Reversible addition fragmentation chain transfer polymerization - RAFT

Reversible Addition–Fragmentation Chain Transfer Synthesis of

Amidine-Based, CO2-Responsive Homo and AB Diblock (Co)Polymers

Comprised of Histamine and their Gas-Triggered Self-Assembly in Water

Jing Yang Quek,1 Peter J. Roth,1 Richard A. Evans,2 Thomas P. Davis,1 Andrew B. Lowe1

1Centre for Advanced Macromolecular Design (CAMD), School of Chemical Engineering, University of New South Wales,

Kensington, Sydney, NSW 2052, Australia2Materials Science and Engineering, CSIRO, Bag 10, Clayton South, VIC 3169, Australia

Correspondence to: A. B. Lowe (E-mail: [email protected])

Received 28 August 2012; accepted 28 September 2012; published online 29 October 2012

DOI: 10.1002/pola.26397

ABSTRACT: Well-defined homopolymers of pentafluorophenyl ac-

rylate (PFPA) and AB diblock copolymers of N,N-dimethylacryla-

mide (DMA) and poly(ethylene glycol) methyl ether acrylate

(PEGA) with PFPA were prepared by reversible addition–fragmen-

tation chain transfer (RAFT) radical polymerization. Three PFPA

homopolymers of different molecular weights were reacted with

the commercially available amidine and guanidine species hista-

mine (HIS) dihydrochloride and L-arginine methyl ester (ARG)

dihydrochloride in the presence of S-methyl methanethiosulfonate

to yield, quantitatively, the corresponding amidine and guanidine-

based acrylamido homopolymers. Both the HIS and ARG homo-

polymers are known to reversibly bind CO2 with, in the case of

the former, CO2 fixation being accompanied with a switch from a

hydrophobic to hydrophilic state. The RAFT synthesis of PFPA-

DMA and PEGA-PFPA diblock copolymers yielded well-defined

materials with a range of molar compositions. These precursor

materials were converted to the corresponding HIS and ARG

block copolymers whose structure was confirmed using 1H NMR

spectroscopy. Employing a combination of dynamic light scatter-

ing and transmission electron microscopy, we demonstrate that

the DMA-HIS and PEGA-HIS diblock copolymers are able to

undergo reversible and cyclable self-directed assembly in aque-

ous media using CO2 and N2 as the triggers between fully hydro-

philic and amphiphilic (assembled) states. For example, in the

case of the 54:46 DMA-HIS diblock, aggregates with hydrody-

namic diameters of about 40.0 nm are readily formed from the

molecularly dissolved state. VC 2012 Wiley Periodicals, Inc.

J Polym Sci Part A: Polym Chem 51: 394–404, 2013

KEYWORDS: amidine; gas responsive; histamine; micelles; re-

versible addition fragmentation chain transfer (RAFT); self-as-

sembly; stimulus responsive

INTRODUCTION Amidines are the dinitrogen analogues ofcarboxylic acids and esters (RA(C¼¼NAR0)ANR002) and assuch contain both imine-like and amide-like functionality.1

Amidines are interesting for various reasons including theirwell-established basicity (common cyclic organobases suchas 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and 1,5-diazabi-cyclo[4.3.0]non-5-ene (DBN) are examples of amidines), bio-logical activity,2 and their ability to complex, in some instan-ces reversibly, triatomic molecules such as CO2 and CS2.

3,4

Indeed, the ability of amidines to trap, in the form of anHCO�3 salt, and release CO2 has led to their use as buildingblocks for the chemical fixation of CO2 into useful organiccompounds such as in the facile synthesis of cyclic carbo-nates5 and the preparation of 1H-quinazoline-2,4-diones—key intermediates in the synthesis of a range of drugs.6

The preparation of amidine-containing (co)polymers inwhich the amidine functionality is located in the backboneor as pendant side groups has been reported. For example,

B€ohme et al.7 described the synthesis and properties of aseries of polyamidines prepared by acid-catalyzed condensa-tion polymerization of 4,40-diaminodiphenylmethane with arange of orthoesters. More recently, Tang et al. described thecondensation polymerization of orthoester diamino mono-mers with dimethyladipimidate dihydrochloride as a route tonew poly(orthoester amidine) copolymers that were able toform polyplexes with DNA plasmid and undergo pH-trig-gered DNA release.8 The direct polymerization of amidine-containing monomers has also been reported although thereare relative few examples with the majority of thosereported being styrenic derivatives. Zhang et al.9 describedthe emulsion polymerization of a new amidine-containingmonomer with styrene, butyl acrylate, methyl methacrylate,and acrylonitrile forming latex particles that were able toundergo reversible coagulation/dispersion by virtue ofswitching the amidine functionality between nonionic andionic states via reversible salt formation with CO2/H2O.

Additional Supporting Information may be found in the online version of this article.

VC 2012 Wiley Periodicals, Inc.

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Costoyas et al.10 also described the emulsion copolymeriza-tion of the styrenic-amidine monomer 4-vinylbenzyl acetonehydrazone with styrene. Kim et al.11 detailed the synthesis ofa molecularly imprinted polymer with cholesterol esteraseactivity prepared by the radical copolymerization of N,N0-diethyl(4-vinylphenyl)amidine with methyl methacrylate andethylene glycol dimethacrylate. More recently, Guo et al.12

described the reversible addition–fragmentation chain trans-fer (RAFT) synthesis of well-defined linear homopolymerscontaining amidine functionality via a post-polymerizationapproach. Trithiocarbonate-mediated polymerization of 4-vinylbenzyl chloride yielded a reactive precursor homopoly-mer that was subsequently treated with NaN3 to give thecorresponding azido-functional polymer. Cu-catalyzedcoupling of the azide functional groups on the polymer withN0-propargyl-N,N-dimethylacetamidine yielded the CO2-repsonsive amidine polymer. Yan et al.13 described the atomtransfer radical polymerization of AB diblock copolymers ofpoly(ethylene oxide) (PEO) with (N-amidino)dodecylacrylamide (ADA) employing a PEO macroinitiator yiedingamphiphilic materials. The PEO-polyADA (PEO-PADA) blockcopolymers were shown to undergo self-directed assemblyin aqueous media forming spherical vesicular structures.Controlling the degree of ionization of the ADA units viatreatment with CO2 enabled the formation of vesicles withvarious sizes since the aggregates expand as the ADA speciesbecome ionized. This ‘‘breathing’’ behavior was shown to becompletely reversible. Allen et al.14 recently detailed the con-trolled radical polymerization of 4-vinylimidazole, formallyan amidine, via RAFT in glacial acetic acid with 4-cyano-4-(ethylsulfanylthiocarbonylsulfanyl) pentanoic acid as theRAFT agent. Homopolymerization was shown to be con-trolled as evidenced by the linear pseudo first order kinetics,molecular weight control, and narrow molecular weight dis-tributions of the resulting polymers. Finally, it is worth not-ing that Patrickios and coworkers15–18 have described thegroup transfer (co)polymerization of 2-(1-imidazolyl)ethylmethacrylate yielded novel materials including homopoly-mers, block copolymers, and networks that exhibit, for exam-ple, catalytic activity in the hydrolysis of p-nitrophenylacetate.15,16

RAFT polymerization is an example of a reversible deactiva-tion radical polymerization (RDRP) process that is mediatedby thiocarbonylthio compounds.19–25 As a polymerizationtechnique RAFT has evolved into arguably the most versatileand robust of the RDRP techniques exhibiting a remarkablydiverse functional group tolerance facilitating the directpreparation of many functional (co)polymers with manyinteresting properties and potential applications including,but not limited to, gold complexation.23,26–36 In recent years,there has been significant interest in combining polymersprepared by RAFT with various highly efficient chemistriesincluding alkyne–azide coupling,37 a range of thiol-basedreactions,38–43 as well as the preparation of functional(co)polymers via post-polymerization modification ofactivated ester precursors such as those based on penta-fluorophenyl (meth)acrylate (PFP(M)A). Indeed, there is an

impressive range of novel applications of PFPA-containing(co)polymers reported in the literature as well as other acti-vated monomers based on PFP esters.44–49 For example,Chua et al.44 recently described the synthesis of a library ofPEG-(meth)acrylamido (co)polymers by the post-polymeriza-tion reaction of PFP(M)A residues with a-amino, x-methoxyfunctionalized poly(ethylene glycol)s. The resulting PEG-amide (co)polymers exhibited significantly improved water-solubility compared to the (meth)acrylic analogues asevidenced by their LCST characteristics and were also shownto possess UCST-type behavior in select low molecularweight alcohols.

Herein we describe the facile preparation of a series of(co)polymers formally containing CO2-responsive amidineand guanidine functionality. Unlike many previous literatureexamples that have required the synthesis of novel amidinemonomers, or the introduction of such functional groupspost-polymerization with novel reactive species, we haveexploited the naturally occuring amidine- and guanidine-containing molecules histamine and L-arginine methyl ester.The RAFT synthesis of PFPA homopolymers and copolymersyields reactive scaffolds that can be quantitatively modifiedto amides via acyl substitution reactions with primaryamine-containing molecules. We describe the quantitativesynthesis of histamine and L-arginine-containing homo- andcopolymers via this post-polymerization modificationapproach. Finally, we show how such CO2-responsive behav-ior can be exploited to induce self-directed assembly in thehistamine-based AB diblock copolymers and that the transi-tion between unimeric and self-assembled states is fullyreversible and cyclable.

EXPERIMENTAL

All reagents were purchased from the Aldrich ChemicalCompany at the highest available purity and used as receivedunless stated otherwise. L-Arginine methyl ester dihydro-chloride was purchased from Chemimpex and used asreceived. 2,20-Azobis(2-methylpropionitrile) (AIBN) wasrecrystallized twice from methanol and then stored at �24�C until needed. Pentafluorophenyl acrylate (PFPA) was pre-pared according to the general procedure described byEberhardt et al.45 N,N-Dimethylacrylamide (DMA) and poly-(ethylene glycol) methyl ether acrylate (PEGA, molar mass ofmonomer ¼ 480 g/mol) was purified by passage over a col-umn of basic alumina to remove inhibitor and then stored at5 �C until needed.

Synthesis of BenzylpropyltrithiocarbonateBenzylpropyltrithiocarbonate (BPTC) was synthesized accord-ing to the general method of Rotzoll et al.50 but with a slightmodification:

To a solution of triethylamine (1.1 g, 10.9 mmol) in chloro-form (10.0 mL) at 0 �C was added propanethiol (0.83 g, 10.9mmol). The reaction solution was stirred for 20 min afterwhich carbon disulfide (1.0 g, 13.1 mmol) was added drop-wise. After stirring for a further 3 h, benzyl chloride (1.8 g,14.4 mmol) was added and the reaction temperature raised

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to 50 �C for 3 h. Chloroform was subsequently removed byrotary evaporation and the crude product purified by silicacolumn chromatography using hexane/ethyl acetate (3:2) aseluent. The target species was isolated as a yellow liquid(1.1 g, 4.3 mmol, 40% yield). 1H NMR (300 MHz, CDCl3, d(ppm): 1.04 (t, J ¼ 7.37 Hz, 3H), 1.68–1.82 (m, 2H), 3.34–3.39 (t, J ¼ 7.21 Hz, 2H), 4.62 (s, 2H), 7.26–7.43 (m, 5H);13C NMR (75 MHz, CDCl3, d (ppm): 13.76, 21.87, 39.21,41.66, 127.88, 128.84, 129.40, 135.25.

RAFT Homopolymerization of PentafluorophenylAcrylateBelow is a typical procedure for the homopolymerization ofPFPA:

To a glass vial (25 mL) equipped with a magnetic stirbarwas added PFPA (2.0 g, 8.4 mmol), BPTC (0.1 g, 0.42 mmol),AIBN (0.014 g, 0.084 mmol), and acetonitrile (4.0 mL). Thevial was sealed with a rubber septum and nitrogen was thenbubbled through the solution for 30 min. The vial was subse-quently immersed in a preheated oil bath set at 70 �C andpolymerization was allowed to proceed for 13 h. The poly-merization was halted by cooling and exposure to air. Poly-PFPA (PPFPA) was isolated by double precipitation in metha-nol. PPFPA was dried in vacuo prior to characterization. Thesame procedure was followed for the synthesis of two addi-tional higher target molecular weight PPFPA homopolymers.In these instances, the [monomer]:[BPTC]:[AIBN] were equalto [40]:[1]:[0.2] and [80]:[1]:[0.2]. Isolated yields were 50,57, and 56%, respectively.

RAFT Homopolymerization of Poly(ethylene glycol)Methyl Ether Acrylate (PEGA)Below is a typical procedure for the homopolymerization ofPEGA:

To a vial, equipped with a magnetic stir bar was addedPEGA (6.0 g, 12.5 mmol), BPTC (0.038 g, 0.156 mmol), AIBN(2.56 mg, 0.0156 mmol) ([monomer]:[CTA]:[AIBN] ¼80:1.0:0.1), and DMF (6.0 mL). The vial was sealed with arubber septum and the solution sparged with N2 for 30 min.The vial was then immersed in a pre-heated oil bath set to60 �C and polymerization was allowed to proceed for 6 h.The polymerization was halted by cooling and exposure toair. The PEGA homopolymer was then purified by dialysisagainst MeOH for 3 days with three changes of solvent perday using 1000 Da MWCO dialysis tubing. MeOH was thenremoved using a rotary evaporator and the PEGA homopoly-mer was dried in a vacuum oven at 40 �C.

Reaction of PFPA Homopolymers with Primary Aminesin the Presence of S-Methyl MethanethiosulfonatePFPA homopolymers were reacted with histamine dihydro-chloride or L-arginine methyl ester dihydrochloride in thepresence of S-methyl methanethiosulfonate (MMTS). Theexample given below highlights the procedure for the reac-tion between PPFPA and histamine dihydrochloride.

To a glass vial equipped with a magnetic stirbar was addedPPFPA (0.28 g, 1.18 mmol PFPA repeat units, 1.0 equiv.),MMTS (53 lL, 10 equiv. with respect to polymer endgroups),

and DMF (2.0 mL). To a second glass vial was added hista-mine dihydrochloride (0.54 g, 2.94 mmol, 2.5 equiv.), DBU(1.06 mL, 7.06 mmol, 6.0 equiv.), and DMF (2.0 mL). Aftercomplete dissolution the solutions were combined and thereaction left to stir overnight at ambient temperature.

Synthesis of AB Dibock Copolymers of PEGA with PFPAIn all instances, PEGA-PFPA AB diblock copoplymers wereprepared using a polyPEGA macro chain transfer agent(CTA). Below is the procedure for the preparation of a PEGA-PFPA block copolymer with a targeted molar composition of80:20, PEGA:PFPA.

To a glass vial equipped with a magnetic stir bar was addedpolyPEGA macroCTA (0.5 g, 1.0 mmol), PFPA (0.062 g, 0.26mmol), AIBN (4.0 mg, 0.024 mmol), and acetonitrile (1.0mL). The vial was then sealed with a rubber spetum. Afterdissolution of all the components the solution was spargedwith N2 for 30 min and then immersed in an oil bath pre-heated to 70 �C. Polymerization was allowed to proceed for6 h. Polymerization was halted by cooling and exposure toair. Acetonitrile was then removed using a rotary evaporator.The AB diblock copolymer was then dissolved in a small vol-ume of toluene and purified by a series of precipitations intohexane. After filtration/isolation with a Buchner funnel, theblock copolymer was dried in a vacuum oven at 40 �C (iso-lated yield: 42%). The same procedure was followed for thepreparation of PEGA-PFPA diblocks with targeted molar com-positions of 65:35 (isolated yield: 45%) and 50:50 (isolatedyield: 46%).

Synthesis of AB Dibock Copolymers of PFPA withN,N-DimethylacrylamideIn all instances, PFPA-DMA AB diblock copolymers were pre-pared with a PPFPA macroCTA. Below is detailed the proce-dure for the synthesis of a PFPA-DMA block copolymer witha targeted molar composition of 20:80, PFPA:DMA.

To a glass vial equipped with a magnetic stir bar was addedpolyPFPA (0.9 g, 3.78 mmol of PFPA), DMA (1.5 g, 15.12mmol), AIBN (4.0 mg, 0.024 mmol), and anisole (3.0 mL).The vial was sealed with a rubber septum and the solutionpurged with N2 for 30 min. Subsequently, the vial wasimmersed in a pre-heated oil bath set at 70 �C and polymer-ization was allowed to proceed for 1.5 h prior to terminationusing a combination of cooling and exposure to air. Blockcopolymers were purified by a series of precipitations inhexane followed by isolation by Buchner filtration and dry-ing in vacuo at 40�C (isolated yield: 26%). The same proce-dures were followed for the preparation of PFPA-DMAdiblocks with targeted molar compositions of 35:65 (isolatedyield: 39%) and 50:50 (isolated yield: 53%).

Reaction of PEGA-PFPA and PFPA-DMA AB DiblockCopolymers with Primary Amines in the Presenceof S-Methyl MethanethiosulfonatePEGA-PFPA/PFPA-DMA diblock copolymers were reactedwith primary amines of either histamine dihydrochloride orL-arginine methyl ester dihydrochloride in the presence ofMMTS. The example below shows the typical procedure used


POLYMER SCIENCE


for the reaction of PEGA-PFPA with histamine dihydrochlor-ide in the presence of MMTS.

To a glass vial equipped with a magnetic stir bar was addedPEGA-PFPA diblock (0.2 g, 0.132 mmol, 1.0 equiv.), MMTS (7lL, 10 equiv. with respect to polymer end groups), and DMF(2 mL). To a separate glass vial was added histamine dihy-drochloride (0.073 g, 0.395 mmol, 3 equiv.), DBU (0.118 mL,0.79 mmol, 6 equiv.), and DMF (2 mL). After complete disso-lution, the solutions were combined and the reaction wasleft to stir overnight at ambient temperature.

In the case of L-arginine methyl ester dihydrochloride, thesame equivalent of reactants was used.

General Purification ProceduresHistamine and arginine modified (co)polymers were purifiedby dialysis using regenerated cellulose membrane with a mo-lecular weight cut off of 3500 g/mol. Dialysis was performedfor three days with solvent changes three times per day. His-tamine modified homopolymers were dialyzed againstMeOH, arginine modifed species against water, statisticalcopolymers against water for the first day, and then MeOHfor the following two days. MeOH was removed by rotaryevaporation, a small volme of water was added and the(co)polymers isolated by lyophilization.

Instrumentation and Sample PreparationNuclear magnetic resonance (NMR) spectra were recordedon a Bruker DPX 300 spectrometer at 300 MHz for hydrogennuclei, 282 MHz for fluorine nuclei, and 75 MHz for carbonnuclei. Chemical shifts are reported in parts per million(ppm). Data are reported as follows: chemical shift [multi-plicity (s ¼ singlet, d ¼ doublet, dd ¼ doublet of doublets,t ¼ triplet, m ¼ multiplet), coupling constants in Hertz,integration]. Measurements were performed in eitherCDCl3, D2O, or CD3OD with the internal solvent signalbeing used as the reference signal (7.26 ppm for CDCl3solutions, 4.79 ppm in D2O solution, and 3.31 ppm forCD3OD). NMR data were evaluated using iNMR 4.0.4 Mes-trelab Research software. Size exclusion chromatography(SEC) was performed on a Shimadzu system with fourphenogel columns in dimethylacetamide (DMAc) as eluentat a flow rate of 1.0 mL/min, and equipped with a refrac-tive index detector. Chromatograms were analyzed usingCirrus SEC software (version 3.0). The system was cali-brated with a series of narrow molecular weight distribu-tion polystyrene (PS) standards with molecular weights inthe range of 580–1,820,000 g/mol. Dynamic light scatter-ing (DLS) measurements were performed on a MalvernZetasizer Nano ZS at a scattering angle of 173�. Data wereanalyzed with Malvern Zetasizer Software version 6.20.Samples were prepared at a concentration of 1 wt %. Inthe case of HIS-based block copolymers, 3.0 mg of samplewas initially dissolved in MeOH (150 lL) prior to dilutionwith distilled water (2850 lL). For ARG-based copolymersthe sample (3.0 mg) was directly dissolved in distilledwater (3.0 mL). All samples were filtered through a 13-mm polytetrafluoroethylene (PTFE) syringe filter of 0.45lm pore size prior to analysis. For gas cycling, samples

were transferred to a glass vial and sealed with a rubberseptum. N2 or CO2 (100% pure from Coregas) was thenbubbled through the solutions for 5 min prior to filteringas detailed above and DLS analysis. Transmission electronmicroscopy (TEM) was conducted on a Jeol 1400 instru-ment with an accelerating voltage of 100 kV. AB diblockcopolymers were initially dissolved in a 95% vol/vol solu-tion of water and methanol. Two drops of the solution (5g/L) were dropped onto a carbon-coated TEM grid on atissue and dried overnight. Collected images were used toobtain an average particle size using the equation Dn ¼P

niDi/P

ni, where Dn is the number-average diameter.

RESULTS AND DISCUSSION

Successful RAFT (co)polymerization requires the use of anappropriate thiocarbonylthio mediating agent.51 Of the fami-lies of RAFT agents both dithioesters and trithiocarbon-ates23,52 are well-documented to be suitable for acrylicand acrylamido monomers. Given this, we first prepared abenzyl functional trithiocarbonate, BPTC. BPTC was pre-pared via standard procedures involving base-mediatedaddition of propanethiol to CS2 followed by a nucleophilicsubstitution reaction with benzyl chloride. The structureof this trithiocarbonate was confirmed using a combina-tion of 1H and 13C NMR spectroscopy (see Supporting In-formation). With this trithiocarbonate inhand, three homo-polymers of PFPA with targeted molecular weights of5000, 10,000, and 20,000 were prepared under standardRAFT conditions (Scheme 1).

Figure 1 shows the SEC traces for the three precursor PPFPAhomopolymers. In all instances, the molecular weight distri-butions are narrow with polydispersity indices (Mw/Mn) inthe range 1.15–1.18. The absolute molecular weights, asdetermined by end-group analysis via 1H NMR spectroscopy,were 6800 (Mn theory 5000) and 10,200 (Mn theory10,000), both of which agree well with the targeted theoreti-cal molecular weights. End-group analysis was not possiblefor the highest molecular weight PFPA homopolymer.

In the next step, the well-defined PFPA homopolymers weretreated with a slight excess of the commercially availableamidine and guanidine species histamine dihydrochloride(HIS) and L-arginine methyl ester dihydrochloride (ARG)under basic conditions, Scheme 1, in an acyl substitutionreaction to yield the target acrylamido derivatives. However,while such reactions are very facile it is noted that the thio-carbonylthio end-group on the PFPA homopolymers are alsosusceptible to aminolysis by these primary amines. Indeed,this is the most common method for effecting end-groupcleavage in RAFT-prepared (co)polymers.38,41 The aminolysisof such thiocarbonylthio species yields the correspondingmacromolecular thiols that are prone to oxidative couplingyielding polymeric disulfides as well as thiolactones in someinstances.53,54 To avoid such undesirable side reactions, thetreatment of the PFPA homopolymers with HIS and ARG wasperformed in the presence of MMTS. MMTS, and variousfunctional derivatives, have been previously shown to behighly effective for trapping macromolecular thiols formed



from end group aminolysis reactions thus eliminating thepossibility of undesirable side-reactions.44,55,56 Successfulconversion of the PFPA homopolymers to the correspondingHIS and ARG acrylamido derivatives was verified using acombination of 1H and 19F NMR spectroscopies. As a repre-sentative example, Figure 2 shows the 1H NMR spectra ofthe 6800 molecular weight precursor PFPA homopolymerand the corresponding HIS and ARG derivatives. As expectedthe 1H NMR spectrum of the PFPA precursor homopolymeris relatively simple with the major resonances being thoseassociated with the polymer backbone visible between d�2.0 and 3.0 ppm. Signals associated with the Ph group atthe a-terminus and the ACH3 group associated with thepropyl group of the thiocarbonylthio species at the x-termi-nus are evident at d �7.5 and 1.0 ppm, respectively. Theanalogous 1H NMR spectra for the higher molecular weightPFPA homopolymers and the HIS and ARG-modified speciescan be found in the Supporting Information. In the case ofthe HIS-modified species (Fig. 2, middle), the spectrum isentirely consistent with a poly(histamine acrylamide)obtained from the direct radical polymerization of the hista-mine acrylamide monomer reported in the literature.57 The1H NMR spectrum of the ARG derivative (Fig. 2, bottom) isconsistent with the predicted spectrum. The Ph end group atthe a-terminus is visible at d �7.2 ppm; however, the re-mainder of the signals falls between d �1 and 4 ppm makingit difficult to obtain any quantitative structural information.

The reversible capture-and-release properties of CO2, in theform of the HCO3

� salt, of both HIS and ARG modified homo-polymers was initially examined in aqueous media. As notedabove, the ability of amidines and guanidines to reversiblybind CO2 in this manner is well documented4,12,13,58 and ishighlighted in Scheme 2. In the case of the HIS-based

homopolymers the reversible fixation of CO2 as a salt wasaccompanied with a hydrophobic-to-hydrophilic transition. Inthe free, non-ionized state, the HIS homopolymers werehydrophobic and exhibited little-to-no solubility in water,whilst in the ionized, or complexed state, the HIS homopoly-mers were readily water-soluble.

In contrast, the ARG-based homopolymers, and specificallythe two higher molecular weight species, did not exhibit anytunable solubility with CO2/N2 cycling and remained whollywater-soluble. While this clearly limits their use as buildingblocks in stimulus-induced assembly applications such guani-dine functionality does have potential in other important

SCHEME 1 Outline for the preparation of homopolymers containing amidine and guanidine functionality via the post-polymeriza-

tion modification of poly(pentafluorophenyl acrylate) homopolymers with histamine and L-arginine methyl ester.

FIGURE 1 SEC traces of three precursor PFPA homopolymers

measured in N,N-dimethylacetamide on a system calibrated

with narrow molecular weight distribution polystyrene

standards.


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areas. For example, Tew and coworkers have demonstratedthat guanidinium-containing (co)polymers exhibit antimicro-bial activity against both Gram-positive and Gram-negativebacterium59,60 as well as being able to serve as efficient pro-tein transduction domain mimics.32,61

The ability of the HIS homopolymers to undergo a reversiblesoluble-to-insoluble transition in water in response to differ-ent gases clearly suggests that HIS could serve as a suitablebuilding block in diblock copolymers for stimulus-inducedself-directed assembly in aqueous media. Given this, in addi-tion to homopolymers of HIS and ARG, a series of precursordiblock copolymers of DMA or poly(ethylene glycol acrylate)(PEGA) with PFPA were also prepared. In the case of thePEGA-based block copolymers syntheses were accomplishedemploying a polyPEGA macroCTA for the subsequent poly-merization of PFPA whilst in the case of the DMA-basedmaterials AB diblocks were prepared with a PPFPA macro-CTA. Both synthetic strategies are shown in Scheme 3 whilea summary of the theoretical target compositions, actualcompositions (as determined by 1H NMR spectroscopy), Mn,and Mw/Mn values for the block copolymers are given inTable 1.

The diblock copolymers were readily prepared with varyingcomposition as summarized in Table 1. All block copolymerswere prepared such that the major component in the materi-als was the permanently hydrophilic species (DMA or PEGA).In all instances, the resulting copolymers had reasonablynarrow molecular weight distributions with polydispersityindices � 1.28. With the PEGA/DMA-PFPA diblock copoly-mers in hand, each was modified with the HIS and ARG dihy-drochlorides in the same fashion as outlined in Scheme 1 forthe PFPA homopolymers. This yielded four new series ofcopolymers, namely the PDMA-b-HIS, PDMA-b-ARG, PEGA-b-HIS, and PEGA-b-ARG diblock species.

Successful modification of the PFPA-based precursor diblockcopolymers was confirmed by 1H and 19F NMR spectroscop-ies and, in all instances, indicated quantitative formation ofthe amidine and guanidine functional block copolymers. As arepresentative example, Figure 3 shows the 1H NMR spec-trum of a DMA-HIS diblock copolymer with a molar

FIGURE 2 1H NMR spectra of a parent PFPA homopolymer (abso-

lute molecular weight: 6800, recorded in CDCl3) (top) and the cor-

responding histamine acrylamido (middle) recorded in CD3OD

and arginine acrylamido (bottom) derivatives recorded in D2O.

SCHEME 2 Reversible CO2 capture and release in amidine and

guanidine functional species via the in situ formation of car-

bonic acid with subsequent protonation and salt formation.



composition of 54:46 with the resonances associated withthe HIS residues highlighted.57

Given the non-responsive nature of the ARG modified(co)polymers only the HIS-based diblock copolymers weresubsequently evaluated as potential CO2-responsive materialscapable of gas-induced assembly–disassembly. Given the abil-ity to tune the solubility of the HIS block it was anticipatedthat when complexed with CO2 in the form of a salt the DMAand PEGA-based diblock copolymers would exist as molecu-larly dissolved unimers while subsequent switching of theHIS solubility with N2 would result in self-directed assemblyyielding nanosized objects such as micelles, Scheme 4 lightgray blocks represent the tunable HIS species while the darkgray block represents a permanently hydrophilic species. Todetermine the ability of the DMA-HIS block copolymers tobehave as theorized in Scheme 4, the 54:46 DMA:HIS diblock

copolymer was analyzed using a combination of DLS andTEM.

The 54:46 DMA-HIS diblock copolymer was initially dis-persed in pure water. Under these conditions, the block co-polymer is amphiphilic and spontaneously formed aggregateswith a measured hydrodynamic diameter (Dh) of 36.2 nmand a corresponding polydispersity of 0.012 [Fig. 4(A) blackline and (B) first data point]. Upon bubbling CO2 through thesolution the HIS residues become ionized and hydrophilicand the aggregates disassemble into unimers with a meas-ured Dh of 3.0 nm. Subsequent N2 bubbling deionizes theHIS residues reconferring amphiphilic characteristics and theblock copolymer self-assembles forming nanosized specieswith measured Dh values essentially identical to the origi-nally formed aggregates (43.4 nm, polydispersity 0.065).This process is readily repeatable and in all instances

SCHEME 3 Synthetic outline for the RAFT synthesis of diblock copolymers of PFPA with DMA and PEGA

TABLE 1 Summary of Target and Actual Molar Compositions

and the Measured Number Average Molecular Weights (Mn)

and Polydispersity Indices (Mw/Mn) for AB Diblock Copolymers

of PFPA with DMA and PEGA

Copolymer

Target

composition

Actual

Compositiona Mnb Mw/Mn

b

PFPA-DMA 20:80 15:85 67,600 1.28

PFPA-DMA 35:65 30:70 49,200 1.17

PFPA-DMA 50:50 46:54 35,900 1.20

PEGA-PFPA 80:20 73:27 17,400 1.21

PEGA-PFPA 65:35 43:57 17,900 1.28

PEGA-PFPA 50:50 33:67 21,000 1.28

a As determined by 1H NMR spectroscopyb As determined by size exclusion chromatography in DMAc.

FIGURE 3 1H NMR spectrum of the PDMA-HIS diblock copoly-

mer with a composition of 54:46, recorded in d4-methanol.


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excellent reproducibility is observed in terms of aggregateand unimer sizes, Figure 4(B).

In the case of the other DMA:HIS diblock copolymers, sim-ilar observations were made with the copolymers exisitingas unimers in the fully doubly hydrophilic state (with CO2

fixation) while aggregates were observed upon N2 sparg-ing (see Supporting Information). For example, in the caseof the 70:30 DMA:HIS block copolymer unimers with ameasured average diameter of 3.93 nm were observed inthe doubly hydrophilic state, while aggregates of 76.9 nm(polydispersity 0.198) were formed after N2 bubbling.Very similar observations were made for the series ofPEGA:HIS block copolymers with unimer sizes of 5.1(73:27 PEGA:HIS), 4.8 (53:77), and 5.0 nm (33:67) beingmeasured while aggregate sizes ranged from 10.2 to 36.1nm. While we have not at this time conducted a thoroughinvestigation of the effect of A and B block lengths on theaggregation process this preliminary data clearly indicatesthat adjusting the balance of the two building blocksshould allow for the tuning of aggregate sizes and, pre-sumably, morphology.

While the DLS data is compelling, the aggregates were alsoanalyzed by TEM and 1H NMR spectroscopy. As an example,Figure 5 shows an unstained TEM image of the 54:46 DMA:-HIS block copolymer. Clearly, the aggregates possess a spher-ical morphology consistent with the formation of polymericmicelles. Additionally, the size distribution appears to be rea-sonably narrow, and the average calculated diameter (34.4nm) is consistent, although lower (as expected), than thosedetermined by DLS.

In the DLS and TEM experiments, it is assumed that the re-versible self-assembly is a direct result of the tunable hydro-philicity/hydrophobicity of the HIS residues while DMAremains in a permanently hydrophilic state. To confirm thatthe aggregation process is due to a change in the solvationstate of the PHIS block 1H NMR spectra were recorded ofthe diblock copolymer in its fully hydrophilic (ionized) stateas well as its amphiphilic state induced by N2 bubbling. Suchsimple NMR experiments have previously been demonstratedto be an effective method for following the relative solvationstate of repeat units in a (co)polymer and to verify antici-pated solution behavior for AB diblock copolymers.23,62–67

Figure 6 shows the 1H NMR spectra, recorded in D2O, of thediblock copolymer in these two different states.

The clearest, and most obvious, difference relates to the shiftand change in intensity of the two peaks associated with thearomatic heterocycle of the HIS residues. In the ionized,hydrophilic state (Fig. 6 black spectrum) the spectrum isidentical to that shown in Figure 3 with all the majorresonances associated with the HIS repeat units clearly visi-ble, although slightly shifted to lower d values due to the dif-ferent NMR solvents. After N2 sparging distinct changes areobserved with these signals. The two aromatic signals shiftto lower ppm values and decrease significantly in intensityand broaden. This is consistent with the dehydration of thesespecies. However, the fact that these two signals are stillpresent implies that there is still significant hydration associ-ated with these species and is due, presumably, to the pres-ence of water molecules via H-bonding associations. In con-trast, the signal associated with the methylene groupadjacent to the ring completely disappears and further con-firms dehydration of the HIS residues. Collectively, the 1HNMR experiment in conjunction with the DLS and TEMresults confirm that self-association into polymeric micellesof the DMA-HIS block copolymer is a direct result of thehydrophilic-to-hydrophobic transition associated with theHIS residues.

FIGURE 4 A: Volume average size distributions for the 54:46

DMA:HIS diblock demonstrating the reversible unimer-to-

aggregate phase transitions as a function of N2 and CO2

bubbling, and (B) a plot of hydrodynamic diameter as a func-

tion of CO2/N2 cycling (line is added to aid the eye).

SCHEME 4 Anticipated reversible self-assembly of a PHIS-con-

taining diblock copolymer with N2 and CO2 serving as the

external triggers for assembly and disassembly, respectively.



CONCLUSIONS

Herein we have described the synthesis and aqueous solutionproperties of novel amidine-based CO2-responsive AB diblockcopolymers that are capable of undergoing reversible self-association into spherical polymeric micelles. The approach

described herein facilitates facile access to gas responsiveamidine-based (co)polymers without the need for monomersynthesis or the preparation of amidine-functional reactivereagents as described previously. Well-defined precursordiblock copolymers composed of permanently hydrophilicDMA or poly(ethylene glycol) methyl ether acrylate werecopolymerized, at different molar ratios, with the protectedester species PFPA via RAFT in the presence of a trithiocar-bonate CTA. Subsequent reaction of the PFPA residues withhistamine dihydrochloride or L-arginine methyl ester dihydro-chloride in the presence of methyl methanethiosulfonateyielded, quantitatively, the corresponding amidine and guani-dine-based block copolymers. Histamine-based copolymerswere able to undergo reversible assembly/disassembly byvirtue of the hydrophobic-to-hydrophilic transition associatedwith the histamine residues upon reversible binding of CO2.Spherical polymeric micelles with measured hydrodynamicdiameters of about 40.0 nm were formed as determined byDLS and TEM.

ACKNOWLEDGEMENTS

JYQ is grateful for financial support via an Australian Postgrad-uate Award (APA). PJR acknowledges partial funding from theAustralian Research Council (DE120101547).

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FIGURE 6 1H NMR spectra, recorded in D2O, of the 54:46

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FIGURE 5 TEM image of aggregates formed from the DMA:HIS, 54:46, diblock copolymer.


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Reversible addition fragmentation chain transfer polymerization - RAFT

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