Thiol-alkyne Chemistry for the Preparation of Micelles with Glycopolymer Corona: Dendritic Surfaces...

Communication

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Thiol-alkyne Chemistry for the Preparation ofMicelles with Glycopolymer Corona: DendriticSurfaces versus Linear Glycopolymer in TheirAbility to Bind to Lectinsa

Jatin Kumar, Antoine Bousquet, Martina H. Stenzel*

M. H. Stenzel, J. KumarCentre of Advanced Macromolecular Design (CAMD), School ofChemical Engineering, The University of New South Wales, NewSouth Wales 2052, AustraliaE-mail: [email protected]. KumarCooperative Research Centre for Polymers, 8 Redwood Drive,Notting Hill, Victoria 3168, AustraliaA. BousquetDepartment of Chemistry, Aarhus University, Langelandsgade140, DK-8000, Aarhus C., Denmark

a Supporting Information for this article is available at Wiley OnlineLibrary or from the author.

A poly(tert-butyl acrylate) (P(tBA)) with a glycodendric endfunctionality with eight glucosemoieties was synthesised in four steps via a combination of esterification, thiol-alkyneconjugation and hetero-Diels–Alder (HDA) cycloaddition. A linear glycopolymer of similarsize and composition was also synthesised in order to compare the protein binding charac-teristics of the polymer with glycodendritic endfunctionality to the linear glycol blockcopo-lymer. The two amphiphilic polymers were self-assembled in water into micelles. Theseparticles were then tested for their abilityto bind to Concanavalin A (Con A). In aturbidity assay, the polymer glycodendronexhibited a significantly faster clusteringrate to the lectin as compared to the linearglycopolymer. In a precipitation assay, it isfound that significantly less glucose resi-due is required for binding per Con A forthe polymer with the glycodendritic end-functionality.

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Introduction

Glycopolymers play a pivotal role in biological interac-

tions[1] anda lot of efforthasbeendevoted in recentyears to

optimise the synthesis of glycopolymers and glycodendri-

mers.[2] Dendrimers have been employed in a varied range

of applications,[3] however,most potential for them appear

to lie in drug delivery[2c,4] and biotechnological applica-

tions.[5] Glycodendrimers allow for multivalent interac-

tions with proteins, similar to the use of multivalent

glycopolymers, thereby expanding theuseof saccharides in

drug delivery mechanism.[1a] What sets glycodendrimers

apart from glycopolymers though, is that studies have

shown that glycodendrimers exhibit better protein inter-

action as compared to linear glycopolymers. Gillies and

library.com DOI: 10.1002/marc.201100331

Thiol-alkyne Chemistry for the Preparation of Micelles with Glycopolymer Corona: . . .

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colleagueshypothesise that this is due to the inherent steric

hindrance of the dendrimer that allows for the saccharides,

which are often terminally functionalised, to present

themselves on the surface of the molecular matrix as

opposed to the case of a glycopolymer where the

saccharides may be within the polymer matrix.[3e]

Previous studies of polymer-based dendrons have

generally involved repetitive multi-stage reactions to

achieve a higher number of branches. With an increase

in the number of steps, the most salient issue is ensuring

that yield is optimised through orthogonal and efficient

reactions.

‘Click’ chemistry techniques are by their very definition

orthogonal, efficient and versatile. Both copper catalysed

azide-alkyne click (CuAAC)[6] aswell as the recentlypopular

thiol-alkene[7] ‘click’ chemistry have been reportedly used

to synthesise dendrimers with high efficiency. There is

also a report of the synthesis of dendrimers by a

combination of CuAAC and traditional esterification

with high yields and efficiency.[8] However, dendrimers

synthesised via the CuAAC technique may ultimately

not be biocompatible due to the toxicity of copper.[9]

Chen et al. synthesised a dendrimer holding 48 terminal

hydroxyl functionalities within 3 steps via a combina-

tion of the thiol-alkyne technique as well as a simple

esterification reaction.[10] In comparison Antoni et al.

required eight steps to synthesise a dendrimer holding

the same number of functionality.[11]

Another polymer structure that has shown to have

promising lectin binding characteristics is a micelle.

Micelles were shown to have better lectin binding abilities

than their linear counterparts.[12] Their spherical shape

resembles that of dendrimers although the size is typically

much larger.

The central question is now if a dendritic structure is

advantageous over a traditional micelles structure or if the

introduction of a dendritic structure on the surface of a

micelle can increase binding with lectins. The aim of this

study is thus to synthesise polymers with glycodendritic

endfunctionality to assess their lectin binding character-

istics as compared to linear glycopolymers. Polymers with

dendritic endfunctionalities have been synthesised earlier

via reversible addition fragmentation chain transfer (RAFT)

polymerisation[13] but the aimof thiswork is to specifically

compare the performance of traditional glycopolymer

micelles with dendritic structures.

For this purpose two different glycopolymers are

envisaged: a poly(tert-butyl acrylate) (P(tBA)) was used

as the hydrophobic block while eight glucose units

represented the hydrophilic part either in linear or

dendritic arrangement. The dendritic amphiphilic block

copolymer was prepared by subsequent steps consisting

of RAFT polymerisation, RAFT-hetero-Diels–Alder (HDA)

reaction, thiol-yne click reaction and an esterification

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Macromol. Rapid Commun.

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step to yield polymers with four alkyne functionalities.

The linear block copolymer was prepared from poly(tert-

butyl acrylate)-block-poly(2-hydroxyethyl acrylate)

(P(tBA)-b-PHEA) synthesised via RAFT followed by ester-

ification to obtain a block copolymer with four alkyne

functionalities. Subsequent functionalisation with b-

thioglucose sodium salt leads to two different polymer

architectures with eight glucose functionalities each

(Scheme 1).

Experimental Section

See Supporting Information for detailed experimental procedure

and analysis.

Results and Discussion

The synthesis of the dendritic structure required an alkyne

endfunctional polymer. While direct synthesis using an

alkyne functional RAFT agent is possible, the postmodifica-

tion using a new bi-functional clicking agent was found to

be a convenient pathway to high endfunctionality. The

unique bi-functional clicking agent was synthesised in a 1-

step reactionwith2,4-trans,trans-hexadien-1-ol (HPYS) and

the 4-oxo-4-(prop-2-ynyloxy)butanoic anhydride (acety-

lene anhydride) as show in Scheme S1 of Supporting

Information. This bi-functional clicking agent can react

directly with the RAFT endfunctional terminal polymer via

HDAreaction converting theRAFTgroup inanefficientway

into an alkyne group.

The synthesis of the dienophile end-chain capable P(tBA)

was achieved by RAFT polymerisation using BPDF as the

RAFT agent and AIBN as a thermal initiator. The molecular

weightwasdeterminedvia 1HNMRandGPC.Twopolymers

of Mn (a) 1 200 g �mol�1 (PDI¼ 1.24/DPn ¼ 7) and (b)

5 600 g �mol�1 (PDI¼ 1.13/DPn ¼ 42) (SEC traces shown in

Figure S1 of Supporting Information) were obtained and

used in further experiments. The purpose of the polymer

with Mn of 1 200 g �mol�1 is only to follow the progress of

the reaction pathway via electrospray ionization mass

spectrometry (ESI-MS) so as to provide further structural

confirmation in addition to 1H NMR through the various

stages of functionalisation.

The two polymers were then reacted with HPYS via

an HDA reaction. TFA acts as a catalyst, and this reaction

was carried out at ambient temperature under vigorous

stirring in chloroform. As the reaction proceeded,

the solution lost its original pink colouration due to

the RAFT end-groups and became colourless due to the

formation of the 3,6-dihydro-2H-thiopyran ring. The

HPYS-P(tBA) conjugates were reacted with thioglycerol

via a thiol-alkyne conjugation reaction via a radical

pathway. The reactants were dissolved in DMF along

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S

SNn

S

SN

O O

Sn S

N

O

OO

OO

OO

O

AIBN, Toluene65 ºC

TFAChloroform25 ºC

Sn S

N

O

OO

O

SS

OHOH

HOOH

DMPA, DMF,N2 DegassedUV (365nm), 4 hr

HSOH

OH

SnS

N

O

OO

O

SS

OO

OO

O

O

O

O

OO

OO

O O

O O

DMAP, TEA,45 ºC, 18 hr,Chloroform

OO

OO

O

OO

(I)(II)

(III)(IV)

O O O O

OO

O O

OO

O

OHOH

OH

HO

SO

OHHO

OH

HO

S

OO

OS

OO

O OHOH

HO

HO

S

O

OHOH

OH

HO

SO

S OO

O OHOHHO

OH

S

OOHOH

HO

HO

SO

O

O

OOO

OHOH

HOOH

S

OOHHO

OH

S

O

O

OO

O

S

S N

O O

OH

HO

H

HO

H

HOHH SH

OH


O O

OO

OH

S S

S

OH

O

OO O

OH

O

OO

OO

OO

O

O

OO

DMAP, TEAChloroform, 50 ºC, 24 hr

65 ºC, AIBN, Toluene

65 ºC, AIBN,DMAc

1)

2)

S S

S

OH

On m OO O

O

O

S S

S

OH

On m

O

OO

OO O

O

O

S S

S

OH

On m

OH

HO

HHO

H

HOHH SH

OH


O

H OH

H

OHH

HOH

H

S

HO

O

H

HO HHO

H HOHH

SH

HO

Scheme 1. Reaction pathway to generate 4-alkyne functionalised P(tBA)-based polymer dendron and the alkyne functionalised linear blockcopolymer P(tBA)-b-PHEA and the subsequent modification to structures with eight glucose units.

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J. Kumar, A. Bousquet, M. H. Stenzel

withasmall amountofDMPA, aphoto-initiatorand reacted

for 2h at 30 8C under UV light at a wavelength of 365nm.

Once this reaction was completed, the product was

precipitated in 50:50 vol% water/methanol to eliminate

all remnant thioglycerol and DMPA and then dried under

vacuum. Since thiols react with alkynes in a 2–1 ratio,



the HPYS-P(tBA) conjugates would have two thioglycerol

units attached terminally, thus affording four hydroxyl

groups at the chain ends.

Subsequent reaction 4-oxo-4-(prop-2-ynyloxy)butanoic

anhydride in the presence of DMAP and TEA over 18h at

45 8C afforded product (IV) (Scheme 1). ESI-MS of the lower

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Figure 1. ESI-MS overlay of (1) both experimental (E), and theoretical (T) masses as well as the degree of polymerisation (DPn) are reported forNaþ charged species. Hþ and double charged (with Naþ) species are denoted by a and b respectively. E: experimental, T: theoretical.


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Mn polymer was taken at various stages of functionalisa-

tion to serve as structural confirmation of the desired

product (Figure 1). The calculated mass was observed to be

close to the theoreticalmass. Other observedpopulations in

the ESI-MS spectra were due to double charged species.

Further structural confirmation via NMR is shown in

Figure 2 for all stages of functionalisation [structures (I)–

(IV) in Scheme 1].

As a control experiment, a linear version of the dendritic

polymer was synthesised. This is achieved by RAFT

polymerisation of tert-butyl acrylate using BPATT as the

RAFT agent and AIBN as a thermal initiator. The product

polymer was analysed for its molecular weight via 1H NMR

and GPC and is found to be 5900 g �mol�1 (PDI¼ 1.15/

DPn ¼ 46). P(tBA)-b-PHEA was then generated via a chain

extension of the homopolymer. The molecular weight

distribution of the P(tBA) homopolymer as well as the chain

extended copolymer is shown in Figure S2 of Supporting

Information. From 1H NMR, the polymer was found to have




anaverageof4.5HEAunitsperpolymer chainby integrating

the two protons (3.80ppm) next to the hydroxyl moieties

against the integraloftheprotonsofthe tert-butylgroups(2–

2.4ppm) as illustrated in Figure S3 of Supporting Informa-

tion. The polymer was then functionalised using a similar

procedure as outlines above resulting a block copolymer

with on average 4.5 alkyne functionalities per chain.

Glycosylation of the linear control as well as the

dendrimer bwas performed via a thiol-alkyne conjugation

process with a b-thioglucose sodium salt. The salt, HCl,

polymer and a small amount of DMPAphoto-initiatorwere

dissolved in DMF. HCl is required to protonate the glucose

salt for the purposes of this reaction. The mixture was

degassed viaN2 sparging and then reacted under UV light at a

wavelength of 365nm for a period of 4h. The product was

dialysed against 50:50 vol% water/methanol. The products,

P(tBA)44-dentritic-Glucose8andP(tBA)46-block-P(HEA-glucose)9,

were analysed via 1H NMR to verify the correct structure

(Figure3andFigure S5of Supporting Information). Only the

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8 7 6 5 4 3 2 1

D

O

OO

O

S

S N

O O

S

S

OO

O

O

OO

O

O

OO

O

O

OO

O

O

O

HOH

H

OH

H

HOH

H

HO

S OH

OH

HOH

H

HHO H

S

OH

OH

OH

H

OHH

HHO HS

HO

O

H

OHH OH

HHHO

H

S

OH

O

H

HOH OH

HHHO

H

S

OHO

HHO

H

OH

H

HHO

H

S

OHO

HHO

H

HO H

HOH

HS

OH

O

HHO

HOH

H

HHO

H

S

OH

AB

C

1 2 3 4 5 67

8E

F G

91011

* impurity from commercial thioglucose

*

Glucose

βDMF

A B G3,10

E

2,7,9

6F

DMFH2O

1,4,5,8,11

D

C

δ / ppm

Figure 3. 1H NMR (d-DMF) structural confirmation of P(tBA)-based dendron with eightglucose functionalities.

8 6 4 2 0

O

OO

O

S

S N

O O

S

S

OO

O

O

OO

O

O

OO

O

O

OO

O

O

F

F,K

3

J

E,I,Ha',12

a

B

32

1E

KJIHGF

GE,I,H

J,KK

JIHGFO

OO

O

S

S N

O O

S

S

OHHO

OH

OH

C AE,G,a'D,F

B

GFE

DCBA

a'a'

O

OO

O

S

S N

O O

n

CH-S ce

d S

N

O O

n

S

bbbba

a

bc

d

e

δ/ ppm

Figure 2. 1H NMR in CDCl3 of the dendritic structures based on P(tBA) with Mn ¼5 600 g �mol�1 providing structural confirmation of the products at the four stagesof dendron synthesis.

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anomeric glucose peak (b in Figure 3) is visible, the

remaining glucose signals are overridden by the water

signal. 1H{13C}HSQC NMR spectra (Figure S4 of Supporting

Information) reveal the missing glucose signals and

especially the H-6a and H-6b are clearly visible.

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� 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinhe

Micelles were prepared by dissolving

the polymers P(tBA)44-dentritic-Glucose8and P(tBA)46-block-P(HEA-glucose)9 in

acetone, followed by the slow addition

of deionised water and the subsequent

removal of acetone via dialysis. The

solutions were assessed for their particle

size by dynamic light scattering, and

their size and morphology via TEM. The

TEM images (Figure S6 of Supporting

Information and insert in Figure 4) and

the number size distribution of the

dynamic light scattering (Figure S7 of

Supporting Information) revealanumber

mean particle diameters via DLS of

133nm for the polymer dendron, and

124nm for the linear control polymer.

The low fraction of glycopolymer in the

block copolymer will result in crew-cut

aggregate, which have a brush-like gly-

copolymer configuration.

Lectin binding assays with Concana-

valin A (ConA) were then carried out to

fully assess and compare the ligation

characteristics of the two different poly-

mers. Two types of studies are performed

to assess two aspects of binding. The first

was a turbidimetry assay to evaluate the

rate at which the glycopolymer binds to

ConA and the extent of interchain cross-

linking due to binding. This analysis was

carried out by mixing a solution of ConA

in HEPES buffered saline into the micelle

solution of both the polymers. The UV

absorbance (at l¼ 420nm) of the solu-

tions was measured against time over

10min (Figure 4).

From the turbimetry assay, an initial

clustering rate constant, K can be

obtained. In the case of the polymer with

the glycodendritic end group, the value is

0.18 AU �min�1 while the value for the

linear glycopolymer is 0.05 AU �min�1. It

needs to be considered that there is an

error associated with this calculation

since significant turbidity can be

observed by the time the solutions are

mixed and placed in the UV/Vis spectro-

meter. The polymer with the dendrimer

end-group has a significantly higher K value despite the

similar overall glucose residue for both solutions.[14] It is

also evident that there is intermolecular cross-linking due

to the ConA binding from the increase in particle size as

obtained from the dynamic light scattering. The number

im www.MaterialsViews.com

Figure 4. Absorbance versus time turbidimetry assays of both the dendrimerb and linear polymermicelle solutionswhen a Con A solution isintroduced.

Figure 5. Correlation of ConA bound to glucose at various glucoseresidues for both the polymer with the dendritic end-group andlinear glycopolymer.


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average particle diameters for the polymer with dendritic

corona and the linear polymer after binding are 269 and

573nm, respectively (Figure S7 of Supporting Information).

The bigger diameter is equivalent to more cross-linking

between the micelles, which is often observed when

binding is slower.[15] Turbidity assay determines the rate

of binding while the precipitation assay quantifies the

number of ConA bound per glucose residue. An excess of

ConA solution was added to solutions with different

polymer concentrations and allowed to bind at an

incubation temperature of 20 8C for 20h. The precipitate

was isolated and the amount of bound ConAwasmeasured

using UV/Vis spectroscopy (at l¼ 280nm). The correlation

bound ConA glucose concentration reveals that the

polymer dendron exhibits better binding characteristics

to ConA with more ConA bound to it as compared to the

linear polymer for similar glucose residues (Figure 5).

In summary, RAFT polymerisation in combination with

RAFT-HDA click chemistry and thiol-yne click chemistry

were used as efficient synthetic procedures to amphiphilic

blockcopolymerstructures, onewitheightglucosemoieties

in a dendritic arrangement and one with nine glucose

units in a linear architecture. Micellisation of both block

copolymers led to micelles with a dendritic glycopolymer

surface and a linear glycopolymer brush structure,

respectively. The turbidity assay and the precipitation

assay found more efficient binding of the dendritic

structure.




Acknowledgements: The authors like to acknowledge theAustralian Research Council for funding and like to thank thestaff from the UNSW NMR facility for their help.

Received: May 17, 2011; Revised: June 21, 2011; Published online:August 19, 2011; DOI: 10.1002/marc.201100331

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Keywords: dendrimers; glycopolymer; lectin; micelles; thiol-yneclick

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Thiol-alkyne Chemistry for the Preparation of Micelles with Glycopolymer Corona: Dendritic Surfaces...

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