.46 (t,

3

J = 2.2 Hz, 2H, acetylenic H), 3.71 (AB q, Δυ = 0.149 ppm, J = 9.0 Hz, 4H, OCH C), 4.14 (d, J = 2.3

2

13

Hz, 2H, CH CCH); C NMR (125.7 MHz, CDCl ) δ 169.1 (CO), 79.6 (CCH), 74.8 (HCC), 71.2

2

3

(

OCH CCH), 58.9 (CH O), 49.6 (Cq), 17.4 (CH ). HR ESI MS: m/z calcd for C H26Na O 425.1571,

2 2 3 22 1 7

found 425.1571.

,4-Bis-2-(2,2’-bis-(2-propynyloxy)methyl)propanoyloxy)ethoxy)benzene (33). Core diol 14 (0.300

g, 2.52 mmol), dry pyridine (5 mL), CH Cl (15 mL), DMAP (0.036 g, 0.30 mmol) and the anhydride 32

1

2

3

New Journal of Chemistry

Page 24 of 30

DOI: 10.1039/C4NJ00992D

(1.52 g, 3.78 mmol) were stirred at rt for 12 h under nitrogen. After work up and purification using

column chromatography (hexanes/ EtOAc; 2:1; R 0.36), the product was obtained as a light brownish

F

1

liquid (0.60 g, 69 % yield): H NMR (500.13 MHz, CDCl ) δ 1.22 (s, 6H, 2CH ), 2.37 (t, J = 2.3 Hz,

3

4

H, acetylenic H), 3.64 (AB q, J = 9.6 Hz, 8H, OCH C), 4.11-4.13 (complex m, 12H, CH CCH and

2 2

1

3

OCH ), 4.42 (t, J = 4.8 Hz, 4H, PhOCH CH ), 6.83 (s, 4H, Ar); C NMR (125.7 MHz, CDCl ) δ 174.2

2

3

(CO), 153.3 (C-Ar), 116.0 (CH-Ar), 79.8 (CCH), 74.5 (HCC), 71.9 (OCH CCH), 66.9 (PhOCH ), 63.2

2 2

(

PhOCH CH ), 58.8 (CH O), 48.3 (C ), 18.0 (CH ). HR ESI MS: m/z calcd for C H NaO 605.2357,

2

q

3

32 38

10

found 605.2374.

Tetramannoside polyester dendrimer 34. Tetraalkyne 33 (0.150 g, 0.257 mmol) and 6-azidohexyl α-

D-mannopyranoside 10 (0.393 g, 1.28) were dissolved in THF (15 mL). To the clear solution was added

sodium ascorbate (0.026 g, 0.134 mmol) and a solution of copper(II) sulfate pentahydrate (0.012 g,

0

.052 mmol) in water (15 mL). The mixture was then vigorously stirred overnight and concentrated

under reduced pressure. Purification using size exclusion chromatography on a Sephadex LH-20 column

o

1

gave the product as an fluffy solid (yield 0.34 g, 73%): mp 122-124 C; H NMR (500.13 MHz, DMSO-

d ) δ 1.06 (s, 6H, 2CH ), 1.22 (m, 8H, N(CH ) CH ), 1.24 (m, 8H, O(CH ) CH ), 1.46 (m, 8 H,

6

3

2 2

2

2 2

2

OCH CH ), 1.78 (p, 8H, J = 7.2 Hz, NCH CH ), 3.25-3.66 (complex m, 32 H, H-2, H-3, H-4, H-5, H-6,

2

H-6’, OCH CC=O), 4.06 (m, 4H, PhOCH ), 4.26 (m, 4H, O=CO-CH ), 4.29 (t, J = 7.1 Hz, 8H,

2

NCH ),4.39 (t, 4H, J = 6.0.3 Hz, OH-6), 4.46 (br s, 8H, CH CN=N), 4.49 (d, 4H, J = 6.0 Hz, OH), 4.57

2

(s, 4H, H-1), 4.63 (d, 4H, J = 4.4 Hz, OH), 4.67 (d, 4H, J = 5.3 Hz, OH), 6.86 (s, 4H, Ar), 8.00 (s, 4H,

1

3

triazole H); C NMR (125.7 MHz, DMSO-d ) δ 174.2 (CO), 153.3 (C-Ar), 145.0 (triaz Cq), 123.6

6

(triazole CH), 115.7 (CH-Ar), 99.7 (C-1), 73.9 (C-5), 71.0 (C-3), 70.4 (C-2), 66.0 (C-4), 71.6, 66.0,

6

4.1, 61.3 (4 x OCH ), 66.2 (2 x CH OPh), 62.6 (2 x CH OC=O), 49.2 (NCH ), 29.6, 28.8, 25.6, 25.1 (4

2

x CH ), 17.5 (2 x CH ). HR ESI MS: m/z calcd for C H Na O /2 924.4298, found 924.4295.

2

3

80 130

2

34

2

4

Page 25 of 30

New Journal of Chemistry

DOI: 10.1039/C4NJ00992D

2

-(p-Toluenesulfonyl)ethyl

2,2’-bis(2,2’-bis-(2-propynyloxymethyl)propanoyloxymethyl)-

propanoate (36) Compound 36 was synthesized as described above in the general procedure for

formation of dendritic esters, using 2-toluenesulfonylethyl 2,2-bis(hydroxylmethylpropanoate (35)

(1.00 g, 3.20 mmol), and anhydride 32 (3.18 g, 7.91 mmol) in dry pyridine (5 mL), and dichloromethane

(15 mL) containing DMAP (0.038 g, 0.320 mmol) at rt for 12 h. After work up and purification using

column chromatography (hexanes/ EtOAc; 2:1; RF 0.36), the product was obtained as a colorless syrup

1

(

1.84 g, 80 % yield): H NMR (500.13 MHz, CDCl ) δ 1.13 (s, 3H, CH ), 1.18 (s, 6H, 2 x CH ), 2.42 (br

3

s, 4H, 4 x CCH), 2.46 (s, 3H, PhCH ), 3.44 (t, 2H, J = 6.3 Hz, SCH ), 3.53-3.62 (m, 8H, 4 x

3

2

OCH CC(C=O)Me), 4.08-4.15 (m, 12H, 4 x CH CCH, and 2 x CH OC=O), 4.44 (t, 2H, J = 6.3 Hz,

2

13

CH OC=O), 7.39 (d, 2H, J = 8.1 Hz, PhH), 7.80 (d, J = 8.2 Hz, 2H, PhH); C NMR (125.7 MHz,

2

CDCl ) δ 173.5 (C=O), 172.3 (C=O), 145.5, 136.5, 130.4, 128.3 (PhC), 79.8 (CCH), 74.9 (HCC), 71.9

3

(

OCH CCH), 65.3 (O=COCH ), 58.8 (4 CqCH O), 58.4 (2 CqCH O), 55.2 (CH S), 48.3 (2 Cq), 46.8

2 2 2 2 2

(

Cq), 21.8 (PhCH ), 18.1 (2 CH ), 17.6 (CH ). HR ESI MS: m/z calcd for C H NaO S 723.2446,

3 3 3 36 44 12

found 723.2437.

2

,2’-Bis(2,2’-bis-(2-propynyloxymethyl)propanoyloxymethyl)propanoic acid (37) Compound 36

(

1.70 g, 2.42 mmol) was dissolved in CH Cl (15 mL) in a flame-dried round-bottom flask equipped

2

-8

with a magnetic stir bar, and 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU) (200 μL, 1.3 x 10 mmol) was

added. The reaction was vigorously stirred for 12 h, then the solution was concentrated to a syrup. The

product was then washed three times with 9:1 hexanes: ethyl acetate (v/v) and residual solvent was

1

removed by concentration to yield a gummy syrup (1.14 g, 91 % yield): H NMR (500.13 MHz, CDCl )

3

δ 1.18 (s, 6H, 2xCH ), 1.32 (s, 3H, CH ), 2.45 (br s, 4H, 4 x CCH), 3.62-3.68 (m, 8H, 4 x OCH Cq),

3

2

13

4

.12-4.18 (m, 8H, CH CCH), 4.32 (AB q, 4H, J =11.4 Hz, Δν = 0.013 ppm, CH OC=O); C NMR

2 2

(

125.7 MHz, CDCl ) δ 177.2 (C=OOH), 173.6 (C=O), 79.7 (CCH), 74.8 (HCC), 71.8 (OCH CCH),

3

2

5

New Journal of Chemistry

Page 26 of 30

DOI: 10.1039/C4NJ00992D

6

5.3 (CH OC=O), 58.7 (CqCH O), 48.3 (2 Cq), 46.9 (Cq), 18.0 (2 CH ), 17.8 (CH ). HR ESI MS: m/z

2 2 3 3

calcd for C H NaO 541.2044, found 541.2033.

2

7

34

10

2

,2’-Bis(2,2’-bis-(2-propynyloxymethyl)propanoyloxymethyl)propanoic anhydride (38) A solution

of acid 37 (1.10 g, 2.10 mmol) and DCC (0.240 g, 1.10 mmol) in dichloromethane (15 mL) was stirred

at rt for 12 h under nitrogen. The resulting mixture was filtered to remove N, N’-dicyclohexylurea and

1

the filtrate was concentrated to a colorless gummy solid (38) (1.0 g, 90 % yield): H NMR (500.13

MHz, CDCl ) δ 1.23 (s, 12H, 4 x CH ), 1.36 (s, 6H, 2 x CH ), 2.46 (br s, 8H, 8 x HCC), 3.62-3.68 (m,

3

1

6H, 8 x OCH C), 4.15-4.17 (m, 16H, 8 x CH CCH), 4.35 (AB q, 8H, J = 11.3 Hz, Δν = 0.023 ppm, 4 x

2 2

13

CCH OC=O); C NMR (125.7 MHz, CDCl ) δ 173.4 (C=O), 167.6 (O=COC=O), 79.7 (CCH), 74.8

2

3

(

HCC), 71.8 (OCH CCH), 64.7 (CH OC=O), 58.8 (CqCH O), 48.42 (Cq), 48.36 (Cq), 18.0 (4 CH ),

2 2 2 3

1

7.3 (2 CH ). HR ESI MS: m/z calcd for C H NaO 1041.4091, found 1041.4101.

3 54 66 19

Octapropargylated polyester dendrimer (39). Core molecule 14 (0.090 g, 0.450 mmol) and

anhydride 38 (1.00 g, 0.980 mmol) dissolved in dry pyridine (5 mL), and dichloromethane (15 mL)

containing DMAP (0.038 g, 0.320 mmol) were stirred at rt for 12 h under nitrogen under the conditions

for formation of dendritic esters. After work up and purification using column chromatography

(hexanes/ EtOAc; 1:1; RF 0.45), the product (39) was obtained as colorless syrup (0.40 g, 73 % yield):

1

H NMR (500.13 MHz, CDCl ) δ 1.18 (s, 12H, 4 x CH ), 1.26 (s, 6H, 2 x CH ), 2.40 (td, 8H, J = 0.8, 2.3

3

Hz, 8 x CCH), 3.56-3.63 (m, 16H, 8 x OCH Cq), 4.11 (d, 16H, J = 2.3 Hz, 8 x OCH CCH), 4, 12-4.14

2

(m, 4H, 2 x PhOCH ), 4.28 (s, 8H, 4 x CH OC=O), 4.43 (1/2 AA'XX' pattern, 4H, 2 x O=COCH ), 6.83

2 2 2

1

3

(

s, 4H, PhH); C NMR (125.7 MHz, CDCl ) δ 173.6 (C=O), 153.2 (PhC), 116.0 (PhCH), 79.7 (CCH),

3

7

4.7 (HCC), 71.9 (OCH CCH), 66.6 (PhOCH ), 65.6 (CH OC=O), 63.7 (PhOCH CH ), 58.8

2 2 2 2 2

(

CqCH O), 48.3 (4 Cq), 46.9 (2 Cq), 18.0 (4 CH ), 17.8 (2 CH ). HR ESI MS: m/z calcd for

2

3

C H NaO 1221.4877, found 1221.4860.

64

78

22

2

6

Page 27 of 30

New Journal of Chemistry

DOI: 10.1039/C4NJ00992D

Octavalent mannose-terminated polyester dendrimer (40) 6-Azidohexyl α-D-mannopyranoside (10)

0.611 g, 2.00 mmol) and octapropargylated polyester dendrimer (39) (0.200 g, 0.160 mmol) were

(

dissolved in THF (15 mL) and sodium ascorbate (0.016 g, 0. 080 mmol) and a solution of copper (II)

sulfate pentahydrate (0.002 g, 0.008 mmol) in water (5 mL) were added. The mixture was stirred

vigorously for 12 h, then concentrated to a syrup. Purification using size exclusion chromatography on

Sephadex LH-20 column gave the product as light yellow fluffy solid (0.40 g, 65% yield): mp became

o

1

transparent at 85 C, then decomposed at 200-205 C; H NMR (500.13 MHz, D O) δ 1.10 (br s, 18H,

2

6

x CH ), 1.24– 1.34 (br s, m, 32H, CH CH CH ), 1.53 (br s, 16H, CH CH O), 1.84 (br s, 16H,

3 2 2 2 2 2

CH CH N), 3.42 – 3.96 (m, 80H, Sugar- H-2, H-3, H-4, H-5, H-6, OCH ), 4.16 (br s, 16H, OCH C),

2

4

.37 (br s, 16H, OCH C), 4.16 (br s, 16H, OCH NNC), 4.55 (br s, 16H, OCH C-N), 4.16 (br s, 8H,

2 2 2

1

3

Sugar- H-1), 6.86 (s, 4H, PhH), 7.93 (br, 8H, H-triazole); C NMR (125.7 MHz, CDCl3) δ 174.6, 173.4

(C=O), 152.8 (PhC), 143.6 (triaz Cq), 124.6 (Triaz CH), 116.5 (PhCH), 99.8 (C-1), 72.8 (C-5), 71.8

(

OCH ), 70.8 (C-3) 70.3 (C-2), 67.6 (OCH ), 66.8 (C-4), 66.0 (PhOCH ), 65.3, 64.2, 64.0, 60.9 (CH O),

2

6

0.9 (CH OC=O), 50.3 (NCH ), 48.4, 46.7 (Cq), 29.5, 28.5, 25.6, 25.0 (CH ), 17.4 (4 x CH ), 17.1 (2 x

2 2 2 3

CH ). HR ESI MS: m/z calcd for C H Na O /3 1236.2452, found 1236.2432.

3

160 262

3

70

SUPPORTING INFORMATION AVAILABLE

1

13

H and C NMR spectra of all compounds prepared.

ACKNOWLEDGEMENTS

We thank Natural Sciences and Engineering Research Council of Canada (NSERC) for support

and NMR-3 of Dalhousie University for NMR time. We thank Prof. David Jakeman and

Stephanie Forget for the use of the preparative HPLC.

2

7

New Journal of Chemistry

Page 28 of 30

DOI: 10.1039/C4NJ00992D

References

1

2

3

4

.

C. K. Cusumano and S. J. Hultgren, Idrugs, 2009, 12, 699-705.

N. Sharon, Biochim. Biophys. Acta, Gen. Subj., 2006, 1760, 527-537.

H. Remaut and G. Waksman, Curr. Opin. Struct. Biol., 2004, 14, 161-170.

T. J. Hannan, M. Totsika, K. J. Mansfield, K. H. Moore, M. A. Schembri and S. J. Hultgren, Fems Microbiol.

Rev., 2012, 36, 616-648.

5

6

7

8

.

T. M. Hooton and W. E. Stamm, Infect. Dis. Clin. N. Am., 1997, 11, 551-581.

B. Foxman and P. Brown, Infect. Dis. Clin. N. Am., 2003, 17, 227-241.

B. Foxman, Infect. Dis. Clin. N. Am., 2014, 28, 1-13.

B. Xie, G. Zhou, S. Y. Chan, E. Shapiro, X. P. Kong, X. R. Wu, T. T. Sun and C. E. Costello, J. Biol. Chem.,

2

006, 281, 14644-14653.

9

1

.

0.

G. R. Nielubowicz and H. L. T. Mobley, Nature Rev. Urol., 2010, 7, 430-441.

J. Bouckaert, J. Berglund, M. Schembri, E. De Genst, L. Cools, M. Wuhrer, C. S. Hung, J. Pinkner, R.

Slättegård, A. Zavialov, D. Choudhury, S. Langermann, S. J. Hultgren, L. Wyns, P. Klemm, S. Oscarson, S.

D. Knight and H. De Greve, Mol. Microbiol., 2005, 55, 441-455.

1

1.

2.

R. U. Lemieux, Chem. Soc. Rev., 1989, 18, 347-374.

G. Roos, A. Wellens, M. Touaibia, N. Yamakawa, P. Geerlings, R. Roy, L. Wyns and J. Bouckaert, ACS Med.

Chem. Lett., 2013, 4, 1085-1090.

1

3.

4.

M. Scharenberg, O. Schwardt, S. Rabbani and B. Ernst, J. Med. Chem., 2012, 55, 9810-9816.

L. M. Nilsson, W. E. Thomas, E. Trintchina, V. Vogel and E. V. Sokurenko, J. Biol. Chem., 2006, 281, 16656-

1

6663.

1

5.

I. Le Trong, P. Aprikian, B. A. Kidd, M. Forero-Shelton, V. Tchesnokova, P. Rajagopal, V. Rodriguez, G.

Interlandi, R. Klevit, V. Vogel, R. E. Stenkamp, E. V. Sokurenko and W. E. Thomas, Cell, 2010, 141, 645-

6

55.

1

6.

7.

8.

H. Wang, G. Min, R. Glockshuber, T. T. Sun and X. P. Kong, J. Mol. Biol., 2009, 392, 352-361.

N. Firon, S. Ashkenazi, D. Mirelman, I. Ofek and N. Sharon, Infect. Immun., 1987, 55, 472-476.

Z. F. Han, J. S. Pinkner, B. Ford, R. Obermann, W. Nolan, S. A. Wildman, D. Hobbs, T. Ellenberger, C. K.

Cusumano, S. J. Hultgren and J. W. Janetka, J. Med. Chem., 2010, 53, 4779-4792.

O. Sperling, A. Fuchs and T. K. Lindhorst, Org. Biomol. Chem., 2006, 4, 3913-3922.

L. Pang, S. Kleeb, K. Lemme, S. Rabbani, M. Scharenberg, A. Zalewski, F. Schaedler, O. Schwardt and B.

Ernst, ChemMedChem, 2012, 7, 1404-1422.

1

2

9.

0.

2

1.

2.

3.

X. Jiang, D. Abgottspon, S. Kleeb, S. Rabbani, M. Scharenberg, M. Wittwer, M. Haug, O. Schwardt and B.

Ernst, J. Med. Chem., 2012, 55, 4700-4713.

M. Scharenberg, X. H. Jiang, L. J. Pang, G. Navarra, S. Rabbani, F. Binder, O. Schwardt and B. Ernst,

ChemMedChem, 2014, 9, 78-83.

A. Bernardi, J. Jiménez-Barbero, A. Casnati, C. De Castro, T. Darbre, F. Fieschi, J. Finne, H. Funken, K. E.

Jaeger, M. Lahmann, T. K. Lindhorst, M. Marradi, P. Messner, A. Molinaro, P. V. Murphy, C. Nativi, S.

Oscarson, S. Penadés, F. Peri, R. J. Pieters, O. Renaudet, J. L. Reymond, B. Richichi, J. Rojo, F. Sansone, C.

Schäffer, W. B. Turnbull, T. Velasco-Torrijos, S. Vidal, S. Vincent, T. Wennekes, H. Zuilhof and A. Imberty,

Chem. Soc. Rev., 2013, 42, 4709-4727.

2

4.

5.

J. Bouckaert, Z. Li, C. Xavier, M. Almant, V. Caveliers, T. Lahoutte, S. D. Weeks, J. Kovensky and S. G.

Gouin, Chem.-Eur. J., 2013, 19, 7847-7855.

M. Touaibia, A. Wellens, T. C. Shiao, Q. Wang, S. Sirois, J. Bouckaert and R. Roy, ChemMedChem, 2007, 2,

1

190-1201.

2

6.

7.

8.

A. Adronov and J. M. J. Fréchet, Chem. Commun., 2000, 1701-1710.

J. K. Twibanire and T. B. Grindley, Polymers, 2014, 6, 179-213.

T. Dutta, N. K. Jain, N. A. J. McMillan and H. S. Parekh, Nanomed.-Nanotechnol., 2010, 6, 815-815.

2

8

Page 29 of 30

New Journal of Chemistry