Synthesis and applications of heterobifunctional poly ... · resistant to hydrolytic degradation or...

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Polymer 49 (2008) 345e373www.elsevier.com/locate/polymer

Feature Article

Synthesis and applications of heterobifunctionalpoly(ethylene oxide) oligomers

M.S. Thompson, T.P. Vadala, M.L. Vadala, Y. Lin, J.S. Riffle*

Department of Chemistry and the Macromolecules and Interfaces Institute, Virginia Polytechnic Institute and State University,

Blacksburg, VA 24061-0212, USA

Received 6 August 2007; received in revised form 17 October 2007; accepted 21 October 2007

Available online 24 October 2007

Abstract

Poly(ethylene oxide) (PEO) oligomers are employed extensively in pharmaceutical and biomedical arenas mainly due to their excellentphysical and biological properties, including solubility in water and organic solvents, lack of toxicity, and absence of immunogenicity. PEOcan be chemically modified and reacted with, or adsorbed onto, other molecules and surfaces. Sophisticated applications for PEO have increasedthe demand for PEO oligomers with tailored functionalities, and heterobifunctional PEOs are often needed. This review discusses the synthesisand applications of heterobifunctional PEO oligomers possessing amine, carboxylate, thiol, and maleimide functional groups.� 2007 Elsevier Ltd.

Keywords: Poly(ethylene oxide), PEO; Polyethylene glycol, PEG; Polyether

Open access under CC BY-NC-ND license.

1. Introduction

Heterobifunctional PEOs have the structure XePEOeY,where X and Y are different functional groups. They can serveas hydrophilic, flexible, biocompatible, and inert spacers withdefined lengths connecting two components. Applicationsinclude linking of macromolecules to surfaces, site-specifictargeting of drugs and liposomes, and functionalization of nano-particles for bioassays and biorecognition [1e15]. Whenbounded to other molecules, PEO typically increases their solu-bility in aqueous media and yields improved circulation times invivo [16e26]. PEO-modified surfaces also display increasedhydrophilicity as well as suppressed protein adsorption, plateletadhesion and macrophage attachment [27e31].

There have been several reviews discussing PEO chemistry,most of which have focused on monofunctional PEO, withonly limited treatment of heterobifunctional polymers [32e36].This review concentrates on synthetic pathways to achieve

* Corresponding author. Tel.: þ1 540 231 8214; fax: þ1 540 231 8517.E-mail address: [email protected] (J.S. Riffle).

0032-3861 � 2007 Elsevier Ltd.doi:10.1016/j.polymer.2007.10.029

Open access under CC BY-NC-ND license.

heterobifunctional oligomers and applications of thesematerials.

2. Synthesis and properties of PEO

PEO is a linear or branched polyether often terminated withhydroxyl groups that are derived from neutralization of the ter-minal ether repeated unit in the chain. PEO oligomers are mostcommonly synthesized via anionic ring-opening polymeriza-tion of ethylene oxide (EO). One desirable characteristic ofPEO is the relatively narrow molecular weight distributionthat can be achieved compared with many other polymers.PEO prepared by anionic ring-opening polymerization gener-ally has a polydispersity (Mw/Mn) less than 1.1 [37].

Anionic ring-opening polymerization of EO can be living innature due to the stability of the propagating species. Suchpolymerizations of EO are often carried out with a hydroxideor alkoxide initiator (Fig. 1). The reactions take place via nucleo-philic attack on an EO methylene to open the ring and form thepropagating species.

Epoxide polymerizations can also be conducted with coordi-nation catalysts, usually in conjunction with an alcohol initiator.

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HO–

OO–CH2CH2HO

OCH2CH2HO x H OCH2CH2HO x CH2 CH2 O–

O++

H+

Fig. 1. Anionic ring-opening polymerization of EO initiated by hydroxide.

346 M.S. Thompson et al. / Polymer 49 (2008) 345e373

Double metal cyanide catalysts are commonly employed to po-lymerize propylene oxide (PO) and sometimes EO. These cata-lysts are well known for their activities in syntheses of highquality poly(propylene oxide) (PPO) with low unsaturation[38e42]. The side reaction that occurs in PO polymerizationscaused by hydrogen abstraction from the methyl side chain ofthe monomer by a basic catalyst can be avoided. This class ofcatalysts was originally discovered by workers at GeneralTire, Inc., with improvements made in other companies includ-ing ARCO, Shell, Asahi Glass, and Bayer [39,40,43,44]. Oneexample of a double metal cyanide catalyst, Zn3[Co(CN)6]2,was utilized by Huang et al. to synthesize random copolymersof EO and PO [45]. Copolymers with various EO/PO composi-tions and with unimodal molecular weight distributions in therange of 1.21e1.55 were obtained.

PEO is clear, colorless, odorless, inert to many chemicalagents, stable against hydrolysis, and nontoxic. Biocompatibil-ity and lack of immunogenicity make PEO an important poly-mer for biomedical applications [20,46]. When bounded to animmunogenic substrate having a desirable function in thebody, PEO tends to reduce or eliminate immune response sothat the organism can tolerate the substance [36]. Another im-portant property of PEO is its solubility in water as well as inmany organic solvents. When bounded to a water insolublecompound, the resulting PEO conjugate generally displaysincreased water solubility or dispersibility [22,47,48].

Since the repeating ether units of PEO are essentially non-reactive, these oligomers must be reacted with, or adsorbedonto, other compounds through terminal or pendant functionalgroups. Examples include PEO with thiol or carboxylic acidend groups that have been adsorbed onto metal or metal oxidesurfaces [11,48e52]. Maleimide functionality on PEO chainends is one example that has been of great interest in theformation of bioconjugates [53e55].

As an aside, although aliphatic polyethers such as PEO areresistant to hydrolytic degradation or attack by nucleophiles or

+

Protected or latent functional group

functionalization

termA

B

X OHO

HO CH2 CH2 O n H

Fig. 2. Synthetic methods to produce heterobifunctional PEO oligomers: direct synth

acids, the relatively labile CeH bonds adjacent to the etheroxygens are susceptible to oxidative degradation througha radical mechanism. This can be especially important in caseswhere one or more of the PEO end groups has been modifiedwith reactive groups such as methacrylate or acrylate thatwould subsequently be exposed to a free radical reaction.Other post-reactions such as the deposition of plasma coatingsmay also lead to PEO degradation due to these aspects.

Two broad methods are commonly employed for synthesiz-ing heterobifunctional PEO oligomers (Fig. 2). The most directis the ring-opening polymerization of EO from a heterobifunc-tional anionic initiator, and this is followed by termination withanother functional moiety. The second method involves partialderivatization of PEO diols, followed by separation of the resul-tant statistical mixtures to isolate the targeted heterobifunc-tional oligomers.

Initiation and polymerization of EO by a heterobifunctionalinitiator so that one of the functional groups reacts with theEO and the other group remains intact is shown in Fig. 2A[32,34,56e58]. Anionic polymerizations of EO from initiatorscontaining a protected functional group such as potassiumbis(trimethylsilyl)amide or (cyanomethyl)potassium, and ter-mination by acidification, have been utilized to prepare hetero-bifunctional PEOs with a hydroxyl group at one end [59e63].The hydroxyl terminus can then be derivatized to producemore complex heterobifunctional polymers such as H2NePEOeCOOH [56]. Other heterobifunctional PEO oligomers have beensynthesized with various end groups such as carboxyl, vinylben-zyl, acetal, pyridyl disulfide, maleimide, and methacryloyl moi-eties by utilizing an appropriate initiator and terminating agent[58,64e68]. When synthesizing heterobifunctional PEOs, thereaction conditions must be completely anhydrous to ensurethe purity of the heterobifunctional product. If water is present,then it will also initiate the EO, producing PEO diols as sideproducts and reducing the anticipated molecular weights ofthe desired heterobifunctional polymers [61]. It is also importantto note that the polymerization of ethylene oxide can be danger-ous and care must be taken when working with toxic and poten-tially explosive gases.

The second method requires alteration of the terminalhydroxyl groups of a,u-dihydroxy-poly(ethylene oxide) (PEOdiol) through a series of reactions, followed by separation ofthe mono-, di-, and un-substituted components (Fig. 2B). Toobtain heterobifunctional oligomers, only a portion of the hy-droxyl end groups are converted to the new functional group,

reactions, X and Y

inating agent, YX CH2 CH2 O n Y

X CH2 CH2 O n Y

X CH2 CH2 O n X

Y CH2 CH2 O n Y

esis of heterobifunctional PEO (A) and end group modification of PEO diols (B).

RH2N

CH2 C OHCH2CH2O xCH2CH2X

O

N

O

O

HO+

CH2 CCH2CH2O xCH2CH2X

O

NH R

N

O

O

CH2 C OCH2CH2O xCH2CH2X

O

DCC

Fig. 3. Formation of an amide linkage via the active succinimidyl ester.

347M.S. Thompson et al. / Polymer 49 (2008) 345e373

so the statistics of these reactions alone lead to low yields.These synthetic approaches are complicated because mostemploy several reaction steps and require post-separations ofchemically similar polymers that differ only in their end groupstructures [61,69,70]. As the molecular weights of the startingPEO diols increase, the chemical and physical differencesamong the mono-, di-, and un-substituted products becomeincreasingly smaller and the heterobifunctional oligomers aretherefore harder to isolate [71]. For these reasons, most hetero-bifunctional PEOs synthesized from PEO diols are either lowmolecular weight oligomers, or they possess ionizable endgroups such as amines or carboxylic acids that enable separa-tions by ion exchange chromatography.

3. Synthesis and applications of XePEOeCOOH

a-Carboxy-u-hydroxy-poly(ethylene oxide) (HOOCePEOeOH) oligomers are important precursors to PEO biocon-jugates. PEO with a carboxylic acid on one end and anotherfunctional group on the other can also be utilized as inter-mediates for other heterobifunctional polymers [72e74]. Animportant example is that a carboxyl terminus of PEO canbe activated by forming highly reactive succinimidyl esters.Many biologically relevant ligands have been covalentlyattached to PEO through amide bonds via these succinimidylester intermediates (Fig. 3) [27,75e78]. When using XePEOeCOOH especially in biomedical applications, it is alsoimportant to consider the stability of heterobifunctionalPEOs linked to substrates via ester bonds. These ester bondscan be hydrolyzed under certain conditions and this phenom-enon can be taken advantage to release biological molecules ofinterest [79].

3.1. Direct synthesis of XePEOeCOOH

3.1.1. Synthesis of (HOOC)1e3ePEOeOHHeterobifunctional PEO with a hydroxyl group on one chain

end and 1e3 carboxylic acid groups on the other have beensynthesized utilizing vinylsilylpropanol initiators (Fig. 4) [80].These initiators containing one, two, or three vinyl groupswere synthesized from 3-chloropropylchlorodimethylsilane,3-chloropropyldichloromethylsilane, and 3-chloropropyltrichlorosilane, respectively, by reaction with vinylmagnesiumchloride (Fig. 5). The alkyl chlorides were then converted tothe corresponding alcohols.

Si K1. THF, RT2. acetic acid

CH2CH2CH2-O

CH2CH2CH2-OSi

Si

xCH2CH2O H

OHSCH2-C-OH

toluene, 80 °CAIBN

Fig. 4. Synthesis of (HO

The alkoxide initiators were prepared by reacting theappropriate vinylsilylpropanol with potassium naphthalide inTHF (1 mol of eOH:0.95 mol of potassium naphthalide). Aslight deficiency of potassium naphthalide ensured that thevinyl groups were preserved during alkoxide formation andpolymerization. The anionic initiator was added to EO andreacted at room temperature, and the polymerizations wereterminated with acetic acid. The ratio of end group protons ob-served via 1H NMR matched the theoretical values, (3:2:2:2,9:2:2:2, and 6:2:2:2 for the mono-, di-, and tri-vinylsilane ini-tiators, respectively) confirming the structures of the heterobi-functional polymers. Molecular weights obtained by 1H NMRand SEC matched well with the targeted values based on themonomer to initiator ratios. Molecular weight distributionswere narrow (�1.13).

Conversions of the terminal vinylsilane moieties into carb-oxylic acids were achieved via eneethiol additions utilizingmercaptoacetic acid and AIBN (Fig. 4). The functionalizationswere monitored via 1H NMR by following the disappearance ofvinyl proton resonances at approximately 6.0 ppm. The NMRresonances of the products corresponded with those reportedby Wilson et al. for tricarboxylic acid terminated PDMS [81].A similar synthesis utilizing cysteamine hydrochloride andAIBN was also carried out to yield (H2N)1e3ePEOeOH [82].

Si

CH2CH2CH2-O CH2CH2O Hx

x

HOOCCH2SCH2CH2

HOOCCH2SCH2CH2

HOOCCH2SCH2CH2

CH2CH2CH2-O CH2CH2O H

OC)3ePEOeOH.

Si

Si

SiSi

Si

Si

1)

2)

3)

Cl

Cl

Cl

CH2CH2CH2 Cl

CH2CH2CH2

CH2CH2CH2 H2O, HMPACH2CH2CH2

CH2CH2CH2

CH2CH2CH2

Cl

I

I

Cl3 MgCl

THF60 °C24 h

60 °C

100 °C

NaHCO3

acetone2 Nal

OH

Fig. 5. Preparation of (1) 3-chloropropyltri-vinylsilane, (2) 3-iodopropyltri-

vinylsilane, and (3) 3-hydroxypropyltri-vinylsilane.

CO

KO CH2 CH2 S K+

O1) n

2) HClCO

HO CH2 CH2 S CH2 CH2 O Hn

Fig. 6. Thiolate-initiated anionic polymerization of EO in the presence of

a weak carboxylate nucleophile.


Heterobifunctional poly(ethylene oxide-b-propylene oxide)(PEO-b-PPO) block copolymers have also been synthesizedutilizing trivinylsilylpropoxide as the initiator [83]. Even un-der mild reaction conditions, the anionic polymerization ofPO leads to some unsaturation at the chain end due to abstrac-tion of a methyl proton on the PO monomer converting it toallyl alkoxide [84e86]. This abstraction reaction is generallyreferred to as a chain-transfer reaction and not only increasesthe unsaturation in the product, but also limits the degree ofpolymerization. Compared with conventional base-catalyzedpolymerization of PO, double metal cyanide catalysts yieldhigh quality PPO with negligible unsaturation and much fasterrates of polymerization even with low catalyst concentrations[42,45]. Thus, the PPO blocks of these diblock copolymerswere prepared in batch polymerizations utilizing the doublemetal cyanide catalyst, zinc hexacyanocobaltate (Impact 3,Bayer), and this yielded heterobifunctional PPO oligomerswith molecular weight distributions in the range of 1.84e1.91. Retention of the desired end groups during polymeriza-tion was confirmed by 1H NMR. These (vinyl)3ePPOeOHoligomers were then utilized as macroinitiators to polymerizeEO anionically, and this was followed by conversion of thevinyl groups to carboxylic acids via eneethiol reactions asdescribed above.

The relatively broad molecular weight distributions obtainedfrom batch reactions utilizing double metal cyanide catalystshave been well documented [42,45,87]. These catalysts are het-erogeneous at least in the initial stages of polymerization, andthey become somewhat solubilized as the reactions proceed.The broadened molecular weight distributions relative to thoseobtained with base-initiated polymers can likely be attributed toa combination of the heterogeneous nature of the catalysts andalso to the very low catalyst levels utilized (i.e., 25e100 ppm)relative to the concentrations of propagating chain ends. Theinitiator to catalyst ratio was 103 or higher for these polymeri-zations. At the beginning of the reaction there are relativelyfew active chains coordinated with the catalyst and a relativelyhigh concentration of monomer. These conditions combined

with a fast rate of propagation relative to chain transfer couldlead to the broadened molecular weight distributions observedfor these batch reactions.

Carboxylate anions have been widely utilized for adsorbingsurfactants or polymers onto the surfaces of magnetite nano-particles [48,88e90]. Copolymer dispersion stabilizers havebeen synthesized that contain carboxylic acids at specificpositions along the backbone of the copolymer, and withcontrol over the number of functional groups in the polymer[48,52]. Magnetite complexes have been reported withtriblock copolymers consisting of a polyurethane anchor blockcontaining carboxylic acid functional groups flanked by PEOand PEO-b-PPO tail blocks adsorbed on their surfaces, andthe copolymers served as dispersion stabilizers in aqueousmedia [48,52]. The synthetic approach utilizing the vinylsilyl-propoxide initiators to prepare carboxylic acid containing PEOdispersion stabilizers does not require a separate anchoringblock such as the urethane segment. These carboxylic acidfunctional oligomers can be adsorbed onto the surfaces ofmagnetite nanoparticles, and the resultant polyetheremagne-tite complexes can be dispersed in water. The nanocomplexesalso offer possibilities for conjugating bioactive molecules tothe free hydroxyl ends on the PEO after complexation to themagnetite surface. Post-conjugation of bioactive moleculesto nanoparticles is of great interest for applications in targeteddrug delivery and for biorecognition of particular cell types.

3.1.2. Synthesis of HOOCePEOeOH via thiol-initiatedanionic polymerization

Zeng and Allen investigated alkoxide and thiolate initiatorswith protected or free carboxylic acid groups for synthesizingheterobifunctional PEOs with a carboxyl group at one chainend and a hydroxyl group at the other (HOePEOeCOOH)[73]. One effective functional initiator was 3-mercaptopro-pionic acid. Dipotassium-3-mercaptopropionate was preparedby reacting 3-mercaptopropionic acid with 2 equiv of potassiumnaphthalide. EO was added to the initiator solution and reactedat 40 �C in THF, and the reactions were terminated with hydro-chloric acid to yield the heterobifunctional PEOs (Fig. 6).Molecular weights ranging from 1000 to 25,000 g mol�1 wereachieved with narrow molecular weight distributions, 1.07e1.15. End group analysis by 1H NMR showed the expectedstructure of the heterobifunctional PEOs, thus confirming thatthe carboxylate did not participate in the polymerizations underthe conditions utilized.

The HOePEOeCOOH oligomers were utilized as macro-initiators for synthesizing a-carboxy-poly(ethylene oxide-b-3-caprolactone) copolymers via a hydrochloric acid-catalyzedcationic polymerization [73]. Poly(3-caprolactone) blockswere synthesized with molecular weights ranging from 2000

CH2 CH CH2 OH

CH2 CH CH2 O CH2 CH2 O CH2x CH2 O C

O

CH2 CH2 C

O

OH

CH2 CH2 CH2 O CH2 CH2 O CH2x CH2 O C

O

CH2 CH2 C

O

OHSC

O

CH3

NCH2 CH2 CH2 O CH2 CH2 O CH2x CH2 O C

O

CH2 CH2 C

O

OHSS

1) Potassium naphthalide2) Ethylene oxide3) Succinic anhydride

hνAIBNThioacetic acid

2,2’-dithiodipyridineN-propylamine

Fig. 7. Synthesis of pyridyleSSePEOeCOOH.


to 9000 g mol�1 and the copolymers had molecular weightdistributions �1.18. Analysis of the block copolymers bySEC showed that no residual unreacted PEO macroinitiatorremained.

3.1.3. Synthesis of NaOOCePEOeNH2 via(cyanomethyl)potassium-initiated PEO

Polymerizations of EO by (cyanomethyl)potassium haveyielded heterobifunctional PEOs with a carboxyl group atone end and an amine at the other (NaOOCePEOeNH2)[56]. The (cyanomethyl)potassium initiator was prepared byreacting an equimolar ratio of potassium naphthalide and ace-tonitrile in THF at room temperature. The initiator was addedto solutions of EO and 18-crown-6 in THF, and the polymeri-zations were conducted at 30 �C. a-Cyano-u-hydroxy-poly(ethylene oxide) oligomers (NCePEOeOH) were synthesizedwith molecular weights ranging from 400 to 5000 g mol�1 andwith molecular weight distributions �1.26.

The hydroxy terminus of NCePEOeOH was converted toa primary amine through a series of three reactions. The firststep was activation of the hydroxyl terminus for nucleophilicdisplacement by reaction with toluene-4-sulfonyl chloride toyield NCePEOeOTs. The tosyl group was displaced with po-tassium phthalimide, and then the phthalimide end group wasremoved by reaction with hydrazine hydrate. SEC confirmedthat the molecular weights of the polymers remained unalteredthrough this series of reactions. The degree of amination was>92% as indicated by titrations.

The cyano group was hydrolyzed in a sodium hydroxidesolution at 80 �C to yield the heterobifunctional NaOOCePEOeNH2. Hydrolysis of the cyano group was confirmed bythe disappearance of the nitrile triple bond absorptionat 2164 cm�1 and the presence of a new absorption at1587 cm�1 corresponding to a carboxylate salt in the infraredspectra. SEC showed unimodal molecular weight distributions,and this suggested that the polymers were not degraded duringhydrolysis of the cyano groups.

To develop a targeted drug delivery vehicle, Zhang et al.utilized the NaOOCePEOeNH2 oligomers as macroinitia-tors for ring-opening polymerizations of g-benzyl-glutamateN-carboxyanhydride to produce block copolymers comprisedof hydrophilic PEO and hydrophobic poly(g-benzyl-L-gluta-mic acid) [72]. In aqueous media these copolymers self-as-sembled into micelles with diameters ranging from 30 to80 nm, and the aggregates had carboxylate groups on theirsurfaces. It was demonstrated that the carboxylate endgroups could be coupled with several biologically importantligands.

3.1.4. Synthesis of HOOCePEOeSHDerivatizations of allyl groups in the presence of a radical

generator to form functionalities such as amino, carboxy, hy-droxy, and thiols are well known [91]. Ishii et al. synthesizeda heterobifunctional PEO with pyridyl disulfide at one chainend and a carboxylic acid at the other (PyridyleSSePEOeCOOH) utilizing allyl alcohol as initiator (Fig. 7) [92]. Allylalcohol was reacted with potassium naphthalide to afford

potassium allyl alkoxide, and this was followed by polymeri-zation of EO. The polymers were terminated with succinicanhydride to produce a carboxylic acid end group. TheallylePEOeCOOH precursors had narrow molecular weightdistributions and their molecular weights were in good agree-ment with the targeted values. The polymer structures wereconfirmed by 1H NMR spectra. The integral ratios of the sig-nals assigned to the methylene protons between the twocarboxyl groups and the signal corresponding to the allylmethylene protons was ca. 2:4, indicating that the functional-ization of the chain end was quantitative.

Radical additions of thioacetic acid did not proceed to com-pletion using AIBN as the initiator at 60 �C. Some couplingbetween chains also occurred under those reaction conditions.By contrast, when the allyl end was modified by the radicaladdition of thioacetic acid under UV irradiation at room tem-perature, the reactions proceeded to completion without anychain coupling. The thio-ester group was cleaved under alka-line conditions to yield a mercaptan. To obtain the mercaptogroup without also cleaving the oxo-ester at the other chainend, reaction conditions were tailored to selectively deprotectthe thio-ester. Thus, an aminolysis reaction was carried out indry THF with a 20:1 excess of n-propylamine relative to thethio-ester. The reactions were conducted in the presence of2,20-dithiodipyridine and monitored by 1H NMR. The peakintensity of the thio-ester group gradually decreased whilethe peak intensity associated with the carboxyl end groupremained constant, indicating that selective reaction of thethio-ester was achieved. The mercapto group was then freeto react with the 2,20-dithiodipyridine to form pyridyleSSePEOeCOOH. The pyridyl disulfide prevented oxidation ofthe mercapto group during storage of the polymer. SECconfirmed that the reaction sequence proceeded withoutdimerization of the PEO chains. The pyridyl disulfideprotecting group was cleaved with dithiothreitol,

CH CH CH2CH2 SHHS

OH

OH, to generate the free mercaptan.


A heterobifunctional PEO with the structure HSePEOeXhas been employed in biosensor chips having tethered PEOchains with functional groups at the free chain ends [92].AldehydeePEOeSH oligomers on the sensors were utilizedto immobilize proteins via reductive amination [49]. However,sufficient protein was not always bounded to the surfacesvia reductive amination due to the protein-repelling natureof PEO. Alternatively, HOOCePEOeSH was tethered tothe sensor surface via the SH end and the carboxyl terminuswas activated with N-hydroxysuccinimide. The active esterwas formed by immersing the surface with the PEOeCOOHtethered chains in a solution of N-hydroxysuccinimide(NHS) and N-ethyl-N0-(3-dimethylaminopropyl)carbodiimide(EDC). Using a surface plasmon resonance sensor, the effi-ciency of protein immobilization via reaction of the N-hydr-oxysuccinimidyl ester with the PEO tethered chains wascompared to immobilization via imine formation, then re-duction, of the aldehyde-ended PEO tethered chains. Ishiiet al. found that the PEO surface with the active ester immo-bilized a much higher amount of IgG than the aldehydeePEOsurface.

Herrwerth et al. synthesized HSePEOeCOOH to function-alize the surfaces of gold substrates [78]. EO was polymerizedutilizing 10-undecen-1-ol and sodium hydride as the initiatorto produce an allylePEOeOH intermediate. The hydroxylterminus was deprotonated with sodium hydride, then reactedwith the sodium salt of chloroacetic acid to form allylePEOeCOOH. This method of attaching a carboxylic acid via anether linkage was more stable than the ester linkage formedfrom the reaction with anhydrides. Thioacetic acid was reactedwith the allyl terminus utilizing UV irradiation with AIBN asthe radical generator. The stability of the ether linkage allowedfor subsequent hydrolysis of the thio-ester to be carried outwith concentrated hydrochloric acid and ethylenediamine-tetraacetic acid to afford the HSePEOeCOOH oligomer.Matrix assisted laser desorption/ionization time-of-flight massspectroscopy (MALDI-TOF MS) showed a molecular weightdistribution of 1.02, indicating that this series of functionaliza-tion reactions proceeded cleanly.

The HSePEOeCOOH polymer formed a densely packedself-assembled monolayer (SAM) on gold [78]. The SAMwas linked to antibodies after activating the carboxylic acidterminus with NHS in the presence of EDC. The resistanceto non-specific protein adsorption remained even after immobi-lization of antibodies on the surface. Even though the SAMwas inert to non-specific adsorption, specific antigens couldeffectively bind to the immobilized receptors. The surfaceproperties of the HSePEOeCOOH monolayer were comparedto an antibody film covalently attached to the surface via(3-aminopropyl)trimethoxysilane (APTMS) and glutaralde-hyde (GA). Although the amount of surface-bounded anti-bodies was higher on the APTMS/GA surface, theperformance in complex protein solutions was significantlyreduced compared to the HSePEOeCOOH SAM. Largeamounts of non-specific proteins adsorbed on the APTMS/GAsurface, and deactivated the binding sites of the surface-bounded receptors.

3.2. Synthesis of XePEOeCOOH via end groupmodification of PEO diols

3.2.1. Synthesis of allylePEOeCOOHVölcker et al. synthesized allylePEOeCOOH from a PEO

diol in four steps [57]. First, the PEO diol was tosylated to ac-tivate the chain ends toward nucleophilic substitution (TsOePEOeOTs). End group analysis via 1H NMR determinedthat the conversion was almost quantitative, and molecularweight distributions obtained from MALDI-TOF MS andSEC remained unchanged as expected.

Selective substitution of one tosylate by an end group ofhigher hydrophilicity in a biphasic environment was unsuc-cessful. Therefore, statistically driven reaction conditionswere employed, and this was followed by separation usingion exchange chromatography on DEAEeSephadex A-25. Al-lyl alcohol was reacted with TsOePEOeOTs in an equimolarratio to obtain a mixture of mono-, di-, and un-substituted olig-omers. An IR spectrum of the mixture showed characteristicabsorptions corresponding to the remaining tosylates as wellas the new allyl end groups. The ratio of tosyl/allyl groups,1.16, was calculated from the ratio of 1H NMR peak integralscorresponding to their respective end groups.

Ethyl mercaptoacetate was reacted with the remaining tosy-lates, and this was followed by hydrolysis of the esters to yielda mixture of allylePEOeallyl, allylePEOeCOOH, andHOOCePEOeCOOH. The allylePEOeCOOH polymer wasisolated from the mixture based on the amount of carboxylicacid functionality by ion exchange chromatography with anoverall yield of w35%. End group analysis via 1H NMR de-termined that the allyl/carboxymethylthio ratio was 1.0. Rela-tively narrow molecular weight distributions were obtained viaMALDI-TOF MS.

3.2.2. Synthesis of a HOOCePEOeOH intermediate anddistearoylphosphatidylethanolamineePEOehydrazide(DSPEePEOeHz)

A heterobifunctional PEO with the structure lipidePEOeXwas utilized to prepare liposomes bearing functional groups ontheir exterior. A heterobifunctional PEO with a DSPE moiety onone chain end and a hydrazide at the other (DSPEePEOeHz)was synthesized from a PEO diol (Fig. 8) [77,93]. First, ethylisocyanatoacetate was utilized to partially introduce carboxylgroups via urethane linkages onto the PEO diol, and this was fol-lowed by hydrolysis of the ethyl esters. A mixture of mono- anddi-functional materials was produced and some unreacted PEOdiol remained. The heterobifunctional HOePEOeCOOH wasseparated from the mixture by ion exchange chromatographyon a DEAEeSephadex A-25 column. Titration of the carboxylgroups gave 97% of the theoretical value.

A boc-protected hydrazide was introduced onto theintermediate by coupling tert-butyl carbazate to the carboxylterminus of HOePEOeCOOH in the presence of dicyclohexyl-carbodiimide (DCC). The hydroxyl end group was then acti-vated toward nucleophilic substitution with disuccinimidylcarbonate (DSC) in the presence of pyridine to yield SCePEOeHz-boc. Mild reaction conditions were important for

CH2 O n H

CH2 O n CO

NH CH2 CO

OH

HO CH2

CH2 O nCH2

HO CH2

O CH2 CH2 O n CO

NH CH2 CO

NH NH CO

OCH3

CH3CH3

COO

N

O

O

O

O

NH

OPO

OO

O

O

O

C17H35

C17H35

O

O

NHNH NH2

O

1) DSPE2) HCl

1) OCNCH2CO2Et/TEA2) H2O/NaOH3) Ion exchange chromatography

1) BocNHNH2/DCC2) DSC/Pyridine

Fig. 8. Synthesis of an amine-terminated lipidePEO copolymer (DSPEe

PEOeHz).


introducing the succinimidyl carbonate due to the potential re-activities of the boc and hydrazide groups. Quantitative incor-poration of succinimidyl carbonate was confirmed by 1HNMR together with titrations of the active acyl groups. Finally,DSPE was conjugated to the chain end via reaction with theactive acyl group in the presence of triethylamine to produceDSPEePEOeHz-boc. The polymer was purified by dialysisagainst saline solution followed by deionized water and then ly-ophilized. Deprotection with acid gave the target amine-func-tional DSPEePEOeHz and was confirmed by the absence ofthe 1H NMR signal associated with the boc.

It has been shown that liposomes modified with methoxyePEOeDSPE exhibited desirable pharmacokinetics and biodis-tributions [94]. A DSPEePEOeHz derivative was utilized toform liposomes bearing hydrazide groups on their surfacesfor introducing targeting moieties [77,93]. DSPEePEOeHzwas incorporated into liposomes prepared from lecithin andcholesterol. The pharmacokinetic behavior of the hydrazide-functional liposomes was compared to their protectedDSPEePEOeHz-boc and methoxyePEOeDSPE analogues.The liposomes were labeled with 67Ga and injected intrave-nously into rats, and their disappearance from the blood streamwas followed by quantifying the 67Ga-label. As anticipated,the pharmacokinetic behavior was essentially the same forthe liposomes bearing methoxy-, hydrazide-, and boc-pro-tected hydrazide groups on the surface. To determine if attach-ment of a targeting moiety on the surface of the functional

CH3

CH3

OCH2

CH2SH 6O

CH2SH 6O

Fig. 9. Structure of (HS

liposomes would affect their pharmacokinetic behavior, lipo-somes containing DSPEePEOeHz were covalently linked toa model ligand, IgG, through the hydrazide groups at theends of the polymer chains [93]. It was determined that cova-lent attachment of IgG to the PEO termini had no adverseeffects on blood circulation times of the liposomes.

3.3. Additional applications of XePEOeCOOH

3.3.1. SPR sensor chipsSurfaces coated with PEO-based materials have shown po-

tential for preventing non-specific adsorption of proteins, andthis is important for pharmaceutical applications and biomed-ical devices. As described previously, PEO bearing a mercaptogroup on one chain end can be utilized to form a SAM on goldsubstrates. For sensor devices, it is advantageous to haveanother functional group at the free PEO chain end to bindwith biological vectors. A carboxylic acid group at the freePEO chain end is typically used because it is easily activatedfor nucleophilic attack with NHS in the presence of a carbodi-imide such as EDC. Gobi et al. produced an immunosensorbased on SPR for detecting insulin utilizing a PEO with twomercapto groups on one chain end and a carboxylic acid onthe other ((HS)2ePEOeCOOH) (Fig. 9) [27]. The heterobi-functional PEO was used to functionalize the surface ofa thin gold film on an SPR chip. The amino groups at theN-termini of the two polypeptide chains on insulin were cova-lently attached to the free carboxylate-functional PEO chainend in the presence of NHS and EDC.

A competitive immunosensing approach was employed fordetecting insulin. An anti-insulin antibody was incubatedwith an analyte solution before being passed over the sensorchip. In the presence of insulin, both the insulin on the sensorsurface and the insulin in solution competitively adsorbed tothe anti-insulin antibody. Therefore, more anti-insulin antibodydetected on the SPR chip corresponded to lower concentrationsof insulin in the analyte solution. This approach employinga PEO spacer produced a sensor chip with high resistanceto non-specific adsorption of proteins. The chip could detectinsulin concentrations as low as one and as high as300 ng mL�1. The active sensor surface could also be regener-ated and reused for more than 25 cycles without significantchange in sensor activity.

3.3.2. Single molecule recognition force microscopySingle molecule recognition force microscopy (SMRFM) is

an atomic force microscopy technique that can measure inter-action forces on the single molecule level. The major compo-nent of SMRFM is a functionalized measuring tip that can

O CH2 CH2 O CH2 CH2 CO

OH3

)2ePEOeCOOH.

C

O

N

O

O

CH2 C

O

O3S CH2 CH2 CO

O CH2 CH2 O n CH2 CHCH3

NHCH2CHCH3

NHCH2CH2SO

OCHCH2

S

NHHN

O

CH2 CO


NHCH2CHCH3

NH C

O

N

O

O

CH2 C

O

O34

N

O

O

CH2 CH2 CO


NHCH2CHCH3

NH C

O

N

O

O

CH2 C

O

O35C

O

NSS CH2 CH2 C

O

O CH2 O nCH2 CHCH3

NHCH2 CH2CHCH3

NH C

O

N

O

O

CH2 C

O

O3

NSS CH2 CH2 C

O


NHCH2CHCH3

NH C

O

CH2 C

O

3 NH CH2 4CH

COOHNCH2COOH

CH2 COOH

Fig. 10. Heterobifunctional PEOs utilized in SMRFM.


specifically interact with a target molecule. Another keycomponent of SMRFM is a flexible spacer for binding ligandsthat allows them to freely move and rotate about the tip withina restricted volume corresponding to the length of the spacer[95]. Since the PEO chain is chemically and physically inert,heterobifunctional PEOs are ideal for this application. Differ-ent functional groups at the PEO chain ends allow for their co-valent reaction onto the AFM tip, and the length of the PEOdefines the rotational mobility.

Riener et al. synthesized several heterobifunctional deriva-tives from a H2NePEOeCOOH intermediate for tip-PEO-probe conjugation [76,95]. These heterobifunctional PEOspossessed an NHS-activated ester on one chain end and eitherbiotin, maleimide, vinylsulfone, or a pyridyl disulfide group atthe other (Fig. 10). Another important PEO derivative wassynthesized having a pyridyl disulfide group on one chainend and a nitrilotriacetic acid (NTA) group on the other.This was successfully used to tether His6-tagged proteins toAFM tips via noncovalent NTAeNi2þeHis6 bridges.

3.3.3. Anticoagulant medical devicesPrevention of blood coagulation on the surfaces of medical

devices is important to avoid complications during the clinicaluse of blood-contacting artificial devices. One method ofreducing blood coagulation is by regulating the activity ofthrombin, a key enzyme in the coagulation process, throughsurface modification of the biomaterial. The biocompatibilityof PEO makes it an ideal candidate for surface functionaliza-tion of medical devices.

Salchert et al. utilized H2NePEOeCOOH and HOOCePEOeCOOH to functionalize polymer coatings with a

benzamidine derivative capable of selectively binding throm-bin at their free termini (Fig. 11), and thus removing the coag-ulant from circulation [96]. The PEO oligomeric spacers werereacted with films of poly(octadecene-alt-maleic anhydride)copolymers. To attach the HOOCePEOeCOOH, the maleicanhydride-functional surface was reacted with 1,4-diaminobu-tane, and this was followed by coupling the polymer in thepresence of EDC and the sodium salt of N-hydroxy-sulfosuc-cinimide (sulfo-NHS). Attachment of the benzamidinederivative to the remaining free carboxylate groups wasaccomplished utilizing EDC and sulfo-NHS. When usingH2NePEOeCOOH, the amine terminus was reacted withthe maleic anhydride-functional surface to form an imide,and then the benzamidine derivative was bounded.

The benzamidine surface density was enhanced when sur-faces were prepared from the heterobifunctional polymercompared to the homobifunctional polymer. The decreasedactivity of the homobifunctional polymer was attributed toPEO bridging, thus leading to less free carboxyl groups onthe surface. Although PEO has the propensity to resist non-specific protein adsorption, these benzamidine-modified sur-faces selectively bound thrombin. The study concluded thatthe benzamidineePEO surfaces had significant potential forscavenging thrombin.

Chen et al. investigated immobilization of amine-containingbiomolecules including oligopeptides, proteins, and glycosami-noglycans (e.g., heparin) on Sylgard polysiloxane elastomers(Dow-Corning) utilizing heterobifunctional PEO spacers[3,75]. The siloxane surface was functionalized with SieHgroups using (MeHSiO)n under acidic conditions. First, theallyl terminus of allylePEOeNHS was grafted to the SieH

2 HCl

O

O

O

O CH2 CH2 O nCH2CHO CH2 C OH

OO

HN

NH2NH C

OCH2 NH2•11

CH2 CH2 CH2 CH2 NH2N

O

O

NH2 CH2 CH2 CH2 CH2 NH2

CH2 CH2 CH2 CH2N

O

O

O CH2 CH2 O nCH2C CH2 C

OONH

NH

NH2NHCCH2NH 11

O

1)

2)

EDC, sulfo-NHS

EDC, sulfo-NHS

O

O

O

O CH2 CH2 O nCH2CH2H2N CH2 CH2 NH C

O

CH2 O CH2 C

O

OH

N

O

O

O CH2 CH2 O nCH2CH2 CH2 CH2 NH C

O

CH2 O CH2 C

O

NH

NH2NHCCH2NH 11

O

HN

H2NNH C

OCH2 NH2•2 HCl112)

EDC, sulfo-NHS

1)

Surface modification using H2N-PEO-COOH

Surface modification using HOOC-PEO-COOH

Fig. 11. Surface functionalization with a benzamidine derivative.


functional surface by hydrosilation in the presence of a platinumcatalyst. Amine-containing biomolecules were then covalentlytethered to the surface through the PEOeNHS active ester. Forcomparison, surfaces with only PEO were prepared by a similarmethod using PEO with an allyl group at one end and an inertmethoxy group at the other. The heparin modified surfacesdemonstrated significantly less thrombin activity as comparedto the methoxyePEO surface, while maintaining resistance tonon-specific protein adsorption [3]. This approach to function-alized surfaces employs a relatively simple procedure that canbe utilized to tailor the surface groups to reject or attracta wide range of target molecules.

4. Synthesis and applications of H2NePEOeX

The higher reactivity of primary amine-terminated PEOcompared to hydroxy-terminated PEO in nucleophilic substitu-tion reactions makes it a widely used derivative for preparingbioconjugates. Low molecular weight drugs, cofactors, peptides,glycoproteins, and biomaterials can be linked with amine-func-tional PEO through amide, sec-amine, urea, and other chemicalbonds [97e101]. Furthermore, amine-terminated PEO caninitiate polymerizations of amino acid N-carboxyanhydridesor lactones/lactides for synthesizing biocompatible blockcopolymers [63,72,102,103].

4.1. Direct synthesis of heterobifunctional H2NePEOeX

4.1.1. Schiff base-initiated polymerization of EOEthanolamine with a protected amine has been utilized to

initiate and polymerize EO to generate a-amino-u-hydroxy-poly(ethylene oxide) (H2NePEOeOH) [59]. The Schiff basewas prepared from benzaldehyde and ethanolamine to preventthe primary amine from reacting with the EO. The anionicinitiator was prepared by reacting the Schiff base of ethanol-amine with an equimolar amount of sodium. The initiatorwas added to EO and the polymerization was carried out at95 �C. The protecting group was removed by acidificationwith hydrochloric acid to yield the H2NePEOeOH.

It is well known that end group functionalization can becarried out by terminating the propagating species of livinganionic polymerizations with a suitable terminating agent.One advantage of this method is that the polymer does notneed to be isolated and then functionalized in a subsequentstep. Huang et al. found that when bromoacetic acid wasused as a terminating agent for PEO that had been initiatedwith N-benzylideneaminoethoxide, the Schiff base prema-turely decomposed during the termination [74]. To avoidthis, two steps were employed to synthesize heterobifunctionalPEO with a carboxylic acid at one chain end. First, a Schiffbase-initiated PEO with a terminal hydroxyl group was

N

NCH2 CH2 O n CH2H2N C

O

NH SO2 NH

Fig. 12. PEO with a primary amine and sulfadiazine moieties.

N

O

O

CH2CH2 CO

CO

ON

O

O

O N

O

O

CH CH CO

CO

ON

O

O

OOH OH

disuccinimidyl succinate (DSS) disuccinimidyl tartrate (DST)

Fig. 14. Structure of DSS and DST.


synthesized. In the second step, the hydroxyl terminus wasdeprotonated with a slight molar excess of sodium in THF,and the mixture was cooled to 0 �C before dropwise additionof a bromoacetic acid solution in THF, and this was followedby refluxing. This produced a heterobifunctional PEO witha Schiff base-protected amine at one chain end and a carb-oxylic acid group at the other, with retention of the originalmolecular weight as determined by SEC.

To produce a heterobifunctional PEO for targeted drugdelivery, a tumor cell targeting agent, sulfadiazine, was cova-lently bounded to the carboxyl end of the PEO (Fig. 12) [74].The carboxylic acid was transformed to the more reactive acidchloride, and this was followed by adding sulfadiazine. TheSchiff base was then hydrolyzed with acetic acid to yield thefree amine, thus producing a polymer with a targeting moietyon one chain end and an amine group at the other for attachingan anti-tumor drug.

4.1.2. Potassium bis(trimethylsilyl)amide-initiatedpolymerization of EO

Potassium bis(trimethylsilyl)amide has been used as aninitiator for polymerizing EO to yield heterobifunctional poly-mers (Fig. 13) [60]. Following polymerization, the protectinggroup was removed by treatment with 0.1 N hydrochloric acid.The resulting polymer was purified by ion exchange chroma-tography. Molecular weights were in close agreement withpredicted values based on the initiator to monomer ratios,and the molecular weight distributions were w1.1. Titrationsconfirmed that each mole of polymer contained 1 mol of amine.

Tessmar et al. utilized the synthesis described above to pro-duce amine-reactive biodegradable diblock copolymers fortissue engineering, where surface-immobilized cell adhesionpeptides or growth factors are needed to control cell behavior[104]. PEO was synthesized with potassium bis(trimethylsilyl)-amide to produce the macroinitiator, H2NePEOeOH. Theblock copolymer, H2NePEOePLAeOH, was prepared byring-opening polymerization of D,L-lactide using stannous2-ethylhexanoate as catalyst. To ensure that only the hydroxylgroup participated in polymerization of the D,L-lactide, the tri-methylsilyl groups were either left intact or glacial acetic acidwas added to convert the amine into a non-reactive ammoniumsalt.

The presence of the amine group was difficult to confirm by1H NMR due to the high molecular weight of the polymer.Thus, an amine-reactive fluorescent dye was reacted with the

NMe3Si

Me3SiK+

O1) n

2) HCl OCH2CH2O nHCH2CH2H2N

Fig. 13. Potassium bis(trimethylsilyl)amide-initiated polymerization of EO.

diblock copolymer as well as with a control copolymer,methoxyePEOePLA, to establish that the dye would not reactwith the hydroxyl terminus of the PLA block. The polymer/dye mixture was analyzed using SEC with a UV detector. Me-thoxyePEOePLA and H2NePEOePLA showed no UV sig-nals at low retention times. However, the H2NePEOePLA/dye produced a significant UV signal indicating that thosepolymer chains had reacted with the dye, and this confirmedthe presence of the amine terminus. The amine chain end ofthe H2NePEOePLA diblock copolymer was transformedinto an NHS-activated ester by reaction with disuccinimidyltartrate (DST) or disuccinimidyl succinate (DSS) (Fig. 14).The capacity for the activated chain ends to react with aminegroups was confirmed by reaction with an amine-functionalfluorescent dye, and subsequent analysis by SEC with a UVdetector as previously described.

Potassium bis(trimethylsilyl)amide was also utilized to syn-thesize H2NePEOeOH polymers to prepare tumor-targetingdrug delivery systems. These heterobifunctional PEO oligo-mers were used to link an anti-tumor drug, chlorambucil, witha tumor-targeting moiety, sulfadiazine (Figs. 15 and 16)[105]. Protection of the primary amine end with benzaldehydewas required before selectively coupling chlorambucil to thehydroxy terminus. The carboxylic acid group on chlorambucilwas covalently reacted with the hydroxy terminus of the poly-mer chain using DCC as a coupling agent, and this was followedby deprotecting the amine with acetic acid to afford H2NePEOeCBL. The sulfadiazine was covalently bounded to theprimary amine terminus by first reacting 2-[N1-2-pyrimidinyl-( p-benzylidene)aminobenzenesulfonamido]ethanol with bis(trichloromethyl)carbonate. Subsequent addition of H2NePEOeCBL to the reaction mixture afforded the target com-pound, SDePEOeCBL. A series of non-targeted controlshaving the same molecular weights as the SDePEOeCBLpolymers were also prepared by covalently coupling monome-thoxy-poly(ethylene oxide) (MPEO) with chlorambucil(MPEOeCBL) using DCC.

To test the activity of chlorambucil bounded to PEO chains,cytotoxic assays were performed on C6 human breast cancercells. The IC50 values for MPEOeCBL, SDePEOeCBL, andfree chlorambucil were in the range of 10�8e10�9 mol L�1, in-dicating that the activity of chlorambucil was preserved in vitro,

C

O

CH2 3 NCH2

CH2

CH2

CH2

Cl

ClHO NH

N

NSO2NH2

chlorambucil (CBL) sulfadiazine (SD)

Fig. 15. Structure of chlorambucil and sulfadiazine.

NH CH2 CH2 O n C

O

CH2 3 NCH2

CH2

CH2

CH2

Cl

ClC

O

OCH2CH2N

N N

SO2H2N

1) DCC2) Acetic acid

C

O

CH2 3 NCH2

CH2

CH2

CH2

Cl

ClHO+O CH2 CH2 O n CH2 CH2 OHCH2CH2NCH

C

O

CH2 3 NCH2

CH2

CH2

CH2

Cl

ClO CH2 CH2 O n CH2 CH2 OCH2CH2H2N+

CHOCH2CH2N

N N

SO2NC

O

Cl

Fig. 16. Synthesis of SDePEOeCBL.


and there were no significant differences among the targetedand non-targeted polymer drugs. The toxicities of the polymerdrugs were tested using TA1 mice. The LD50 for the polymer-bounded chlorambucil with or without the targeting moietywas 10e16 times higher than for the chlorambucil control.

In vivo studies were conducted using Lewis lung carcinomaimplanted in mice. The anti-tumor activities of SDePEOeCBL were higher than the corresponding MPEOeCBLcontrols but slightly lower than chlorambucil. Since the SDePEOeCBL and MPEOeCBL polymer drugs showed nosignificant difference in activity in vitro, this suggested thatthe increased activity of the SDePEOeCBL in vivo couldbe attributed to the targeting moiety, sulfadiazine.

4.1.3. Potassium TDMA-initiated polymerization of EOA protected initiator, N-2-(2,2,5,5-tetramethyl-1-aza-

2,5-disilacyclopentyl)-ethylmethylamine (TDMA), was syn-thesized to prepare a heterobifunctional H2NePEOeOH(Fig. 17) [106]. The polymerization was carried out by firstreacting TDMA with an equimolar amount of potassium naph-thalide in THF, and then the EO was polymerized at room tem-perature. Termination of the reaction and deprotection of thesilylamine were accomplished by adding a few drops of aceticacid. 1H NMR spectra in DMSO-d6 showed that the peak ratioof signals corresponding to the hydroxyl end and methyleneprotons adjacent to the primary amine were 1:2, indicatingthat the polymer had one hydroxyl and one primary amine

O1) n

2) Acetic acidN

Si

SiN K

+ OCH2CH2O nHCH2CH2NCH3

CH2CH2NH2

Fig. 17. TDMA-initiated polymerization of EO.

terminus. The molecular weights determined by SEC matchedwell with the targeted values controlled with monomer toinitiator ratios, and the molecular weight distributions wererelatively narrow (�1.32). The results demonstrated that poly-merization of EO with the TDMA initiator took place withoutcleavage of the protecting group. MALDI-TOF MS showedsignals corresponding to the EO repeated unit plus the molec-ular weight of the two functional ends, thus providing evi-dence for purity of the heterobifunctional material.

4.1.4. (Cyanomethyl)potassium-initiated PEOHeterobifunctional PEOs have also been prepared by poly-

merizing EO initiated with (cyanomethyl)potassium [107].The synthesis of NCePEOeOH was carried out in a similarmanner as previously described [56]. Following polymeriza-tion, the cyano group was reduced with lithium aluminumhydride to afford H2NePEOeOH.

A triblock copolymer, poly(g-benzyl-L-glutamic acid-b-ethylene oxide-b-3-caprolactone) (PBLGePEOePCL), wassynthesized with modifications to the procedure describedabove [63]. A nitrile-terminated block copolymer, NCePEOePCL, was synthesized with the (cyanomethyl)potassiuminitiator in THF by the sequential polymerization of EO and3-caprolactone (3-CL). The nitrile was converted to a primaryamine by catalytic hydrogenation using palladium on carbonor Raney nickel to obtain H2NePEOePCLeOH. End groupanalysis via 1H NMR confirmed complete conversion of thecyano group. Catalytic hydrogenation was chosen over othermethods to convert the cyano group to an amine to avoid deg-radation of the relatively labile PCL ester linkages. Analysisby SEC before and after hydrogenation did not show any sig-nificant changes in molecular weight or molecular weightdistribution.


4.1.5. AllylePEOeOH intermediates for synthesisof H2NePEOeOH

Another approach to synthesize heterobifunctional PEOthat does not involve protecting and deprotecting primaryamines is to utilize a heterobifunctional initiator containinghydroxyl functionality and another functional group, such asallyl, that is unreactive during the polymerization. Cammaset al. synthesized a heterobifunctional PEO utilizing an allylalcohol initiator to yield allylePEOeOH with a narrowmolecular weight distribution (1.1) [61]. Molecular weightsobtained from SEC and 1H NMR were in good agreementwith those targeted.

A radical addition reaction was employed to introducea primary amine onto the allyl terminus by reacting with 2-aminoethanethiol hydrochloride (cysteamine hydrochloride)utilizing AIBN as the radical generator. The molecular weightsand molecular weight distributions determined by SEC wereunaffected, indicating that the radical addition reactions tookplace without significant side reactions. This synthetic ap-proach is extremely versatile due to the ability to convert theallyl terminus into a wide variety of functional groups througheneethiol additions [91].

4.1.6. Synthesis of vinylbenzylePEOeNH2Matsuya et al. constructed a core-shell fluorescent nano-

sphere with reactive PEO tethered chains on the surface fordetecting proteins in a time-resolved fluorometric immuno-assay [108]. A key component was a heterobifunctional PEOthat could be tethered to the surface and still possess a reactivemoiety for binding a biological vector. Vinylbenzyl alcohol(VBA) was shown to be a suitable initiator for EO to producean amine-functional PEO macromonomer (Fig. 18) [109]. Ini-tiation with VBA was a preferred method as opposed to obtainthe vinylbenzyl group by termination with vinylbenzylbromide because it was difficult to maintain the primary aminogroup quantitatively with the latter approach. VBA wasreacted with a stoichiometric amount of potassium naphthalidein THF to produce the alkoxide. After adding EO, the mixturewas stirred at room temperature for two days. Triethylaminewas added to the reaction and the mixture was poured intoa solution of methanesulfonyl chloride (MsCl) in THF to yielda MsOePEOeVBA intermediate. A primary amine was intro-duced onto the activated chain end by reacting MsOePEOeVBA with aqueous ammonia at room temperature for twodays to produce the H2NePEOeVBA macromonomer.Molecular weights of the macromonomers determined bySEC closely matched the predicted values based on the

O

CH2CCHH2C CH2 O K+

1) n

2) Ms-Cl, TEA

Aqueous ammonia CHH2C

Fig. 18. Synthesis of V

monomer to initiator ratios, and the molecular weight distribu-tions were relatively narrow (e.g., 1.20). However, some highmolecular weight impurities were observed by SEC, and thesewere attributed to small amounts of vinyl oligomerizationduring alkoxide formation. It was reasoned that the coupledproducts could initiate EO and lead to high molecular weightby-products.

Functionalized nanospheres were prepared in four steps.First, the H2NePEOeVBA was employed as a macromonomerand surfactant in a suspension radical polymerization of styreneto produce a coreeshell nanosphere. Nanospheres produced bythis method had PEO tethered chains on their surfaces bearingprimary amino groups at the free chain ends. The second stepwas to incorporate fluorescent europium chelates into the nano-psheres via physical entrapment. Conversion of the aminogroups into thiol-reactive moieties was carried out by reactingN-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carbox-ylate with the amine termini of the PEO chains on the nano-spheres to produce maleimide-functional surfaces (Fig. 19).Lastly, a biological vector, an anti-human a-fetoprotein (AFP)Fab0 fragment, was covalently bounded to the nanosphere sur-face via reaction of thiols with the maleimides. The fluorescentnanospheres bearing anti-human AFP Fab0 fragments were uti-lized for an immunoassay of AFP, and zeptomolar detectionwas reportedly achieved. Even with the antibody bounded tothe surface, non-specific binding was practically negligible.

4.2. Synthesis of H2NePEOeX via end groupmodification of PEO diols

4.2.1. Synthesis of H2NePEOeCOOHHeterobifunctional PEOs were prepared from PEO diols

with a carboxylic acid on one chain end and a primary amineon the other [110]. A PEO diol was chlorinated with a limitingamount of thionyl chloride in refluxing toluene. Introductionof ester-protected carboxylic acid groups onto the remaininghydroxyl termini was accomplished by reaction with an excessof ethyl isocyanatoacetate. An excess of sodium azide wasreacted with the chlorinated chain ends, then the carboxylateesters were deprotected by hydrolysis with an aqueoussolution of sodium hydroxide to produce the carboxylic acidend groups. The N3ePEOeCOOH intermediate was separatedfrom the mixture of products based on the number of carbox-ylic acid groups by ion exchange chromatography on DEAEeSephadex. It is also worth noting that PEO chains with differ-ent amounts of ionic groups could be separated by TLC onsilica gel using a 10:2:1 mixture of isopropanol, concentrated

H CH2 O OCH2CH2O nCH2CH2 S

O

O

CH3

CH2 O NH2CH2CH2O nCH2CH2

BAePEOeNH2.

O CH2 CH2 O n CH2 CH2 OBPSN

O

O

O CH2 CH2 O n CH2 CH2 OBPSH2N

O CH2 CH2 O n CH2 CH2 OBPSHNboc

O CH2 CH2 O n CH2 CH2 OHHNboc

O CH2 CH2 O n CH2 CH2 BrHNboc

HO CH2 CH2 O n CH2 CH2 OH SiCl

PhPht-Bu+

1) BPS-Cl 2) Chromatography

N OH

O

O

HO CH2 CH2 O n CH2 CH2 OBPS+

TPP/DIAD

Hydrazine hydrate

Boc2O/TEA

TBAF

CBr4/TPP

CF3COOH

O CH2 CH2 O n CH2 CH2 BrH2N

Fig. 20. Synthesis of H2NOePEOeBr.

N

O

O

NH2

NH2NH2

NH2

NH

H2N

H2N

H2N

N

O

O

CH2 C

O

O N

O

O

N

O

ON

O

O

N

O

O

N

O

O

NO

O

N

O

O

N

O

O

Fig. 19. Synthesis of maleimide-functional nanospheres.


aqueous ammonium hydroxide, and water. The azide was con-verted to a primary amine by catalytic hydrogenation to affordthe target H2NePEOeCOOH.

4.2.2. Synthesis of aminooxyePEOeBrA PEO diol was converted by a series of reactions and sep-

arations to yield a-aminooxy-u-bromo-poly(ethylene oxide)(H2NOePEOeBr) spacers for bioconjugates. The ease ofoxime ether formation reportedly made this a superior methodfor targeting aldehydes and ketones compared to reductiveamination (Fig. 20) [111]. The oxime ether linkage is stableat physiological pH.

A PEO diol was partially silylated with tert-butyldiphenyl-silyl chloride (BPS-Cl) and the intermediate was separated bycolumn chromatography. The remaining hydroxyl terminiwere then derivatized in a Mitsunobu coupling reactionwith N-hydroxyphthalimide in the presence of triphenylphos-phine and diisopropyl azodicarboxylate (DIAD),

CO

N N CO

CHCH3

CH3H3C

H3CCH , to afford the phthalimido ether. The

phthalimide end group was removed via hydrazinolysis toproduce the aminooxy-terminated polymer.

Prior to conversion of the silyl ether, the aminooxy termi-nus was protected by reacting it with di-tert-butyldicarbonate(boc2O) in the presence of triethylamine. Introduction of thebromide was accomplished by desilylation utilizing tetra-n-butylammonium fluoride (TBAF). The alcohol to bromidetransformation was completed by reaction with carbon tetra-bromide and triphenylphosphine at 0 �C, and this wasfollowed by deprotecting the boc-aminooxy moiety to yieldH2NOePEOeBr.

4.2.3. Synthesis of folate-targeted PEO carboplatinanalogues

Carboplatin (Fig. 21) is a chemotherapy drug that is mostcommonly used to treat ovarian and lung cancers but maybe used to treat other types of cancer as well. Several hetero-bifunctional PEOs were synthesized from PEO diols to inves-tigate the pharmacokinetic properties of PEO with a folic acid

moiety (Fig. 21) on one chain end and a carboplatin analogueon the other [16]. Folate-targeted PEO carriers are desirablebecause PEO conjugates are known to improve blood circula-tion times of low molecular weight drugs, and the folate termi-nus could also improve cell permeation by taking advantage offolate receptor-mediated endocytosis.

N

N N

N

H2N

OH

CH2NH C

ONH CH

C

CH2 CH2 CO

OH

OHO

O

Pt

O

O

O

NH3

NH3

Carboplatin Folic acid

Fig. 21. Structure of carboplatin and folic acid.


The first step toward folate-targeted PEO carboplatin ana-logues was to synthesize a mono-protected PEO diamine.This is a versatile starting material, allowing attachment ofa wide range of functional groups to either terminus via stableamide bonds. The first step was to activate the hydroxyltermini of the PEO diol by reaction with an excess of metha-nesulfonyl chloride to afford MsOePEOeOMs. ThenMsOePEOeOMs was dissolved in a concentrated solutionof aqueous ammonia to displace the methanesulfonyl endgroups and form the PEO diamine, H2NePEOeNH2. An equi-molar ratio of fluorenylmethyl chloroformate (fmoc) and PEOdiamine was reacted to yield a mixture of un-protected, mono-protected, and bis-protected PEO diamines. The mixture wasseparated on a Sephadex CM-25 cation exchange columnwith a yield of w30% of the mono-protected product. Endgroup analysis by 1H NMR showed that the ratio of signalscorresponding to the methylene protons adjacent to the un-protected amine terminus and those corresponding to themethylene protons adjacent to the fmoc-protected amineterminus were 2:2. Fluorescently labeled PEOs were preparedto determine whether the folate terminus affected accumula-tion of PEO in cells that had a high density of folate receptors(Fig. 22). An active ester of folic acid (FAeNHS) was reactedwith the fmoc-PEOeNH2 to yield fmoc-PEOeFA, and thiswas followed by removal of the fmoc group by reactionwith piperidine. The regenerated amine was then reactedwith fluorescein isothiocyanate (FITC) to yield FITCePEOeFA. A non-targeted, fluorescently tagged PEO control with

O CH2 CH2 O nCH2 CH2 NHCH2CH2NHFA fmoc

O CH2 CH2 O nCH2 CH2 NHCH2CH2NH2 fmoc

FA-NHSIon exchange chromatography

1) Piperidine/H2O2) FITC

O

O

OH

HOO

NHCS

O CH2 CH2 O CH2 CH2HNCH2CH2NHFA

Fig. 22. Synthesis of FITCePEOeFA.

a capped amine was also synthesized by reacting benzyl chloro-formate (Cbz) with one amine terminus and subsequentlyreacting with FITC to give CbzePEOeFITC.

PEO carriers that could release platinum once inside thecell were synthesized by platinating a dicarboxylate ligandon one chain end and attaching a fluorescent tag for monitor-ing cell uptake on the other. The carboplatin analogues weresynthesized by first reacting fmoc-PEOeNH2 with di-tert-butyl 2-(3-succinylaminopropyl)-malonate (MAL(tBu)2). Theprotecting group was removed and the amine was reactedwith FAeNHS as described above to give FAePEOeMAL-(tBu)2. The tert-butyl protecting groups were removed bytreatment with trifluoroacetic acid and transformed to thesodium salt by titration with a solution of sodium hydroxideto give FAePEOeMAL(Na)2, and this was followed by react-ing with cis-[Pt(NH3)2(D2O)2]

2þ(NO3)2 to yield the carbopla-tin analogue, FAePEOePt (Fig. 23). The platination wasmonitored by 1H NMR. Signals corresponding to the twomethylenes adjacent to the malonate shifted significantly uponplatination, indicating that the reaction was complete. ACbzePEOePt control was also synthesized in a similar fashion.

The study showed that non-targeted CbzePEOePt wasfour times more effective at inhibiting cell growth thancarboplatin alone and one and a half times more effectivethan FAePEOePt. The unexpected lower activity of FAePEOePt was attributed to neutralization or blocking of the Pt.

5. Synthesis and applications of HSePEOeX anddisulfideePEOeX

PEO oligomers with a mercapto or pyridyl disulfide groupat one chain end and another functional group at the other areuseful for preparing bio-interfaces. Heterobifunctional mer-capto or pyridyl disulfide-ended PEOs can be utilized for func-tionalizing gold and silver surfaces to construct reactive PEObrush layers. One such application is in the synthesis ofsurface-functionalized gold nanoparticles for colloidal sensorsystems in biological fluids [112]. It is well known that mod-ifying a surface with tethered PEO chains can dramaticallydecrease non-specific interactions of biopolymers [28,113e115]. A heterobifunctional PEO was required to constructgold nanoparticles possessing both sufficient colloidal stabilityand biological functionality for bioassays [11,49].

Due to the capacity for pyridyl disulfide moieties to reactwith thiols, heterobifunctional PEOs bearing a pyridyl disul-fide group can serve as linking agents [116]. Another usefulproperty of the pyridyl disulfide moiety is that it releases

OPt

O

O

O

NH3

NH3O CH2 CH2 O n CH2 CH2 NHCH2CH2NHFA C

O

CH2 CH2 C

O

NH CH2 3

O CH2 CH2 O nCH2 CH2 NH2CH2CH2NHfmoc

O

O

O

O

C

O

CH2 CH2 C

O

NH CH2 3

t-Bu

t-BuOH

O CH2 CH2 O nCH2 CH2 NHCH2CH2NHfmocO

O

O

O

C

O

CH2 CH2 C

O

NH CH2 3

t-Bu

t-Bu

O CH2 CH2 O nCH2 CH2 NHCH2CH2NHFAO

O

O

O

C

O

CH2 CH2 C

O

NH CH2 3

t-Bu

t-Bu

+

ECI

1) H2O/Piperidine 2) FA-NHSIon exchange chromatography

1) TFA 2) NaOH3) cis-[Pt(NH3)2(D2O)2]

2+(NO3)2Ion exchange chromatography

Fig. 23. Synthesis of FAePEOePt.


thiopyridone upon reaction with free mercapto groups(Fig. 24). Thiopyridone can be quantified to determine thenumber of PEO oligomers bounded to a molecule or surface[116,117].

5.1. Direct synthesis of HSePEOeX

5.1.1. Synthesis of formylePEOeSH and formylePEOeSSepyridyl

A heterobifunctional PEO containing a formyl group anda hydroxyl group was synthesized by polymerizing EO withan initiator that contained an acetal, potassium 3,3-diethoxy-propoxide [68]. The initiator was formed by reactinga stoichiometric amount of potassium naphthalide with 3,3-di-ethoxypropanol in THF, and this was followed by polymeriza-tion. The polymers, acetalePEOeOH, had narrow molecularweight distributions, w1, and the molecular weights obtainedfrom 1H NMR and SEC were in good agreement with the

HS-R+

S SCH2X CH2 CH2 O n CH2 R NHS+

NS SCH2X CH2 CH2 O n CH2

Fig. 24. Release of thiopyridone from PEO bearing a pyridyl disulfide moiety

upon reaction with a free mercapto group.

molecular weights calculated from monomer to initiator ratios.The acetal terminus was deprotected to form the aldehyde byreaction with hydrochloric acid.

Modifications to the method described above were em-ployed to synthesize acetalePEOeSH and acetalePEOeSSepyridyl (Fig. 25) [58]. The polymerizations were terminatedwith methanesulfonyl chloride to produce an activated chainend. Capping with the methanesulfonyl moiety was w98%based on end group analysis by 1H NMR. Displacement ofthe methanesulfonyl ester was achieved by reaction withpotassium O-ethyldithiocarbonate. End group analysis via 1HNMR showed that conversion of the methanesulfonyl esterto the ethyldithiocarbonate was almost quantitative, 97%.Generation of the thiol was achieved by cleaving the dithiocar-bonate terminus with n-propylamine. Transformation of themercapto terminus to the pyridyl disulfide derivative wascarried out by reacting the thiol-functional polymer with2-pyridyl disulfide, and the conversion was 68% as determinedby 1H NMR end group analysis.

The heterobifunctional acetalePEOeSH was used to con-struct a colloidal sensor system based on the reversible aggre-gation of gold nanoparticles induced by bivalent ligands [11].PEO chains were tethered to the surfaces of the gold nanopar-ticles, allowing the acetal ends to protrude into solution. ThePEO brush layer on the surface greatly enhanced the stabilityof the nanoparticles in various media such as deionized water,phosphate buffers, serum-containing media, and organic sol-vents. In order to attach a molecular probe to the distal endof the PEO-coated gold nanoparticles, the acetal terminuswas converted to an aldehyde by immersion in an aqueous

O1) n

2) CH3SO2Cl

3) CS

O CH2 CH3K+S

CH CH2 CH2 OEtO

EtOK

+

CH CH2 CH2 OEtO

EtOCH2 CH2 O CH2 CH2 SHn

N-propylamine

2-PDS

CH CH2 CH2 OEtO

EtOCH2 CH2 O CH2 CH2 Sn

NS

S CS

O CH2 CH3CH CH2 CH2 OEtO

EtOCH2 CH2 O CH2 CH2n

Fig. 25. Synthesis of acetalePEOeSH and acetalePEOeSSepyridyl.


solution of hydrochloric acid at pH 2. p-Aminophenyl-b-D-lac-topyranoside and p-aminophenyl-b-D-mannopyranoside wereimmobilized on the surfaces of the gold nanoparticles viareductive amination of the aldehyde at the distal end of thePEO chains.

The capacity for these particles to function in a sensor wastested by reaction with a bivalent galactose-binding lectin,Ricinus communis agglutinim (RCA120). Additions of RCA120to the functionalized gold nanoparticles were monitored byUV-visible spectroscopy. The dispersed particles were red,and as the particles aggregated due to crosslinking withRCA120, the color gradually changed from red to purple.The aggregated gold nanoparticles could be redispersed byadding D-galactose and separated from the dispersion by

O CH2 CH2 O nCH2CH2CHEtO

EtOC

O CH2 CH2 O nCH2CH2CHEtO

EtOCH2 CH2 O K

A

B

O CH2 CH2 OCH2CH2CHEtO

EtOCHn

O CH2 CHCH2CH2CHEtO

EtO

Fig. 26. Synthesis of acetalePEOeSS

centrifugation. The particles could also be redispersed inbuffer solution, then re-aggregated by adding RCA120. Thisprocess was repeatable over several cycles of aggregationand redispersion. Calibration curves enabled detection ofRCA120 concentrations as low as 1 ppm.

Heterobifunctional PEOs having both mercapto and alde-hyde groups were synthesized for functionalizing gold elec-trode surfaces on biosensors or biocatalysts [118]. AnacetalePEOeOH precursor was synthesized as describedabove, then terminated with N-succinimidyl-3-(2-pyridyldi-thio)-propionate to produce acetalePEOeSSepyridyl(Fig. 26A). The hydroxyl terminus was also converted toa thiol by condensation with an excess of thioglycolic acidto prepare acetalePEOeSH (Fig. 26B). End group analysisvia 1H NMR confirmed the polymer structure and incorpora-tion of end groups. Gold electrode surfaces were coated withthe heterobifunctional PEOs, and this was followed by hydro-lysis of the acetal under acidic conditions to form an aldehyde.

Immobilization of cytochrome c (cyt. c), an important elec-tron transport protein, on a gold electrode surface was studiedusing aldehydeePEOeSH and aldehydeePEOeSSepyridyl.The aldehydeePEOeSH formed a monolayer on the goldelectrode. The redox response of cyt. c when covalentlybounded to the gold electrode by the aldehydeePEOeSHpolymer was demonstrated by cyclic voltammetry. This indi-cated that the polymer could be utilized to functionalize elec-trodes. Although no redox response was observed when thealdehydeePEOeSSepyridyl polymer was used to bind cyt.c to the electrode, it was observed that this polymer functionedas a good promoter for the electron transfer between cyt. c inphosphate buffer solution and a gold electrode. The lack ofredox response when using aldehydeePEOeSSepyridyl wasattributed to low surface concentrations of the PEO chainson the surfaces of the gold electrode.

H2 CH2 ON

S SCH2CH2CO

NS SCH2CH2C

O

ON

O

O

+ +

2 CH2 OH + CH2 SHCO

HO

2 O n CH2 CH2 O CH2 SHC

O

epyridyl and acetalePEOeSH.


5.1.2. Synthesis of benzaldehydeePEOeSSepyridylSynthesis of a heterobifunctional PEO with a benzaldehyde

at one chain end and a pyridyl disulfide at the other was car-ried out by polymerizing EO using potassium 4-(diethoxyme-thyl)benzylalkoxide (PDA) as an initiator (Fig. 27) [119]. Thebenzaldehyde moiety was utilized to avoid self-condensationof PEO chains due to an aldol reaction with aldehyde-func-tional PEO oligomers wherein the aldehyde had an a-hydro-gen. Another advantage of benzaldehyde is that its reactionwith an amine produces a sufficiently stable imine that doesnot require further reduction. The initiator was preparedby reducing 4-(diethoxymethyl)benzaldehyde with sodiumborohydride, and this was followed by metalation of 4-(dieth-oxymethyl)benzyl alcohol with potassium naphthalide. Potas-sium 4-(diethoxymethyl)benzylalkoxide was then used as aninitiator for ring-opening polymerization of EO. The reactionwas terminated with methanesulfonyl chloride to produce anactivated chain end. The molecular weight distribution wasw1.03 and the molecular weight closely matched the expectedvalue.

Introduction of pyridyl disulfide onto the chain end beganwith quantitative displacement of the methanesulfonyl esterby reaction with O-ethyldithiocarbonate. Transformation ofthe ethyldithiocarbonate to the pyridyl disulfide was carriedout by deprotecting the mercapto group with n-propylaminein the presence of 2-pyridyl disulfide (2-PDS). The signalsin the 1H NMR spectrum corresponding to the O-ethyldithio-carbonate moiety were no longer observed after the reactionand new signals assigned to the pyridyl disulfide were present.The ratio of integrals corresponding to the pyridyl disulfideand benzylacetal end groups in the 1H NMR spectrum showedthat the conversion of O-ethyldithiocarbonate to the pyridyldisulfide was 99%. Finally, treatment with an aqueous solutionof hydrochloric acid to deprotect the benzylacetal moietyafforded the benzaldehydeePEOeSSepyridyl product.

CH2 OCHOO

CH2CH2

CH2CH2 K+

O1) n

2) CH3SO2Cl

3) S CS

O CH2CH3K+

CS

O CH2CH3CH2 CH2 O n CH2CH2 OCHOO

CH2CH2

CH2CH2 CH2 S

1) N-propylamine

2) 2-PDS

3) HCl

NSCH2 CH2 O n CH2CH2 OCH CH2 S

O

Fig. 27. Synthesis of PEO with both a benzaldehyde and pyridyl disulfide

moieties.

5.2. Synthesis of HSePEOeX via end groupmodification of homobifunctional PEO

5.2.1. Synthesis of HSePEOeOH from an allylePEOeOHintermediate

Protein-resistant SAMS were prepared on gold substratesby chemisorption of low molecular weight PEO oligomers(DP w 10) having a thiol on one end and a hydroxyl groupat the other [120]. The heterobifunctional polymer was pre-pared in three steps. First, reaction of 11-haloundec-1-eneand sodium hydroxide with an excess of PEO diol produceda mixture of mono-, di-, and un-substituted PEO oligomers.The mixture was separated by chromatography on silica gelto obtain the allylePEOeOH. Free radical addition of thioace-tic acid to the allyl terminus was carried out under UV irradi-ation with AIBN to yield PEO with a thioacetate on one chainend and a hydroxyl group at the other. The final step in prepar-ing HSePEOeOH was methanolysis of the thioacetate in thepresence of HCl to generate the thiol.

Ellipsometry was utilized to monitor adsorption of proteinson three different surfaces by measuring increases in thicknessof the SAMs before and after exposure to separate solutionscontaining avidin, hexokinase, or pyruvate kinase. SAMsformed from HSePEOeOH showed no significant increasein thickness while SAMs produced using HSe(CH2)11eCH3showed significant increase. Appearance of a nitrogen signalin the X-ray photoelectron spectra (XPS) confirmed that theincreases in thickness were due to protein adsorption. Theseresults indicated that the SAMs formed from HSePEOeOHresisted non-specific protein adsorption.

5.2.2. Synthesis of pyridyleSSePEOeNHS froma PEO diamine

A PEO diamine (H2NePEOeNH2) was utilized for syn-thesizing PEO oligomers with pyridyl disulfide and NHS func-tional groups (Fig. 28) [116]. End group asymmetry wasintroduced by reacting N-succinimidyl-3-(2-pyridyldithio)pro-pionate (SPDP) with an excess of the PEO diamine. The mono-substitued PEO (pyridyleSSePEOeNH2) was separated fromthe statistical mixture of the mono-, di-, and un-substitutedPEO oligomers by chromatography on silica gel. The terminalamine was then reacted with glutaric anhydride in the presenceof pyridine to introduce a carboxylic acid onto the other chainend. Finally, pyridyleSSePEOeNHS was produced by cou-pling the carboxyl group with NHS using DCC to form theactive ester. The signals in the 1H NMR corresponding tothe pyridyl disulfide and NHS terminals were present in theexpected ratio of 4:4.

Haselgrubler et al. demonstrated that these oligomers couldbe utilized to form liposomes with antibodies bounded to theirsurfaces [116]. Reactivities of the pyridyleSSePEOeNHSoligomers were confirmed by coupling the NHS active esterwith bovine IgG, and this was followed by modifying thepyridyl disulfide end through reaction with a polyclonal sheepantibody (anti-HSA) bearing free thiol functionality. The reac-tivities of the heterobifunctional polymer were similar to theSPDP analogues (Fig. 28).

C

O

N

O

O

CH2 C

O

O3N

SS CH2 CH2 C

O


NHCH2CHCH3

NH


NH2CH2CHCH3

H2N

NSS CH2 CH2 C

O


NH2CH2CHCH3

NH

C

O

CH2 C

O

OH3N

SS CH2 CH2 C

O


NHCH2CHCH3

NH

1) SPDP2) Chromatography on silica gel

O

O

O

NHS/DCC

Fig. 28. Synthesis of pyridyleSSePEOeNHS.


PyridyleSSePEOeNHS oligomers were also used to pre-pare liposomes with bovine IgG bounded to their surfaces. Anamino-functional lipid, 1,2-bis(myristoylphosphatidyl)ethanol-amine (DMPE), was coupled to the NHS terminus of pyridyleSSePEOeNHS. Thus, liposomes bearing pyridyl disulfidefunctionality were prepared from a blend of egg yolk phospha-tidylcholine and the DMPEePEOeSSepyridyl polymer. Thepyridyl disulfide groups on the surfaces of the liposomes werereacted with a mercapto-functional bovine IgG. Fluorescentlytagged anti-HSA containing a free thiol was also coupled tothe surface of liposomes in a similar manner using the lipids1-palmitoyl-2-oleoylphosphatidylcholine and asolectin. Theseresults demonstrated the utility of heterobifunctional polymersfor attaching targeting moieties to liposomes.

5.2.3. Synthesis of pyridyleSSePEOebiotin fromPEO diamines

To investigate binding of biotinePEO conjugates to avidin,Kaiser et al. synthesized a heterobifunctional PEO with biotinat one end and a pyridyl disulfide group at the other (Fig. 29)[117]. The pyridyl disulfide could be utilized to attach biomol-ecules to the biotinePEOeSSepyridyl, but in this study thepyridyl disulfide was used as a chromophoric marker. Whenit is reacted with a free thiol, the pyridyl disulfide releasesthe chromophoric marker, 4-thiopyridone, thus enabling quan-tification of biotinePEO conjugates.

A stoichiometric amount of a PEO diamine was reactedwith di-tert-butyldicarbonate (boc2O) to produce a statisticalmixture of products, then the mixture was separated bycolumn chromatography on either silica or Sephadex C-25(depending on the length of the PEO) to obtain boc-NHePEOeNH2. The free amine terminus was reacted with a bio-tineNHS active ester to form an amide linkage. The boc pro-tecting group was removed with formic acid to regenerate anamine terminus, and the polymer was purified by ion exchangechromatography.

The amine terminus was converted to a pyridyl disulfide byfirst reacting the oligomer with 3,30-dithio(succinimidylpropi-onate). The product was reduced with an excess of 1,4-dithio-threitol to yield biotinePEOeSH. The final product, biotinePEOeSSepyridyl, was obtained by reacting the thiol terminuswith 4,40-dithiodipyridine, and the oligomer was purified byion exchange chromatography.

The potential for avidin binding to biotinePEO was testedby utilizing biotinePEOeSSepyridyl to investigate the stoi-chiometry and metastability of bounded avidin. The avidinebiotinePEO conjugates had dissociation kinetics and half-livessimilar to those previously reported for spacers with 7e27atoms [121e124]. These results confirmed that biotinePEOis a good ligand for avidin.

5.2.4. Polymer supported synthesis of pyridyleSSePEOeOH from PEO diols

A solid-phase method for synthesizing heterobifunctionalPEO derivatives was developed by Bettinger et al. to avoidthe lengthy and often complicated separations that are usuallyemployed when starting from homobifunctional PEOs(Fig. 30) [125]. A PEO diol was activated at one chain endbefore being bounded to the polymer support. Diglycolic an-hydride was reacted with a large excess of PEO diol to obtainmono- and un-substituted products. Without further purifica-tion, the carboxylic acid was transformed to the active esterby reacting with NHS and DCC. After removing the ureaby-product, the mixture of active and inactive PEOs wasgrafted onto an aminomethylated poly(styrene-co-divinylben-zene) solid-phase resin. The resin was washed with dichloro-methane and methanol to remove PEO diol and leave onlyPEO that was reacted onto the resin through amide linkages.The washing procedure was repeated after each syntheticstep to ensure the purity of the resin-bounded PEO.

The hydroxyl terminus of the resin-bounded PEO (resinePEOeOH) was activated by reacting with an excess of

1)

Ion exchange chromatography

2)

Ion exchange chromatography

S NS

NHHN

O

4CH2 CO


NHCH2CHCH3

NH SCH2CH2C

O


NH2CH2CHCH3

H2N

1) Boc2OIon exchange chromatography


NHCH2CHCH3

H2N boc

1) Biotin-NHS 2) Formic acid Ion exchange chromatography

S

NHNH

O

4CH2 CO


NH2CH2CHCH3

NH

N

O

O

N

O

O

CH2 C

O

OCH2SSCH2CH2C

O

O

+

S

NHHN

O

4CH2 CO


NHCH2CHCH3

NH N

O

O

CH2 C

O

OCH2SSCH2CH2C

O

N SS N

HS CH2 CH CH CH2 SHOH

OH

Fig. 29. Synthesis of biotinePEOeSSepyridyl.


toluene-4-sulfonyl chloride. The resin was washed to removeexcess reagents and the presence of the tosylate was confirmedby IR spectroscopy and by gel-phase 13C NMR. The activatedresinePEOeOTs was stable for up to four months at �20 �C.A wide variety of heterobifunctional PEOs could be synthe-sized from this starting material.

A thiol was introduced by reacting a stoichiometricamount of potassium O-ethyldithiocarbonate with the res-inePEOeOTs. An excess of potassium O-ethyldithiocarbon-ate lead to complete cleavage of the PEO from the resin. Thedisappearance of signals corresponding to the tosylate endgroup and appearance of two new resonances correspondingto the thiocarbonate end group in the 13C NMR spectrashowed complete conversion of the end groups. The thio-carbonate was aminolyzed with propylamine under anhydrousconditions to avoid cleavage of the functionalized PEOfrom the resin. Complete conversion of the end group withoutoxidation to form the disulfide was confirmed by 13C NMR.The thiol was protected by reacting with 2,20-dithiodipyridineto yield resinePEOeSSepyridyl. The final step was cleavageof the functionalized PEO from the resin with a mildbasic polar solvent (tetrahydrofuran/methanol/triethylamine).The pyridyleSSePEOeOH was obtained in a global yieldof 65%.

5.2.5. Synthesis of XePEOeSAc from PEO diolsPEO diols were utilized to prepare several heterobifunc-

tional conjugates with a protected mercapto group (thioace-tate) on one end and either a hydroxyl, aldehyde, amine, orazide at the other [70]. The first step in these syntheses wasactivation of one chain end of a 1500-Mn PEO diol with a equi-molar amount of toluene-4-sulfonyl chloride. Tosylation ofboth chain ends was minimized by tosylation in the presenceof silver oxide with a catalytic amount of potassium iodide[126]. The protected mercapto group was introduced by nucleo-philic displacement of the tosylate with potassium thioacetate toyield HOePEOeSAc. This polymer served as an intermediatefor synthesizing several heterobifunctional PEO oligomertypes via derivatizations of the hydroxyl end.

Introduction of an azide moiety was achieved by activatingthe hydroxyl end with methanesulfonyl chloride followed byreaction with sodium azide. The hydroxyl terminus wasconverted to an aldehyde in two steps. Sodium hydride wasused to form the alkoxide chain end, and this was reactedwith 3-bromo-1,1-dimethoxypropane. The acetal terminuswas then converted to the aldehyde using Amberlyst-15(acidic) resin. Benzylamine was reacted with the aldehyde byreductive amination while leaving the thioacetate group intact.This demonstrated that aldehydeePEOeSAc could be

O O

O

O

n H

CH2O n HC

OCH2OCH2C

O

NH

NSSHO CH2 CH2O n CH2 CH2

CH2O n CH2C

OCH2OCH2C

O

NH CH2 O CH3SO

O

S CS

O CH2 CH3

O CH2

O CH2

O CH2 CH2O n CH2C

OCH2OCH2C

O

NH CH2

NSSCH2O n CH2C

O

CH2OCH2C

O

NH CH2

SH

O CH2

O CH2 CH2O n CH2C

OCH2OCH2C

O

NH CH2

+HO

O CH2 CH2O n HC

OCH2OCH2C

O

HONH +

NHS/DCC

CH3SO

OCl

S CS

O CH2 CH3K+

Propylamine

TFH/MeOH/TEA

2,2’-dithiodipyridine

Pyridine

CH2 OCH2

Fig. 30. Polymer supported synthesis of pyridyleSSePEOeOH.


selectively linked to a molecule through the aldehyde function-ality, and then the mercapto group could be deprotected forlater use.

A similar series of reactions was used to convert thehydroxyl terminus to an amine. The hydroxyl terminus wasdeprotonated with sodium hydride and reacted with fmoc-protected 3-aminopropyl bromide, and this was followed bydeprotecting the amine with pyridine. The reactivity of theamine was demonstrated by adding benzaldehyde via reduc-tive amination. The amine was also used to form a peptidebond by reacting with the active NHS ester of boc-protectedlysine.

Several types of reactions were carried out to demonstratethe utility of these heterobifunctional PEOs in the field of bio-compatible nanoparticles and bioconjugation. A three-stageproc

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