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Synthesis and Evaluation of Clickable Block Copolymers for TargetedNanoparticle Drug DeliverySiyan Zhang, Kiat Hwa Chan, Robert K. Prud'homme, and A. James Link*,,

Departments ofChemical and Biological Engineering and Molecular Biology, Princeton University, Princeton, New Jersey 08540,United StatesInstitute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, 138669, Singapore

*S Supporting Information

ABSTRACT: Polymeric nanoparticles with multifunctional capabilities,including surface functionalization, hold great promise to addresschallenges in targeted drug delivery. Here, we describe a concise, robustsynthesis of a heterofunctional polyethylene glycol (PEG), HO-PEG-azide. This macromer was used to synthesize polylactide (PLA)-PEG-

azide, a functional diblock copolymer. Rapid precipitation of thiscopolymer with a hydrophobic cargo resulted in the generation ofmonodisperse nanoparticles with azides in the surface corona. Todemonstrate conjugation to these nanoparticles, a regioselectivelymodified alkyne-folate was employed as a model small molecule ligand,and the artificial protein A1 with an alkyne moiety introduced byunnatural amino acid substitution was selected as a model macromolecular ligand. Using the copper-catalyzed azidealkyneligation reaction, both ligands exhibited good conjugation efficiency even when low concentrations of ligands were used.

KEYWORDS: nanoparticles, click chemistry, bioorthogonal reactions, bioconjugation

INTRODUCTION

In an effort to drive innovation in medical therapy employing

nanotechnology, the NCI developed the Cancer Nano-technology Plan (NIH Publication Number 04-5489, 2004).Among the opportunities identified in the plan are multifunc-tional therapeutics, which combine therapy and diagnostics;enable targeting, control delivery, and release; and monitoreffectiveness. A variety of nanocarriers have been introducedincluding liposomes, microgels, and inorganic and polymericcarriers.1,2 A universal requirement of nanocarriers is aprotective, biocompatible coating to prevent prematureclearance by the reticulo-endothelial system (RES).3 Mostoften, these protective layers are polyethylene glycol (PEG)polymer chains.4 These coatings improve the circulation time ofnanocarriers and also present a handle for the attachment oftargeting ligands to the surface corona of nanoparticles. The

addition of surface functionalization opens up the possibility ofcreating cell-specific or even disease-specific nanocarriers.59

Our focus has been on polymeric nanocarriers withmultifunctional capabilities that can be efficiently made withrapid precipitation and block copolymer-directed kineticassembly, a process termed Flash NanoPrecipitation (FNP)10

(Figure 1). The advantage in using block copolymers is that theprotective PEG layer is an integral part of the nanocarrier,simplifying the overall nanocarrier design. The surfacefunctionalization of polymer nanoparticles continues to be achallenge in large part due to the inefficiency of conventional

bioconjugation strategies such as aminecarboxylic acidcoupling and thiolmaleimide reactions.5 When using these

bioconjugation methods, a large excess of the targeting ligand isoften used to ensure suitable levels of functionalization.11 This

becomes problematic if the targeting ligand is a costly reagent,such as an antibody or other protein. Newer bioconjugationstrategies such as the azidealkyne click reaction1214 promiseto enable more efficient labeling of the surface corona ofpolymeric nanoparticles. Azidealkyne click chemistry is bio-orthogonal,12 can be carried out in an aqueous environment atphysiological temperatures, and also has high efficiency.Ultimately, both small molecules and macromolecules willhave utility as targeting ligands, and azide and alkyne functionalgroups can be readily added to both types of ligands.

Here, we have developed a robust, high-yielding synthesis ofan asymmetric HO-PEG-azide polymer that is expected to be

broadly useful for the synthesis of many different azide-functionalized nanocarriers. Using this azide-PEG, we gen-erated a polylactide (PLA) copolymer, PLA-PEG-azide, and

demonstrated that this copolymer formed monodispersenanoparticles using FNP (Figure 1). We evaluated theefficiency of azidealkyne bioconjugation to these particles

with both an alkyne-labeled small molecule ligand, alkyne-folate, and a model alkyne-labeled protein ligand and found that

both ligands were efficiently conjugated to the nanoparticleseven with low ligand loadings.

Received: February 8, 2012Revised: June 10, 2012Accepted: June 26, 2012Published: June 26, 2012

Article

pubs.acs.org/molecularpharmaceutics

2012 American Chemical Society 2228 dx.doi.org/10.1021/mp3000748| Mol. Pharmaceutics 2012, 9, 22282236
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MATERIALS AND METHODS

Synthesis of Heterofunctional HO-PEG-Azide. Syn-thesis of Allyl-PEG-OH. The synthesis of -allyl--hydroxylPEG was modified from a procedure previously reported byHiki and Kataoka.15 To generate a radical anion solution ofpotassium naphthalene, naphthalene (2.37 g, 16 mM) wasdissolved in 100 mL of dry THF under argon.16 Potassium

metal (0.63 g, 18 mM) was quickly added into the solution, andwithin 5 min of stirring, the mixture turned dark green. After 2h of reaction, the concentration of the naphthalenide radicalanion was determined to be 0.145 M by titration with 0.25 MHCl. Allyl alcohol (2.3 mmol, 135 L) and potassiumnaphthalenide (2 mmol, 13.8 mL) were added into 60 mL ofdry THF. Ethylene oxide gas (13.3 g, 302 mmol) was slowlyintroduced into the reaction flask and allowed to stir overnightat room temperature. THF was then removed via rotavap, andthe residue was dissolved in CH2Cl2 (20 mL) and addeddropwise to diethyl ether (600 mL) to yield a white precipitate.The polymer was recovered by filtration, washed with moreether (3 50 mL), and dried in vacuo to yield allyl-PEG-OH asa white solid (12.4 g, 93% yield, MW 5135 g/mol fromNMR).

1H NMR (500 MHz, CDCl3, in ppm): 3.63 [m, O(CH2)2O, PEG backbone], 4.00 (d, OCH2CHCH2), 5.14a nd 5 .2 6 ( d, OCH2 CHCH2 ) , 5 .8 45 . 93 ( m,OCH2CHCH2).

13C NMR (500 MHz, CDCl3, inppm): 62.5 (CH2CH2OH), 71.0 [O(CH2)2O, PEG

backbone], 72.5 (CH2CHCH2), 117.4 (CH2CHCH2), 135.0 (CH2CHCH2).

Synthesis of Allyl-PEG-Cl. A mixture of allyl-PEG-OH (5.0 g,0.97 mmol) and SOCl2 (2.32 g, 19.5 mmol) in benzene (50mL) was refluxed for 6 h. The reaction mixture was thenevaporated in vacuo, followed by coevaporation with more

benzene (3 50 mL) to remove residual SOCl2. The residue

was dissolved in CH2Cl2 (20 mL) and added dropwise todiethyl ether (500 mL) with vigorous stirring. The whiteprecipitate was filtered, washed with ether (3 50 mL), anddried in vacuo to furnish allyl-PEG-Cl (4.8 g, 0.93 mmol; 96%

yield) as a white solid.1H NMR (500 MHz, CDCl3, in ppm): 3.63 [m, O

(CH2)2O], 3.74 (t, CH2CH2Cl), 4.00 (d, OCH2CHCH2), 5.145.26 (dd, OCH2CHCH2), 5.845.93 (m,OCH2CHCH2). The peak for CH2CH2Cl is notobserved, and it is likely coincident with the large peak at 3.63. 13C NMR (500 MHz, CDCl3, in ppm): 43.0 (CH2Cl),71.0 [OCH2)2O, PEG backbone], 117.4 (CH2CHCH2).

Synthesis of HO-PEG-Cl. To allyl-PEG-Cl (4.8 g, 0.93mmol) in anhydrous THF (50 mL) at 20 C a solution of 9-BBN in THF (3.74 mL, 1.87 mmol; 0.5 M) was injected. Thismixture was allowed to stir for 24 h, after which it was reacted

with 12 mL of a 1:2 mixture of aqueous 5 M NaOH andaqueous 30% H2O2 and allowed to stir for a further 48 h at 20C. The reaction was then quenched with K2CO3 (1 g), diluted

with CH2Cl2 (800 mL), and dried with excess Na2SO4/MgSO4.The solids were filtered, and the filtrate was concentrated to 20mL, which was then added dropwise to ether (600 mL) with

vigorous stirring. The white precipitate was filtered, washedwith ether (3 50 mL), and dried in vacuo to furnish HO-PEG-Cl (4.2 g, 0.82 mmol; 88% yield) as a white solid. Asexpected, the bulky hydroboration reagent 9-BBN led toregioselectivity toward the terminal carbon, although traceamounts (

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(40 mg, 0.098 mM) in 2 mL of toluene and D,L-lactide (0.49 g,3.4 mM) was added into the PEG solution, and thepolymerization reaction was carried out at 70 C under argonovernight. After toluene was removed, the residue was dissolvedin 5 mL of CH2Cl2 and precipitated dropwise into 100 mL ofdiethyl ether. The white precipitate was recovered byfiltration,

washed 3 with 10 mL of ether, and dried in vacuo to yieldPLA-b-PEG-aizde copolymer (0.9 g, 0.09 mmol, 91% yield,PEG MW 5000, PLA MW 5000, PDI = 1.23).

1H NMR (500 MHz, CDCl3, in ppm): 1.55 (d, CH3 onthe PLA backbone), 1.90 (quintet, CH2CH2CH2OPLA),3.39 (t, CH2CH2N3), 4.22 (t, CH2CH2OPLA), 4.35(quartet, CHOH at the end of the PLA chain), 5.15 (quartet,CH on the PLA backbone).

Synthesis of PLA-OH. As in the synthesis of the diblockcopolymer, lactide was recrystallized from toluene and dried,

while Sn(Oct2) was distilled before use. D,L-Lactide (6.3 g, 44mmol) was slowly dissolved in 30 mL of toluene at 90 C. Theinitiator 1-octanol (0.141 g, 1.1 mmol) and Sn(Oct2) (0.441 g,1.1 mmol) in 8 mL of toluene was added to the lactide solution,and the temperature was lowered to 70 C for polymerization.The target molecular weight (MW) was 5800 g/mol, and theextent of polymerization was monitored via NMR. After 25 h ofreaction, the residue was dissolved in 10 mL of CH2Cl2 andprecipitated in 200 mL of 1:9 water/methanol. A white gooeyprecipitate was formed near the bottom of the flask, and thesupernatant was easily decanted. After two more washes with20 mL of 1:9 water/methanol, the precipitate was dried in

vacuo to yield PLA-OH as a white solid (3.5 g, MW 8600 g/mol, 0.41 mM, 55.6% yield). The final product showed a higheraverage MW than the target MW because some of the shorterpolymer chains likely were not precipitated and lost in the stepsof precipitation and washing.

1H NMR (500 MHz, CDCl3, in ppm):1.47 (d, CH3 onthe PLA backbone), 4.03 (t, OCH2(CH2)6CH3), 4.27(quartet, CHOH at the end of the PLA chain), 5.06 (quartet,CH on the PLA backbone). The average MW was estimatedusing integrations at 4.03, 4.27, and 5.05 ppm. GPC shows aPDI of 1.29.

Regioselective Synthesis of ()-Alkyne-Folate. Syn-thesis of tert-Butyl (2S)-2-[(tert-Butoxycarbonyl)amino]-5-oxo-5-(prop-2-yn-1-ylamino) Pentanoate [Boc-Glu(alkyne)-OtBu]. To a solution of Boc-Glu-OtBu (1.00 g, 3.30 mmol) inCH2Cl2 (5 mL) was added ethyl dimethylaminopropylcarbodiimide (EDC) (0.758 g, 3.95 mmol), followed bypropargylamine (0.200 g, 3.63 mmol). The mixture was stirredat 20 C for 2 h, followed by extraction with H2O (3 5 mL)and drying with MgSO4. After the drying agent was filtered, thefiltrate was concentrated and chromatographed through silicagel with hexane/CH2Cl2 to furnish Boc-Glu(alkyne)-O

tBu

(0.810 g, 2.38 mmol; 72.2% yield) as a colorless oil.1H NMR (500 MHz, CDCl3, in ppm): 1.48 (d, CH3,18H), 2.21 (s, CCH), 2.27 and 2.32 (t, CH2CH2), 4.09(t, CH2CCH), 4.19 (sextet, CH), 6.56 (s, NH, 2H). Themass of Boc-Glu(alkyne)-OtBu was also confirmed by ESI-MS,

with observed masses of 341.2 (+H) and 363.2 (+Na).Synthesis of (TMG)2 ()-Alkyne-Folate. A solution of Boc-

Glu(alkyne)-OtBu (0.289 g, 0.849 mmol) in TFA (3 mL) wasstirred at 20 C for 12 h, after which the solvent was removedin vacuo. The deprotected ()-alkyne-glutamic acid wasdissolved in DMSO (2.6 mL), along with pteroyl azide(0.260 g, 0.772 mmol) and tetramethylguanidine (0.385 mL,3.09 mmol), and stirred at 20 C for 48 h. The synthesis of

pteroyl azide from folic acid was described in detail by Luo etal.17After 2 days of reaction, the mixture was filtered through a

wad of Celite, and the filtrate was added dropwise into acetone(30 mL) with vigorous stirring. The yellow precipitate wasfiltered, washed with ether (3 10 mL), and dried in vacuo tofurnish (TMG)2 ()-alkyne-folate (0.421 g, 0.594 mmol; 77.0%

yield) as an orange solid. See Figure 2 below for the structure

and numbering for NMR assignments.

1H NMR (DMSO-d6, ref. 2.54 ppm, in ppm): 8.54 (s, 1H,H-5), 8.42 (t, 3J= 5.5 Hz, 1H, H-22), 7.58 (d, 3J= 8.7 Hz, 2H,

H-11/13), 6.94 (t,

3

J = 5.4 Hz, 1H, H-8), 6.65 (t,

3

J = 8.7 Hz,H-10/14), 4.44 (d, 3J= 4.3 Hz, 2H, H-7), 4.02 (q, 3J= 5.6 Hz,1H, H-17), 3.79 (dd, 3J= 5.3 Hz, 4J= 2.4 Hz, 2H, H-23), 3.04(t, 4J= 2.4 Hz, 1H, H-26), 2.84 (s, 24H, H-28), 2.201.99 (m,2H, H-20), 2.031.65 (m, 2H, H-19). 13C NMR (DMSO-d6,ref. 40.5 ppm, in ppm): 175.2 (C-18), 173.4 (C-21), 166.9(C-15), 166.2, 162.2 (C-27), 159.7, 157.9, 151.7 (C-9), 148.7(C-5), 147.8 (C-6), 129.4 (C-11/13), 129.2, 123.1 (C-12),112.4 (C-10/14), 82.5 (C-25), 73.8 (C-24), 54.9 (C-17), 47.1(C-7), 41.4 (C-28), 33.0 (C-20), 29.9 (C-19), 28.8 (C-23). Themass of the compound was confirmed by ESI-MS withobserved masses of 479.0 (+H) and 501.2 (+Na).

Ethynyl-Phenylalanine Synthesis. 4-Ethynyl-L-phenyl-alanine hydrochloride was synthesized using (S)-4-iodopheny-lalanine (5 g, 17 mmol) as a starting material (purchased fromChem-Impex International Inc.). The detailed procedure wasdescribed by Lei et al.18 and Kayser et al.19 Briefly, the acidmoiety of 4-iodo-L-phenylalanine was protected by a methylester and isolated as the hydrochloride salt (4.75 g, 14 mmol,81% yield) by reacting with SOCl2 and methanol. A Boc group

was added to protect the amine using di-tert-butyl dicarbonate,which resulted in N-t-Boc-4-iodo-L-phenylalanine methyl ester.This compound (3 g, 7.4 mmol) was then reacted withPdCl2(PPh3)2, CuI, ethynyltrimethylsilane, and triethylamine toinstall the alkyne group. The trimethylsilyl and methyl esterprotecting groups were removed by LiOH/H2O. Lastly, theBoc group was removed by reacting with a solution of HCl/ethyl acetate to yield 4-ethynyl-L-phenylalanine hydrochloride(0.94 g, 4.2 mmol, 56% overall yield from doubly protected

iodo-Phe starting material). An overview of the synthesis isprovided in Figure S1 in the Supporting Information.

1H NMR (500 MHz, CD3OD, in ppm): 3.20 (dd,diastereotopic H), 3.35 (dd, diastereotopic H), 3.56 (s,CCH), 4.29 (dd, H on the carbon of the amino acid),7.34 (d, 2 aromatic H's further away from the triple bond), 7.51(d, 2 aromatic H's closer to the triple bond).

Characterization. 1H and 13C NMR spectra were recordedon 500 MHz Bruker AVANCE. Mass spectrometry data wereobtained using Agilent 6220 Accurate-Mass Time-of-Flight LC/MS. The polydispersity index of the diblock copolymer andPLA homopolymer was determined via gel permeationchromatography (GPC). Two 30 cm Polymer Laboratories

Figure 2. Structure of (TMG)2 ()-alkyne-folate.

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PLgel Mixed-C columns and a Waters 410 refractive indexdetector were used with THF as the mobile phase.

Recombinant A1 Expression and Purification. Theartificial protein A1 was first described, expressed, and purified

by Petka et al.20 A1 is an -helical protein that dimerizes toform a leucine zipper. The plasmid encoding A1 and an N-terminal His-tag in a pQE9 vector was a kind gift from theTirrell group at Caltech. Because the native A1 protein doesnot include any phenylalanine (F) residues, a mutation of amethionine (M) residue to F was introduced near the Nterminus of the protein (Figure S2 in the SupportingInformation), creating the plasmid pQE9-A1(M1F). Prof. JinMontclare at NYU Poly kindly provided us with a pQE30plasmid encoding a phenylalanyl-tRNA synthetase mutant(PheRS*) that is capable of incorporating ethynyl-Phe inresponse to Phe codons in Escherichia coli.21 The gene encodingPheRS* was cut out and moved into pQE80 using therestriction enzyme NheI. The gene for the mutant A1 piece wasmoved from pQE9 into pQE80-PheRS* using EcoRI and BlpI.The resulting pQE-80-A1(M1F)-PheRS* plasmid was trans-formed into the Phe auxotrophic strain AFIQ cells,22 whichcontain a pLysIq plasmid to ensure tight control of recombinantprotein expression.

To express A1 incorporating ethynyl-Phe (alkyne-A1), AFIQcells were grown to midlog phase (OD600 0.8) in M9 media(25 mL) with 20 amino acids and then shifted to M9 media

with only 19 amino acids (lacking Phe). Ethynyl-Phe was addedin the culture to a final concentration of 250 mg/L, and proteinexpression was induced with 1 mM isopropyl -D-1-thiogalactopyranoside (IPTG) at 37 C for 4 h. Cells werelysed, and the protein was purified under denaturing conditionsin 8 M urea on a Ni-NTA column (Qiagen) according to themanufacturer's recommendations. A sodium dodecyl sulfatepolyacrylamide gel electrophoresis gel was used to assess thepurity of the fractions. All elution fractions containing thealkyne-A1 protein were combined and dialyzed against

ultrapure water with four changes of the dialysis water in aslidealyzer (Pierce) with a 3500 Da MW cutoff to remove urea.The protein solution was then lyophilized and redissolved in250 L of sterile PBS (137 mM NaCl, 2.68 mM KCl, 4.29 mMNa2HPO4, and 1.47 mM KH2PO4, pH 7.4). The finalconcentration of alkyne-A1 was determined by BCA assay to

be 180 g/mL in PBS, resulting in a protein yield of around 2mg per liter of culture.

Wild-type A1 containing all natural amino acids wasexpressed in DH10B cells and purified as described above.DH10B cells were grown to midlog phase in 10 mL of LB andinduced with IPTG for 3 h at 37 C. After purification, dialysis,and lyophilization, the dried A1 protein was dissolved in 400L of PBS, and the concentration was determined to be 980

g/mL. The protein yield for native A1 is 39 mg/L.Nanoparticles Formation via FNP. Azide-decorated

nanoparticles with PLA-OH homopolymer encapsulated inthe core as a model cargo were generated using FNP. In atypical experiment, a water-miscible THF stream containing 10mg/mL PLA-PEO-azide and 10 mg/mL PLA-OH (Mw 8.6k)homopolymer at 12 mL/min was rapidly mixed with three

water streams each at 40 mL/min using a multi-inlet vortexmixer23 to form PLA-PEO-azide nanoparticles. THF wasremoved through membrane dialysis with 60008000 Mwcutoff following assembly. The size of the nanoparticles wasmeasured using dynamic light scattering (DLS). The averagediameter was around 75 nm (see Figure 3).

Nanoparticle Conjugation to Small Molecule orMacromolecule through Click Chemistry. Clicking Folateto Nanoparticles. Folate conjugation was carried out in 3.4 mLof PBS solution (pH 7). Nanoparticles (2 mg/mL total mass)

with 90 M azide functionality were mixed with 90 M alkyne-folate, 100 M CuSO4, 2000 M sodium ascorbate, and 200M water-soluble ligand bathophenanthrolinedisulfonic acid(BPDA) in a 20 mL scintillation vial. The catalytic Cu(I)species for azidealkyne cycloaddition was generated in situthrough reduction of Cu(II) by sodium ascorbate. After it wasshaken overnight, the reaction mixture was first dialyzed (Slide-

A-Lyzer Mini, 7k mol wt cutoff) against 100 M diethylenetriamine pentaacetic acid (DTPA) and refreshed four times(one hour each) to chelate Cu ions and remove excess clickreagents. Afterward, the mixture was dialyzed against pure 1PBS (pH 7.4) to remove DTPA and refreshed four times(one hour each). The size of nanoparticles remained stableafter folate conjugation, as measured by DLS (Figure 3). Theamount of folate attached to the nanoparticles was assessedusing UVvis spectroscopy (Nanodrop).

Clicking Protein A1 to Nanoparticles. Protein conjugationto nanoparticles was carried out in 107 L of PBS.Nanoparticles (1 mg/mL PLA-PEO-azide in solution,providing a 45 M concentration of azides) were mixed withalkyne-A1 (10 M, A1 with only natural amino acids was usedfor a control reaction) in the presence of sodium ascorbate(2300 M), tris(hydroxypropyl)triazolylmethyl-amine(THPTA) (230 M), and CuSO4 (120 M) and allowed to

react at RT with occasional mixing by pipet. THPTA is a water-soluble ligand for click chemistry developed and generouslydonated by the Finn group at the Scripps Research Institute.24

After 24 h of reaction, the 100 L mixture was microdialyzedagainst pure water using a 0.05 m pore VMWP membrane(Millipore) for 30 min, followed by a 0.025 m pore VSWPmembrane (Millipore) three times for 30 min to remove clickreagents and any proteins not conjugated to the nanoparticle.To determine the amount of protein conjugated to theparticles, 1 L each of PBS, nanoparticles (NP), NP plus allclick reagents (without any protein), a dialyzed reactionmixture of NP and alkyne-A1, as well as a dialyzed mixtureof NP and normal A1, were tested using a spot assay. The

Figure 3. Size of nanoparticles measured by DLS before and afterconjugation. No size increase was observed upon conjugation of folicacid. A 32 nm increase in average diameter was observed afterconjugation to the protein A1.



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nanoparticle/protein samples were spotted on a nitrocellulosemembrane, which was subsequently blocked with low-fat milkin PBS/Tween, washed with PBS/Tween, and probed with ananti-His antibody (Sigma) to semiquantitatively estimate thelevel of protein attached to the nanoparticles. A serial dilutionof alkyne-A1 was also spotted (1 L per spot) to serve as acalibration curve for the estimation of the extent ofconjugation.

RESULTS

Heterofunctional PEG Synthesis. To assemble apolymeric nanoparticle with a click functional handle on thesurface corona, the synthesis of an amphipilic diblockcopolymer with a click group on the hydrophilic chain isnecessary (Figure 1). We chose to build a PEG-b-PLA polymerfor nanoparticle assembly due to the biocompatibility of both

blocks. Our strategy was to synthesize a heterofunctional HO-PEG-azide blockfirst and use it as a macroinitiator for lactidepolymerization.25 This PEG block has one hydroxyl end, whichinitiates ring-opening polymerization, and an azide end, which

will eventually be used for ligand conjugation by azidealkyne

click chemistry. Because commercially available PEGs are oftendiols, the synthesis of heterofunctional PEGs from thesestarting materials has been investigated. Heterofunctional PEGsynthesis from PEG-diol has been carried out throughdifferential substitution;26 however, this approach suffers fromlow yields and often requires additional column separation.27

Additionally, PEGs synthesized in this fashion are restricted inlength to molecular weights of 2000 g/mol or less.28 Thedownstream use of our materials is for drug delivery, and Grefet al. have shown that a PEG molecular weight of at least 5000g/mol is desired to provide efficient protection of thenanocarriers during systemic administration.29 We followedHiki and Kataoka15 to synthesize allyl-PEG-OH, with anestimated MW of 5100 g/mol by NMR (Scheme 1). The OH

end of allyl-PEG-OH was convertedfi

rst to allyl-PEG-Clfollowed by a conversion of the allyl group back to a hydroxylgroup via hydroboration generating HO-PEG-Cl. This useful

intermediate was readily converted to HO-PEG-azide via asimple SN2 displacement of the chloride with sodium azide.

Carbon NMR was used to monitor the change of terminalfunctional groups on the PEG chain during each synthetic step.Hydrogen atoms near the terminal groups often have similarshift on proton NMR as the PEG backbone, which make themdifficult to analyze. After chlorination with SOCl2, a peak at 62ppm representing the carbon next to the terminal OH group(CH2CH2OH) disappeared, and a new peak showed up at 42ppm, representing the carbon next to Cl (CH2CH2Cl). Theallyl group exhibits peaks at 117 and 132 ppm (CH2CHCH2), which disappear upon hydroboration along with theemergence of a peak at 62 ppm, representing a carbon bearing anew OH group (CH2CH2OH). Upon treatment with NaN3,the resonance at 42 ppm disappeared and was replaced by apeak at 51 ppm (CH2CH2N3).

PLA-PEG Block Copolymer Synthesis. Ring-openingpolymerization of lactides was carried out essentially asdescribed before30 except using HO-PEG-azide as a macro-initiator to make the block copolymer PLA-PEG-azide (Scheme2). A similar synthesis was carried out using 1-octanol as aninitiator to synthesize homopolymer PLA-OH, which was usedas a model hydrophobic cargo during nanoparticle assembly.

While the block copolymer was readily recovered due to theinsolubility of the PEG block in ether, precipitation of the lowmolecular weight PLA-OH homopolymer was more challeng-ing. After testing multiple solvent systems, PLA-OH homopol-

ymer was found to be most cleanly precipitated in 1:9 water/MeOH instead of pure methanol as used previously.30,31 Wehypothesize that the addition of water allowed for the removalof any unreacted monomer and short chains.

Nanoparticle Assembly. FNP is a robust way to assemblenanoparticles with desired sizes, high cargo loading, andadjustable coverage of surface functional moieties.10,11,3234

By mixing an equal mass concentration of diblock PLA-PEG-azide and homopolymer PLA-OH, we were able to generate

stable particles with an average diameter of 70 nm and narrowpolydispersity (Figure 3). For drug delivery purposes, it has

been reported that particles from 50 to 200 nm in size performthe best in terms of circulation in the bloodstream.2While smallparticles (200 nm) can

be easilyfiltered by the spleen. Thus, the size of our polymericnanoparticles is within the optimal range for drug deliveryapplication.

Small Molecule Conjugation to Polymeric Nano-particles. With azide-functionalized polymeric nanoparticlesin hand, we sought to study the conjugation of a model smallmolecule ligand to the nanoparticles. We chose to study folate,a vitamin that has been proposed as a targeting ligand to cancer

cells.35 As a vitamin, folic acid is essential for cell proliferationand maintenance. Normal folate uptake is through a low affinityreduced folate carrier36 or proton-coupled folate transporter.37

Because folate receptors are only overexpressed on cancer cellsand activated macrophages, folic acid has been widely used as aligand in targeted delivery.35 We chose folic acid as a modelexample of a small molecule ligand that can be conjugated toour polymeric nanoparticles through click chemistry.

Folic acid has two carboxylic acid moieties (Scheme 3).Previously, it has been demonstrated that folate conjugationcan only be done through its -COOH, not the -COOH, tomaintain the biological activity of folate and have the folatereceptor-mediated endocytosis proceed successfully.38,39 To

Scheme 1. Synthesis of Heterofunctional HO-PEG-Azide



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engineer an alkyne-folate, one possible route is to react folicacid with an amine-bearing alkyne reagent such as propargyl-amine and then separate the -modified compound from the -modified compound. This method requires extensive separation

by HPLC. Instead, we chose to develop a novel regiospecificsynthesis of an ()-alkyne-folate modified only at the -position.Luo et al. described an efficient synthesis of pteroyl azide, a

building block of the regiospecific folate.17 By reacting aprotected glutamic acid with propargylamine followed byremoval of the protecting groups, we obtained a building block

bringing in regiospecific alkyne functionality (Scheme 3).Reacting pteroyl azide with ()-alkyne-glutamic acid yielded()-alkyne-folate.

The click reaction between azide-functionalized nanoparticlesand alkyne-folate was carried out at room temperature inaqueous solution since the azidealkyne click chemistry canproceed readily under these conditions. A control reaction wassimultaneously carried out with unmodified natural folic acid.Unreacted folate and click chemistry reagents were removed bydialysis. The absorbance at 280 nm was used to assess theamount of folate conjugated to the nanoparticle based on acalibration curve. Under a 1:1 alkyne:azide reaction conditions,10% of the folate was successfully conjugated onto thenanoparticle (Figure 4). We also used DLS to measure thesize of the nanoparticles following conjugation (Figure 3), but

because the folate is small relative to the particle, no discernible

size increase was observed. Considering the mild conjugationconditions and the relatively high hydrophobicity of alkynefolate, a 10% extent of labeling was impressive. With a typicalnanoparticle carrying 500 polymer chains,11,34 each particle has

Scheme 2. Synthesis of PLA-b-PEG-Azide and PLA-OHa

aRing-opening polymerization of D,L-lactide was initiated by either HO-PEG-azide or 1-octanol.

Scheme 3. Synthesis of ()-Alkyne-Folatea

aA retrosynthetic analysis of folic acid demonstrates that the molecule can be broken into pteroyl azide and a glutamic acid derivative (block arrow).The regioselective modification of the ()-COOH of folic acid was achieved by reacting ()-alkyne-glutamic acid with pteroyl azide.

Figure 4. UV absorbance of nanoparticles clicked with folic acid. Allmeasurements were made after extensive dialysis. The amount ofcovalently attached folate was determined by the difference betweenthe 280 nm absorbance of the NP + alkyne-FA sample and the NP+ FA sample, using the calibration curve shown in the inset.



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50 folate ligands, making these particles an attractive startingpoint for targeting studies.

Macromolecule Conjugation to Polymeric Nano-particles. As the use of antibodies and other biologics for

both targeting and treatment becomes more prevalent,5 wewanted to demonstrate that our nanoparticle system is not onlycompatible with small molecule ligands but also macro-molecular ligands. We chose an artificial protein as a modelmolecule for this purpose. The protein A1 is a short helicalprotein that dimerizes to form a leucine zipper.20 Because analkyne group is not naturally present in any of the naturalamino acids, we introduced an alkyne group to A1 through theincorporation of ethynyl-Phe, an unnatural amino acid(structure given in Figure S1 in the Supporting Information).In the presence of a phenylalanyl-tRNA synthetase variant,ethynyl-Phe can replace phenylalanine in recombinant proteins

when expressed in a phenylalanine auxotrophic strain AFIQ.21

The native A1 protein does not contain any phenylalanineresidues, so we mutated a single methionine residue near the Nterminus of A1 to phenylalanine (Figure S2 in the SupportingInformation). This modification results in an A1 protein thatcontains only a single alkyne moiety. The rationale for placing

the ethynyl-Phe residue at the end of the protein was to makethe alkyne moiety more accessible in the click reaction. Alkyne-

A1 and native A1 (as a control) were both reacted with azidenanoparticles at a ratio of 1 alkyne per 5 PEG azides. Theconjugation reaction was carried out at RT overnight andfollowed by dialysis to remove unreacted protein and otherreagents. Using DLS, we observed that alknye-A1-clickednanoparticles showed an average diameter increase of 32 nm,indicating the successful conjugation of protein A1 (Figure 3).The A1 protein consists of a 42 aa helix flanked by twounstructured ends (14 aa and 16 aa long). The helical portionof the protein contributes an increase of 6.3 nm in particleradius upon conjugation. Thus, a 16 nm increase in radius isreasonable taking into account both the helix and the random

coil ends. The presence of the random coil protein segmentsmay also contribute to the slight widening of the particle sizedistribution after conjugation. Nanoparticles treated with native

A1 and click reagents did not show an increase in size,illustrating that the conjugation was attributable to the presenceof the alkyne group. The extent of conjugation was semi-quantitatively addressed using a spot blot technique using anantibody against the histidine tag at the N terminus of the A1protein. After dialysis, spots were observed only for alkyne-A1clicked nanoparticles (Figure 5). Only a very faint spot wasobserved for the control reaction sample with normal A1,indicating that without any alkyne group, A1 was notconjugated with the nanoparticle and was removed by dialysis.

A series of different concentrations of alkyne-A1 representing

100% conversion down to 6% were also spotted, creating acalibration curve for comparison (Figure 5). From thesemeasurements, we estimate that the extent of alkyne-A1conjugation was close to 100%, resulting in each nanoparticlecarrying 100 A1 proteins. This high loading of protein on thenanoparticle is consistent with the large size increase that weobserved.

DISCUSSION

Here, we have described a novel synthesis for heterofunctionalPEG bearing an azide group for subsequent modifications byclick chemistry. Our synthesis is robust and high-yielding anddoes not require separations techniques besides precipitation.

An intermediate on the way to generating HO-PEG-azide, HO-PEG-Cl, should be useful in generating a wide variety ofdifferent heterofunctional PEGs. As an application of the HO-

PEG-azide polymer, we generated ampiphilic polylactide (PLA)block copolymers by ring-opening polymerization from thePEG macromer. This fully biocompatible block polymer wasused to assemble nanoparticles that can be used for drugdelivery. While it has been amply demonstrated that cappedmonomethyl PEGs can be used as macroinitiators to createpolylactide-b-PEG block copolymers,40,41 functionalizable blockcopolymers have been much more challenging to produce.42,43

Perhaps the most important result of our study is thedemonstration of the power of the azidealkyne clickchemistry as a tool to functionalize nanoparticles formedfrom these block copolymers. We find that conjugation ofeither alkyne-labeled small molecules or proteins to the PEGlayer of these nanoparticles proceeds to high (in some casesnearly quantitative) extents of reaction without a large excess ofligand and under mild, aqueous conditions. The near 100%efficiency of conjugation of azidecopolymer to alkyneprotein with 1:5 (protein:polymer) stoichiometry is a majoradvance relative to conventional bioconjugation techniques.Previous work in our group using PEGmaleimide conjugationto single thiol groups on the protein BSA resulted in onlyapproximately 10% conversion at 1:1 stoichiometry. Completeconversion of the PEG maleimide was predicted to require over100:1 stoichiometry of protein to PEG at 30% reactive PEGgroups on the nanoparticle surface corona. Clearly for antibodycoupling, this high concentration of antibody to drive thereaction is undesirable. The reason for the difficulty ofconversion for these dense PEG layers lies in the fact thatthe reactive PEG end is not readily accessible to the protein

since it dynamically diffuses throughout the PEG layer as hasbeen demonstrated by Grest and Murat.44 Coupling chemistriessuch as maleimide:thiol11 or activated ester:amine45 faceoxidation of the thiol groups and ester hydrolysis, respectively,over the course of the reaction, leading to decreasedconversion. In contrast, the azide and alkyne groups suffer nodeactivation and, therefore, can proceed to high conversionseven for slow, hindered end coupling reactions. With advancesin the techniques for unnatural amino acid incorporation,4649

our approach can be extended to many different recombinantproteins that can be produced with alkyne-bearing amino acids.In addition, one could imagine inverting the polarity of thechemistry such that the alkyne moiety is placed on the

Figure 5. Spot blot of nanoparticles after conjugation using anti-Hisantibody. Top: A serial dilution of the alkyne-A1 protein on spot blot.A concentration of 10 M represents 100% conversion uponconjugation. Bottom: Signal of protein A1 was only observedabundantly when alkyne-A1 was conjugated to nanoparticles.



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polymeric nanoparticle and azide moieties are added to theligand. Xie et al. have recently reported a concise synthesis ofalkyne-PEG-OH using 3-trimethylsilyl-2-propargyl alcohol(TMSP).50 This polymer can be used to initiate lactidepolymerization and yield PLA-PEG-alkyne. Azide groups can beadded to small molecule ligands of interest and can beintroduced into proteins via the use of unnatural amino acidssuch as azidohomoalanine51,52 and azidonorleucine.5355As wehave demonstrated here, the bioorthogonality and mildconditions of the azidealkyne click reaction make it apowerful tool for the functionalization of nanocarriers.

ASSOCIATED CONTENT

*S Supporting Information

Three additional figures. This material is available free of chargevia the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*A207 Engineering Quadrangle, Princeton, New Jersey, 08540,United States. Tel: 609-258-7191. Fax: 609-258-0211. E-mail:

[email protected]

The authors declare no competing financial interest.

ACKNOWLEDGMENTS

We thank Bryan Beckingham for the help with GPC. We thankM. G. Finn (Scripps) and Jin Montclare (NYU Poly) forsharing materials. This work was supported by a seed grantfrom the Princeton Center for Complex Materials.

REFERENCES(1) Farokhzad, O. C.; Langer, R. Impact of Nanotechnology on Drug

Delivery. ACS Nano 2009, 3, 1620.

(2) Petros, R. A.; DeSimone, J. M. Strategies in the design ofnanoparticles for therapeutic applications. Nature Rev, Drug Discovery2010, 9, 615627.

(3) Stolnik, S.; Dunn, S. E.; Garnett, M. C.; Davies, M. C.; Coombes,A. G. A.; Taylor, D. C.; Irving, M. P.; Purkiss, S. C.; Tadros, T. F.;Davis, S. S.; Illum, L. Surface Modification of Poly(Lactide-Co-Glycolide) Nanospheres by Biodegradable Poly(Lactide)-Poly-(Ethylene Glycol) Copolymers. Pharm. Res. 1994, 11, 18001808.

(4) Jokerst, J. V.; Lobovkina, T.; Zare, R. N.; Gambhir, S. S.Nanoparticle PEGylation for imaging and therapy. Nanomedicine 2011,6, 715728.

(5) Algar, W. R.; Prasuhn, D. E.; Stewart, M. H.; Jennings, T. L.;Blanco-Canosa, J. B.; Dawson, P. E.; Medintz, I. L. The ControlledDisplay of Biomolecules on Nanoparticles: A Challenge Suited toBioorthogonal Chemistry. Bioconjugate Chem. 2011, 22, 825858.

(6) Chacko, A. M.; Hood, E. D.; Zern, B. J.; Muzykantov, V. R.Targeted nanocarriers for imaging and therapy of vascularinflammation. Curr. Opin. Colloid Interface Sci. 2011, 16, 215227.

(7) Fay, F.; Scott, C. J. Antibody-targeted nanoparticles for cancertherapy. Immunotherapy 2011, 3, 381394.

(8) Franzen, S. A comparison of peptide and folate receptor targetingof cancer cells: from single agent to nanoparticle. Expert Opin. Drug

Delivery 2011, 8, 281298.(9) Lawlor, C.; Kelly, C.; O'Leary, S.; O'Sullivan, M. P.; Gallagher, P.

J.; Keane, J.; Cryan, S. A. Cellular targeting and trafficking of drugdelivery systems for the prevention and treatment of MTb.Tuberculosis 2011, 91, 9397.

(10) Johnson, B. K.; Prud'homme, R. K. Flash NanoPrecipitation oforganic actives and block copolymers using a confined impinging jetsmixer. Aust. J. Chem. 2003, 56, 10211024.

(11) Gindy, M. E.; Ji, S.; Hoye, T. R.; Panagiotopoulos, A. Z.;Prud'homme, R. K. Preparation of Poly(ethylene glycol) ProtectedNanoparticles with Variable Bioconjugate Ligand Density. Biomacro-molecules 2008, 9, 27052711.

(12) Best, M. D. Click Chemistry and Bioorthogonal Reactions:Unprecedented Selectivity in the Labeling of Biological Molecules.

Biochemistry 2009, 48, 65716584.(13) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. A

stepwise Huisgen cycloaddition process: Copper(I)-catalyzed regiose-lective ligation of azides and terminal alkynes. Angew. Chem.-Int. Ed.2002, 41, 25962599.

(14) Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.; Sharpless, K.B.; Finn, M. G. Bioconjugation by copper(I)-catalyzed azide-alkyne [3+ 2] cycloaddition. J. Am. Chem. Soc. 2003, 125, 31923193.

(15) Hiki, S.; Kataoka, K. A facile synthesis of azido-terminatedheterobifunctional poly(ethylene glycol)s for click conjugation.

Bioconjugate Chem. 2007, 18, 21912196.(16) Freeman, P. K.; Hutchinson, L. L. Alkyllithium Reagents from

Alkyl-halides and Lithium Radical Anions. J. Org. Chem. 1980, 45,19241930.

(17) Luo, J.; Smith, M. D.; Lantrip, D. A.; Wang, S.; Fuchs, P. L.Efficient syntheses of pyrofolic acid and pteroyl azide, reagents for theproduction of carboxyl-differentiated derivatives of folic acid. J. Am.Chem. Soc. 1997, 119, 1000410013.

(18) Lei, H. Y.; Stoakes, M. S.; Herath, K. P. B.; Lee, J. H.;Schwabacher, A. W. Efficient synthesis of a phosphinate bis-amino acidand Its use in construction of ampiphilic peptides. J. Org. Chem. 1994,59, 42064210.

(19) Kayser, B.; Altman, J.; Beck, W. Alkyne bridged alpha-aminoacids by palladium mediated coupling of alkynes with N-t-Boc-4-iodophenylalanine methyl ester. Tetrahedron 1997, 53, 24752484.

(20) Petka, W. A.; Harden, J. L.; McGrath, K. P.; Wirtz, D.; Tirrell, D.A. Reversible hydrogels from self-assembling artificial proteins. Science1998, 281, 389392.

(21) Kirshenbaum, K.; Carrico, I. S.; Tirrell, D. A. Biosynthesis ofproteins incorporating a versatile set of phenylalanine analogues.Chembiochem 2002, 3, 235237.

(22) Datta, D.; Wang, P.; Carrico, I. S.; Mayo, S. L.; Tirrell, D. A. Adesigned phenylalanyl-tRNA synthetase variant allows efficient in vivo

incorporation of aryl ketone functionality into proteins. J. Am. Chem.Soc. 2002, 124, 56525653.(23) Liu, Y.; Cheng, C. Y.; Prud'homme, R. K.; Fox, R. O. Mixing in a

multi-inlet vortex mixer (MIVM) for flash nano-precipitation. Chem.Eng. Sci. 2008, 63, 28292842.

(24) Hong, V.; Presolski, S. I.; Ma, C.; Finn, M. G. Analysis andOptimization of Copper-Catalyzed Azide-Alkyne Cycloaddition forBioconjugation. Angew. Chem.-Int. Ed. 2009, 48, 98799883.

(25) Cohn, D.; Younes, H. Biodegradable PEO/PLA blockcopolymers. J. Biomed. Mater. Res. 1988, 22, 9931009.

(26) Li, J.; Crasto, C. F.; Weinberg, J. S.; Amiji, M.; Shenoy, D.;Sridhar, S.; Bubley, G. J.; Jones, G. B. An approach to heterobifunc-tional poly(ethyleneglycol) bioconjugates. Bioorg. Med. Chem. Lett.2005, 15, 55585561.

(27) Levy, D. E.; Frederick, B.; Luo, B.; Zalipsky, S. Heterobifunc-tional PEGs: Efficient synthetic strategies and useful conjugation

methodologies. Bioorg. Med. Chem. Lett. 2010, 20, 68236826.(28) Bouzide, A.; Sauve, G. Silver(I) oxide mediated highly selective

monotosylation of symmetrical diols. Application to the synthesis ofpolysubstituted cyclic ethers. Org. Lett. 2002, 4, 23292332.

(29) Gref, R.; Luck, M.; Quellec, P.; Marchand, M.; Dellacherie, E.;Harnisch, S.; Blunk, T.; Muller, R. H. 'Stealth' corona-corenanoparticles surface modified by polyethylene glycol (PEG):Influences of the corona (PEG chain length and surface density)and of the core composition on phagocytic uptake and plasma proteinadsorption. Colloids Surf., B 2000, 18, 301313.

(30) Jalabert, M.; Fraschini, C.; PrudHomme, R. E. Synthesis andcharacterization of poly(L-lactide)s and poly(D-lactide)s of controlledmolecular weight. J. Polym. Sci., Part A: Polym. Chem. 2007, 45, 19441955.


http://pubs.acs.org/mailto:[email protected]:[email protected]://pubs.acs.org/

7/27/2019 Mp 3000748

9/9

(31) Kaihara, S.; Matsumura, S.; Mikos, A. G.; Fisher, J. P. Synthesisof poly(L-lactide) and polyglycolide by ring-opening polymerization.

Nature Protoc. 2007, 2, 27672771.(32) Gindy, M. E.; Panagiotopoulos, A. Z.; Prud'homme, R. K.

Composite block copolymer stabilized nanoparticles: Simultaneousencapsulation of organic actives and inorganic nanostructures.

Langmuir2008, 24, 8390.(33) Zhang, S. Y.; Adamson, D. H.; Prud'homme, R. K.; Link, A. J.

Photocrosslinking the Polystyrene Core of Block-Copolymer Nano-particles. Polym. Chem. 2011, 2, 665671.(34) Zhang, S. Y.; Prud'homme, R. K.; Link, A. J. Block Copolymer

Nanoparticles as Nanobeads for the Polymerase Chain Reaction. NanoLett. 2011, 11, 17231726.

(35) Xia, W.; Low, P. S. Folate-Targeted Therapies for Cancer. J.Med. Chem. 2010, 53, 68116824.

(36) Matherly, L. H.; Hou, Z. J.; Deng, Y. J. Human reduced folatecarrier: Translation of basic biology to cancer etiology and therapy.Cancer Metastasis Rev. 2007, 26, 111128.

(37) Zhao, R. B.; Min, S. H.; Wang, Y. H.; Campanella, E.; Low, P. S.;Goldman, I. D. A Role for the Proton-coupled Folate Transporter(PCFT-SLC46A1) in Folate Receptor-mediated Endocytosis. J. Biol.Chem. 2009, 284, 42674274.

(38) Caliceti, P.; Salmaso, S.; Semenzato, A.; Carofiglio, T.;Fornasier, R.; Fermeglia, M.; Ferrone, M.; Pricl, S. Synthesis and

physicochemical characterization of folate-cyclodextrin bioconjugatefor active drug delivery. Bioconjugate Chem. 2003, 14, 899908.

(39) Wang, S.; Luo, J.; Lantrip, D. A.; Waters, D. J.; Mathias, C. J.;Green, M. A.; Fuchs, P. L.; Low, P. S. Design and synthesis of In-111DTPA-folate for use as a tumor-targeted radiopharmaceutical.

Bioconjugate Chem. 1997, 8, 673679.(40) Kim, S. Y.; Shin, I . G.; Lee, Y. M. Preparation and

characterization of biodegradable nanospheres composed of methoxypoly(ethylene glycol) and DL-lactide block copolymer as novel drugcarriers. J. Controlled Release 1998, 56, 197208.

(41) Ko, J. T.; Lee, J. H.; Kim, J. M.; Kim, M. S.; Rhee, J. M.; Lee, H.B.; Khang, G. Development of Biodegradable Methoxy Poly-(ethyleneglycol)-polyesters Diblock Copolymers as Drug Carrier forImplantable Disc. Tissue Eng. Regener. Med. 2006, 3, 158168.

(42) Petersen, M. A.; Yin, L.; Kokkoli, E.; Hillmyer, M. A. Synthesis

and characterization of reactive PEO-PMCL polymersomes. Polym.Chem. 2010, 1, 12811290.(43) Ji, S.; Zhu, Z.; Hoye, T. R.; Macosko, C. W. Maleimide

Functionalized Poly(epsilon-caprolactone)-block-poly(ethylene gly-col) (PCL-PEG-MAL): Synthesis, Nanoparticle Formation, andThiol Conjugation. Macromol. Chem. Phys. 2009, 210, 823831.

(44) Grest, G. S.; Murat, M. Structure of grafted polymeric brushes insolvents of varying qualityA molecular dynamics study. Macro-molecules 1993, 26, 31083117.

(45) Torchilin, V. P.; Levchenko, T. S.; Lukyanov, A. N.; Khaw, B. A.;Klibanov, A. L.; Rammohan, R.; Samokhin, G. P.; Whiteman, K. R. p-nitrophenylcarbonyl-PEG-PE-liposomes: fast and simple attachment ofspecific ligands, including monoclonal antibodies, to distal ends ofPEG chains via p-nitrophenylcarbonyl groups. Biochim. Biophys. Acta,

Biomembr. 2001, 1511, 397411.(46) Budisa, N. Prolegomena to Future Experimental Efforts on

Genetic Code Engineering by Expanding Its Amino Acid Repertoire.Angew. Chem.-Int. Ed. 2004, 43, 64266463.

(47) Link, A. J.; Tirrell, D. A. Reassignment of Sense Codons In Vivo.Methods 2005, 36, 291298.

(48) Wang, L.; Schultz, P. G. Expanding the genetic code. Angew.Chem.-Int. Ed. 2005, 44, 3466.

(49) Voloshchuk, N.; Montclare, J. K. Incorporation of unnaturalamino acids for synthetic biology. Mol. Biosyst. 2010, 6, 6580.

(50) Xie, Y.; Duan, S.; Forrest, M. L. Alkyne- and 1,6-elimination-succinimidyl carbonate-terminated heterobifunctional poly(ethyleneglycol) for reversible Click PEGylation. Drug Discoveries Ther. 2010,4, 240245.

(51) Kiick, K. L.; Saxon, E.; Tirrell, D. A.; Bertozzi, C. R.Incorporation of azides into recombinant proteins for chemoselective

modification by the Staudinger ligation. Proc. Natl. Acad. Sci. U.S.A.2002, 99, 1924.

(52) Link, A. J.; Tirrell, D. A. Cell surface labeling of Escherichia colivia copper(I)- catalyzed [3 + 2] cycloaddition. J. Am. Chem. Soc. 2003,125, 1116411165.

(53) Link, A. J.; Vink, M. K. S.; Agard, N. J.; Prescher, J. A.; Bertozzi,C. R.; Tirrell, D. A. Discovery of aminoacyl-tRNA synthetase activitythrough cell-surface display of noncanonical amino acids. Proc. Natl.

Acad. Sci. U.S.A. 2006, 103, 10180

10185.(54) Abdeljabbar, D. M.; Klein, T. J.; Link, A. J. An EngineeredMethionyl-tRNA Synthetase Enables Azidonorleucine Incorporationin Methionine Prototrophic Bacteria. Chembiochem 2011, 12, 16991702.

(55) Abdeljabbar, D. M.; Klein, T. J.; Zhang, S. Y.; Link, A. J. A SingleGenomic Copy of an Engineered Methionyl-tRNA Synthetase EnablesRobust Incorporation of Azidonorleucine into Recombinant Proteinsin E. coli. J. Am. Chem. Soc. 2009, 131, 1707817079.



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