Water-soluble dendritic-conjugated polyfluorenes: Synthesis, characterization, and interactions with DNA

Water-Soluble Dendritic-Conjugated Polyfluorenes:Synthesis, Characterization, and Interactions with DNA

MINGHUI YU, LIBING LIU, SHU WANG

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry,Chinese Academy of Sciences, Beijing 100080, People’s Republic of China

Received 7 May 2008; accepted 27 August 2008DOI: 10.1002/pola.23051Published online in Wiley InterScience (www.interscience.wiley.com).

ABSTRACT: Three generation of Boc-protected dendritic-conjugated polyfluorenes(Boc-PFP-G0-2) were synthesized by Suzuki coupling 1,4-phenyldiboronic ester withdendritic monomers that were synthesized through generation-by-generationapproach. The gel permeation chromatography (GPC) analyses showed that theweight-average molecular weight (Mw) of Boc-PFP-G0-2 was in the range of 11,400–20,400 Da with the polydispersity index (PDI) in the range of 1.32–1.96. Treatmentof Boc-protected polymers with 6 M HCl in dioxane yielded cationic dendritic-conju-gated polyfluorenes (PFP-G0-2). They were soluble in common polar solvents such asDMSO, DMF, and water with absorption maxima between 345 and 379 nm. The solu-tions of PFP-G0-2 in water were highly fluorescent with emission maxima between416 and 425 nm. Because higher generation dendrons could prevent the formation ofp-stacking aggregates of backbones of conjugated polymer, the fluorescence quantumefficiencies (QEs) of PFP-G0-2 enhance as the dendritic generation grew. The interac-tions between 25 mer double-stranded DNA (dsDNA) and PFP-G0-2 were studiedusing ethidium bromide (EB) as fluorescent probe. The electrostatic bindings of PFP-G0-2 with dsDNA/EB complex result in displacement of EB from DNA double helix tothe solution accompanying by a quenching of EB fluorescence. The PFP-G2 withhighest generation of dendritic side chains possessed a highest charge density andcould form most stable complex with dsDNA. VVC 2008 Wiley Periodicals, Inc. J Polym Sci

Part A: Polym Chem 46: 7462–7472, 2008

Keywords: conjugated polymers; dendritic-conjugated polymers; DNA; fluorescence;optical properties; synthesis

INTRODUCTION

In recent years, water-soluble conjugated poly-mers have received much attention due to theirpotential applications in the field of sensitivechemical and biological sensors. In comparison

with small molecule counterparts, these polymersexhibit exceptional fluorescence quenching orenergy transfer efficiencies in the presence ofoppositely charged acceptors and therefore resultin the amplification of optical signals for thetransduction of biological recognizing events.1

Recently, we and others have reported novel fluo-rescent chemical and biological sensors based onwater-soluble conjugated polymers to detect metalions, DNA, RNA, enzymes, and proteins in aque-ous media.2 These systems benefit from the sensi-tivity of optical signals from conjugated polymers,

Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 46, 7462–7472 (2008)VVC 2008 Wiley Periodicals, Inc.

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

Correspondence to: S. Wang (E-mail: [email protected])

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however, a major problem with these conjugatedpolymers concerns their tendency to form aggre-gations in aqueous solution. Water-soluble conju-gated polymers are rigid-rod like molecules inwhich the backbones are hydrophobic moieties,whereas the charged side chains are hydrophilicones. The resulting amphiphilic characteristicslead to different aggregation structures in differ-ent polar solvents. In aqueous media, the inter-chain interactions of the conjugated polymerslead to form tight aggregates, which results influorescence quenching due to p-stacking betweenthe backbones of conjugated polymers.3 Theaggregations lead to issues of decreased sensitiv-ity of the biosensors. Therefore it is necessary toenhance the fluorescence quantum efficiencies(QEs) of the water-soluble conjugated polymers inaqueous solution. As promising materials thewater-soluble dendritic-conjugated polymers havebeen highlighted to overcome this drawback.4

Dendronized polymers contain central linearpolymeric cores and peripheral dendronizedgroups.5 The site isolation of the polymer back-bone is achieved by self-encapsulation in a dendri-tic matrix. The dendrons have regularly andrepeatedly branched structures that can preventthe aggregation of the polymers in some extentdue to the steric repulsions between them. As thegeneration of the dendrons increases, the siteisolation effect between the wedges is more obvi-ously, and the aggregation tendency of the con-

jugated backbone reduces evidently, hence, thefluorescent quantum efficiency of the polymerimproves greatly. Among dendronized polymers,the dendritic-conjugated polyfluorenes are deemedto promising materials due to their stabilities andhigh quantum efficiencies.6

The work in the field of gene delivery has dem-onstrated that the double-stranded DNA (dsDNA)with high density of negatively charged phos-phate groups can form rather stable polyelectro-lyte complexes with synthetic polycations byelectrostatic interactions.7 One of the chiefrequirements for these complexes as the genevehicles is the binding stability. In this regard,the dendrimers, such as polyamidoamine andpoly(propylene imines), have been found to bepotential nonviral materials for highly efficientgene delivery.8 Thus, water-soluble conjugatedpolymers with peripheral cationic charges couldprovide a suitable interface for studying genedelivery except for serving as signal transducersin biosensors.

In this contribution we designed and synthe-sized a family of cationic water-soluble dendritic-conjugated polyfluorenes carrying peripheralcharged amino groups (PFP-G0-2, see Scheme 1for their chemical structures). The fluorescencequantum efficiencies were enhanced as increasingthe generation of the side-chain dendron. Thestudies on the complex formations of PFP-G0-2

with dsDNA reveal that the higher cationic

Scheme 1. Chemical structures of cationic water-soluble dendritic conjugated poly-fluorenes (PFP-G0-2).

WATER-SOLUBLE DENDRITIC CONJUGATED POLYFLUORENES 7463

Journal of Polymer Science: Part A: Polymer ChemistryDOI 10.1002/pola

charge density on the side-chain dendronimproves the stability of the dendritic-conjugatedpolymer/DNA complex.

EXPERIMENTAL

Material

All chemicals were purchased from Arcos or AlfaAesar and were used as received. The 1-(30-N-(tert-butyloxycarbonyloxy)-amino)-propyl-4-meth-ylphenylsulfonate9 and 2,7-dibromo-9,9-bis(40-hydroxyphenyl)-fluorene (6)10 were synthesizedaccording to the procedures in the literatures.THF was distilled from sodium using benzophe-none as indicator for dryness. Dichloromethanewas stored over sodium hydride and filteredbefore use, other reagents and solvents wereused without further purification. Oligonucleo-tides were purchased from Shanghai Sangon Bio-logical Engineering Technology and Service andtheir concentrations were determined by meas-uring the absorbance at 260 nm in 160-lL quartzcuvette.

Instrumentation

The 1H-NMR and 13C-NMR spectra were recordedon a Bruker Avance 400 MHz spectrometer. Massspectra were recorded on Bruker Biflex MALDI-TOF spectrometer. Elemental analyses were car-ried out with a Flash EA1112 instrument. The gelpermeation chromatography (GPC) measure-ments were performed on Water-410 systemagainst polystyrene standard with THF as theeluent. UV–vis absorption spectra were taken ona JASCO V-550 spectrophotometer and the fluo-rescence spectra were measured using Hitachi F-4500 fluorometer with a Xe lamp as excitationsource.

3,5-Bis[30-(tert-butoxycarbonylamino)propoxy]-Benzyl Alcohol (2)

To a mixture of 3,5-dihydroxyl-benzyl alcohol (1)(4.2 g 30 mmol), K2CO3 (20.73 g, 150 mmol) andcatalytic quantity of 18-crown-6 in 200 mLacetone was added 1-(30-N-(tert-butyloxycarbo-nyloxy)-amino)-propyl-4-methylphenylsulfonate(20.73 g, 63 mmol). The resulting mixture wasstirred for 3 days at 70 �C. After cooling to theroom temperature, the mixture was poured intodistilled water and extracted with dichlorome-thane. The solvent was removed and the residue

was purified by silica gel column chromatographywith petroleum ether/ethyl acetate (5:2) as eluentto afford a colorless liquid (9.98 g, yield 73%).

1H-NMR (400 MHz, CDCl3): d (ppm) 1.44 (m,18H), 1.95 (m, 4H), 3.31 (m, 4H), 3.98 (t, 4H, J ¼13.2), 4.61 (s, 2H), 4.76 (br, 2H, NH), 6.36 (s, 1H),6.51 (s, 2H). 13C-NMR (100 MHz, CDCl3): d (ppm)28.3, 29.5, 38.7, 66.1, 68.3, 79.8, 100.7, 107.3,139.7, 155.6, 159.6. MALDI-TOF: 477.8 [MþNa].Anal. calcd for C23H37O6N2Br: C, 60.77; H, 8.43;N, 6.16. Found: C, 61.23; H, 8.51; N, 5.83.

3,5-Bis[30-(tert-butoxycarbonylamino)propoxy]-Benzyl Bromide (3)

A solution of compound 2 (9.75 g, 21.5 mmol), trie-thylamine (2.99 g, 30 mmol), and methansul-phonyl chloride (3.71 g, 32.3 mmol) in 150 mLanhydrous THF was stirred at 0 �C for 30 min,and then warmed to room temperature andstirred for 2 h. Then lithium bromide (11.25 g,107.3 mmol) was added and allowed to stirring for16 h. The mixture was poured into distilled waterand extracted with dichloromethane. The solventwas removed, and the residue was purified bysilica gel column chromatography with petroleumether/ethyl acetate (3:1) as eluent to give a whitesolid (8.15 g, yield 92%).

1H-NMR (400 MHz, CDCl3): d (ppm) 1.44 (m,18H), 1.98 (m, 4H), 3.32 (m, 4H), 3.99 (t, 4H, J ¼12.7 Hz), 4.49 (s, 2H), 4.72 (br, 2H, NH), 6.36 (s,1H), 6.52 (s, 2H). 13C-NMR (100 MHz, CDCl3): d(ppm) 28.4, 29.5, 33.4, 37.9, 65.8, 79.2, 101.5,107.6, 139.3, 155.9, 160.0. MALDI-TOF: 540.2[MþNa]. Anal. calcd for C23H38O7N2: C, 53.39; H,7.21; N, 5.41. Found: C, 53.83; H, 7.16; N, 5.32.

Second Generation Benzyl Alcohol (4)

To a mixture of 3,5-dihydroxyl-benzyl alcohol (1)(0.85 g, 6.1 mmol), K2CO3 (4.22 g, 30.5 mmol) andcatalytic quantity of 18-crown-6 in 200 mL ace-tone was added compound 3 (6.46 g, 12.5 mmol).The resulting mixture was stirred for 2 days at70 �C. After cooling to the room temperature, themixture was poured into distilled water andextracted with dichloromethane. The solvent wasremoved and the residue was purified by silica gelcolumn chromatography using petroleum ether/ethyl acetate (2:3) as eluent to give a colorless liq-uid (3.81 g, yield 62%).

1H-NMR (400 MHz, CDCl3): d (ppm) 1.44 (m,36H), 1.94 (m, 8H), 3.30 (m, 8H), 4.01 (t, 8H, J ¼13.7 Hz), 4.63 (s, 2H), 4.77 (br, 2H, NH), 4.96 (m,

7464 YU, LIU, AND WANG


4H), 6.39 (s, 2H), 6.51 (s, 1H), 6.55 (m, 4H), 6.61(m, 2H). 13C-NMR (100 MHz, CDCl3): d (ppm)28.3, 29.4, 38.7, 65.7, 68.4, 69.9, 79.2, 100.8101.9,105.8, 107.6, 138.9, 139.7, 156.1, 159.8, 160.1.MALDI-TOF: 1035.8 [MþNa]. Anal. calcd forC53H80O15N4: C, 62.81; H, 7.96; N, 5.53. Found:C, 63.26; H, 7.87; N, 5.45.

Second Generation Benzyl Bromide (5)

A solution of compound 4 (3.6 g, 3.56 mmol), tri-ethylamine (0.495 g, 5 mmol) and methansul-phonyl chloride (0.62 g, 5.4 mmol) in 50 mL anhy-drous THF was stirred at 0 �C for 30 min, andthen warmed to room temperature and stirred for2 h. Then lithium bromide (1.87 g, 17.6 mmol)was added and allowed to stirring for 16 h. Themixture was poured into distilled water andextracted with dichloromethane. The solvent wasremoved and the residue was purified by silica gelcolumn chromatography using petroleum ether/ethyl acetate (1:1) as eluent to afford a colorlesssolid (2.96 g, yield 77%).

1H-NMR (400 MHz, CDCl3): d (ppm) 1.44 (m,36H), 1.95 (m, 8H), 3.31 (m, 8H), 3.99 (t, 8H, J ¼11.8 Hz), 4.41 (s, 2H), 4.51 (br, 2H, NH), 4.95 (m,4H), 6.41 (s, 2H), 6.48 (s, 1H), 6.56 (m, 4H), 6.62(m, 2H). 13C-NMR (100 MHz, CDCl3): d (ppm)28.4, 29.4, 33.5, 38.7, 65.8, 69.9, 79.1, 100.9102.0,105.9, 108.1, 138.9, 139.8, 156.0, 159.9, 160.1.MALDI-TOF: 1115.8 [MþK]. Anal. calcd forC53H79O14N4Br: C, 59.12; H, 7.40; N, 5.21. Found:C, 59.56; H, 7.56; N, 5.34.

General Procedure for the Synthesis ofMonomer 7–9

To a mixture of 2,7-dibromo-9,9-bis(40-hydroxy-phenyl)-fluorene (6) (1 equiv), K2CO3 and cata-lytic quantity of 18-crown-6 in acetone was added1-(30-N-(tert-butyloxycarbonyloxy)-amino)-propyl-4-methylphenylsulfonate, 3 or 5 (2 equiv). Theresulting mixture was stirred for 2 days at 70 �C.After cooling down to the room temperature, themixture was poured into distilled water andextracted with dichloromethane. The solvent wasremoved and the residue was purified by silica gelcolumn chromatography using petroleum ether/ethyl acetate as eluent to yield colorless solids.

Monomer 7: yield 76 %. 1H-NMR (400 MHz,CDCl3): d (ppm) 1.43 (m, 18H), 1.97 (m, 4H), 3.30(m, 4H), 3.95 (t, 4H, J ¼ 11.7Hz), 4.72 (br, 2H,NH), 6.76 (d, 4H, J ¼ 7.8 Hz), 7.05 (d, 4H, J ¼ 8.1Hz), 7.46 (d, 4H, J ¼ 8.5 Hz), 7.56 (d, 2H, J ¼ 7.9

Hz). 13C-NMR (100 MHz, CDCl3): d (ppm) 28.4,29.4, 38.0, 65.7, 79.2, 114.4, 121.5, 121.8, 128.9,129.2, 130.8, 136.6, 137.8, 153.6, 156.0, 157.8.MALDI-TOF: 861.3 [MþK]. Anal. calcd forC41H46O6N2Br2: C, 59.86; H, 5.64; N, 3.40. Found:C, 60.34; H, 5.53; N, 3.45.

Monomer 8: yield 81 %. 1H-NMR (400 MHz,CDCl3): d (ppm) 1.43 (s, 36H), 1.95 (m, 8H), 3.30(m, 8H), 3.99 (t, 8H, J ¼ 12.6 Hz), 4.73 (br, 4H,NH), 4.93 (s, 4H), 6.38 (s, 2H), 6.54 (s, 4H), 6.84(d, 4H, J ¼ 9.7 Hz), 7.04 (d, 4H, J ¼ 9.6 Hz), 7.47(d, 4H, J ¼ 9.2 Hz), 7.57 (d, 2H, J ¼ 7.7 Hz). 13C-NMR (100 MHz, CDCl3): d (ppm) 28.4, 29.4, 37.9,53.8, 65.8, 69.8, 79.2, 100.8, 105.8, 114.7, 121.6,129.0, 130.8, 132.0, 133.0, 136.8, 137.8, 139.3,153.5, 156.0, 157.8, 160.1. MALDI-TOF: 1419.8[MþK]. Anal. calcd for C71H88O14N4Br2: C, 61.74;.H, 6.42; N, 4.06. Found: C, 62.17; H, 6.37; N, 4.12.

Monomer 9: yield 52 %. 1H-NMR (400 MHz,CDCl3): d (ppm) 1.44 (m, 72H), 1.96 (t, 16H), 3.31(m, 16H), 4.00 (t, 16H, J ¼ 11.6 Hz), 4.78 (br, 8H,NH), 4.95 (s, 12H), 6.40 (s, 4H), 6.56 (s, 10H), 6.66(s, 4H), 6.84 (d, 4H, J ¼ 8.3 Hz), 7.04 (d, 4H, J ¼8.9 Hz), 7.47(d, 4H, J ¼ 8.7 Hz), 7.64 (d, 2H, J ¼7.3 Hz). 13C-NMR (100 MHz, CDCl3): d (ppm)28.4, 29.4, 37.9, 54.9, 65.8, 69.9, 79.2, 100.9101.6,105.9, 106.4, 114.8, 121.6, 121.8, 128.4, 129.0,129.2, 130.8, 132.0, 137.9, 139.1, 139.3, 153.6,156.0, 157.9, 159.8, 160.1, 160.2. MALDI-TOF:2535 [MþK]. Anal. calcd for C131H172O30N8Br2: C,62.97; H, 6.94; N, 4.48. Found: C, 63.39; H, 6.82;N, 4.32.

General Procedure for the Synthesis ofBoc-Protected Dendritic-Conjugated Polymers(Boc-PFP-G0-2)

A mixture of the monomers 7, 8, or 9 (1 equiv),5,5-dimethyl-2-(4-(5,5-dimethyl-1,3,2-dioxabor-inan-2-yl)phnyl)-1,3,2-dioxaborinane (1 equiv) intoluene and 2.0 M aqueous K2CO3 solution wasdegassed, and then catalytic quantity ofPd(PPh3)4 was added under nitrogen atmosphere.The reaction mixture was stirred at 90 �C undernitrogen for 48 h. After cooling down to room tem-perature, 50 mL water was added and wasextracted with chloroform. After the organic sol-vent was removed, the residue was precipitated inmethanol. The crude polymers were purified byprecipitation from chloroform intomethanol againand dried under vacuum to give brown solids.

Boc-PFP-G0: yield 58 %. 1H-NMR (400 MHz,CDCl3): d (ppm) 1.43 (br, 18H) 1.93 (br, 4H), 3.28(br, 4H), 3.89 (br, 4H), 4.77 (br, 2H, NH), 6.76 (br,



4H), 7.14 (br, 4H), 7.56 (br, 8H), 7.83 (br, 2H). 13C-NMR (100 MHz, CDCl3): d (ppm) 28.4, 29.5, 37.9,65.7, 79.2, 114.4, 121.5, 121.8, 128.9, 129.2, 129.6,130.8, 132.6, 136.6, 137.8, 153.6, 156.0, 157.8.

Boc-PFP-G1: yield 48 %. 1H-NMR (400 MHz,CDCl3): d (ppm) 1.40 (br, 36H), 1.91 (br, 8H), 3.27(br, 8H), 3.96 (br, 8H), 4.80 (br, 4H, NH), 4.91 (br,4H), 6.36 (br, 2H), 6.53 (br, 4H), 6.89 (br, 4H), 7.21(br, 4H), 7.63 (br, 8H), 7.84 (br, 2H). 13C-NMR(100 MHz, CDCl3): d (ppm) 28.4, 29.5, 37.9, 55.4,65.7, 69.9, 79.1, 100.8, 105.9, 114.5, 120.5, 124.6,126.5, 127.4, 128.5, 129.2, 132.6, 138.2, 138.9,139.4, 140.1, 152.7, 156.0, 157.6, 160.1.

Boc-PFP-G2: yield 39 %. 1H-NMR (400 MHz,CDCl3): d (ppm) 1.42 (br, 72H), 1.92 (br, 16H),3.27 (br, 16H), 3.95 (br, 16H), 4.82 (br, 8H, NH),4.91 (br, 12H), 6.36 (br, 4H), 6.52 (br, 12H), 6.61(br, 4H), 6.82 (br, 6H), 7.64 (br, 8H), 7.70 (br, 2H).13C-NMR (100 MHz, CDCl3): d (ppm) 28.4, 29.3,37.8, 54.9, 65.9, 69.9,79.2, 100.8, 101.7, 105.9,106.4, 114.8, 121.7, 121.9, 128.3, 129.0, 129.2,129.6, 130.8, 132.0, 132.7, 137.9, 139.2, 139.3,153.5, 156.1, 157.8, 159.7, 160.1, 160.2.

General Procedure for the Synthesis of PFP-G0-2

To a solution of Boc-PFP-G0-2 in 5 mL 1,4-diox-ane was added 6 M aqueous HCl solution. Afterstirring for 24 h at room temperature, the solventwas removed and 10 mL of acetone was added toprecipitate the product as brown solids.

PFP-G0: yield 91%. 1H-NMR (400 MHz,DMSO-d6): d (ppm) 1.97 (br, 4H), 2.93 (br, 4H),3.99 (br, 4H), 4.72 (br, 6H, NH), 6.87 (br, 4H), 7.09(br, 4H), 7.69 (br, 5H), 7.92 (br, 5H), 8.07 (br, 6H,NH3

þ). PFP-G1: yield 85%. 1H-NMR (400 MHz,DMSO-d6): d (ppm) 1.97(br, 8H) 2.93 (br, 8H), 4.00(br, 8H), 4.94 (br, 4H), 6.45 (br, 2H), 6.58 (br, 4H),6.94 (br, 4H), 7.20 (br, 4H), 7.49-7.73 (br, 10H),7.97 (br, 12H, NH3

þ). PFP-G2: yield 88%. 1H-NMR (400 MHz, DMSO-d6): d (ppm) 1.98(br, 16H)2.94 (br, 16H), 4.01(br, 8H), 4.94 (br, 12H), 6.46(br, 4H), 6.59 (br, 8H), 6.95 (br, 4H), 7.21 (br, 4H),7.59-7.62 (br, 10H), 7. 96 (br, 24H, NH3

þ).

RESULTS AND DISCUSSION

Synthesis of the Monomers

The synthesis of the monomers is shown inScheme 2. Reaction of 3, 5-dihydroxybenzyl alco-hol (1) with 1-(30-N-(tert-butyloxycarbonyloxy)-amino)-propyl-4-methylphenylsulfonate in thepresence of potassium carbonate and catalytic

quantity of 18-crown-6 in acetone gave 3,5-bis[60-(tert-butoxycarbonylamino)propoxy]-benzyl alco-hol (2) in 73% yield. Benzyl bromide (3) wasobtained by reaction of benzyl alcohol (2) withmethansulphonyl chloride in the presence of trie-thylamine following by treatment with lithiumbromide in 92% yield overall. Reaction of benzylbromide (3) with 3, 5-dihydroxybenzyl alcohol (1)in the presence of potassium carbonate and cata-lytic quantity of 18-crown-6 in acetone affordedsecond-generation benzyl alcohol (4) in 62% yield.As similar preparation as that of compound 3, sec-ond-generation benzyl alcohol (4) was convertedto the corresponding benzylbromide (5) in 77%yield. 2,7-Dibromo-9,9-bis(40-hydroxyphenyl)-fluo-rene (6) was prepared according to previouslyreported method.10 The dendritic monomers 7, 8,and 9 were synthesized by respective reactions ofcompound 6 with 1-(30-N-(tert-butyloxycarbony-loxy)-amino)-propyl-4-methylphenylsulfonate,compound 3 and 5 in the presence of potassiumcarbonate and catalytic quantity of 18-crown-6 inacetone. After purification by normal silica gelchromatography, pure dendritic monomers 7, 8,and 9 were obtained in yield of 76, 81, and 52%respectively.

Synthesis and Characterization of Dendritic-Conjugated Polymers

The preparation route of Boc-protected polyfluor-enes (Boc-PFP-G0-2) is shown in Scheme 3. Theywere synthesized by Suzuki coupling11 betweenone equivalent of monomers 7, 8, or 9 and 1,4-phenyldiboronic ester in the presence of 2.0 Maqueous K2CO3 and Pd(PPh3)4 in toluene. Toreach a high reaction temperature, the toluenewas chosen as the solvent. The crude polymerswere purified by precipitation from chloroforminto methanol twice to give brown solids. TheseBoc-protected polyfluorenes are readily dissolvedin common organic solvents such as dichlorome-thane, chloroform, and THF. Their structureswere characterized by 1H-NMR and 13C-NMRspectroscopies as shown in Figure 1(a,b), whereBoc-PFP-G1 was given as an example. The gelpermeation chromatography (GPC) analysesagainst polystyrene standard show that theweight-average molecular weight (Mw) of Boc-PFP-G0-2 is in the range of 114,00–20,400 Dawith the polydispersity index (PDI) in the rangeof 1.32–1.96. The molecule weight data are sum-marized in Table 1. The Boc-PFP-G0-2 wastreated with 6 M HCl in 1,4-dioxane to deprotect



Scheme 2. Synthesis of the monomers. Reaction conditions: (a) Boc-NH-(CH2)3O-Tos, K2CO3, acetone, 18-crown-6, 70 �C, 73%; (b) (i) MsCl, Et3N, THF, 0 �C, (ii) LiBr,25 �C, 92%; (c) 1, K2CO3, acetone, 18-crown-6, 70 �C, 62%; (d) (i) MsCl, Et3N, THF,0 �C, (ii) LiBr, 25 �C, 77%; (e) Boc-NH-(CH2)3OTos, K2CO3, acetone, 18-crown-6,70 �C, 76%; (f) 3, K2CO3, acetone, 18-crown-6, 70 �C, 81%; (g) 5, K2CO3, acetone, 18-crown-6, 70 �C, 52%.

Scheme 3. Synthesis of PFP-G0-2. Reaction conditions: (a) 2.0 M K2CO3, toluene,Pd(PPh3)4, 85

�C; (b) 6 M HCl, 1,4-dioxane, room temperature.



the Boc groups to provide water-soluble PFP-G0-2.The 1H-NMR spectroscopy of PFP-G1 was givenin Figure 1(c). In comparing with that of Boc-PFP-G1 [Fig. 1(a)], we can find that the peakintensities of Boc (dCH3 ¼ 1.40) and NH-Boc(dN-H ¼ 4.70) groups in Boc-PPF-G1 reduced

obviously and the peak intensity of NH3þ group

(d ¼ 7.96 ppm) in PPF-G1 appeared clearly.These results indicate that the protected Bocgroups are gotten rid of entirely upon treatmentwith HCl. The PFP-G0-2 show good solubilitiesin water. Since 1H-NMR spectroscopy can beused to study polymer conformations and givesrich information about structural properties, the1H-NMR spectroscopies of the PFP-G0-2 werealso measured in D2O. Noted that the character-istic signals of these polymers in 1H-NMR spec-troscopies are broadened into the baseline inD2O (Fig. S1). This possibly results from theirtight aggregates in water, where the chains expe-rience little tumbling motion within the time-scale of the NMR experiment.3d

Optical Properties of Dendritic-ConjugatedPolymer PPF-G0-2

The cationic polymer PFP-G0-2 displayed goodsolubilities in polar solvents such as DMSO, DMF,and water. They exhibited bright blue fluorescen-ces under UV lamp. The optical properties of thePFP-G0-2 are summarized in Table 1. The meas-urements were performed in aqueous solution. Asshown in Figure 2(a), PFP-G0 and PFP-G1 ex-hibit maximum absorption at 375–379 nm, corre-sponding to the p-p* transition of the conjugatedunits. Noted that the maximum absorption ofPFP-G2 is blue-shifted to 345 nm, which resultsfrom the lower polymerization degree, Dp ¼ 4, cal-culated from GPC measurements. The PFP-G0-2

displays an absorption at 279 nm originated frombenzyl groups on the dendron, at which the ab-sorbance increases as the dendritic generationgrows. A similar result has been reported byothers in the literature.6c The extinction coeffi-cients (e) of PFP-G0-2 decrease as a function ofdendritic generation. As shown in Figure 2(b), theemission spectra of PFP-G0 and PFP-G1 inaqueous solution exhibit similar emission spectrawith maxima at 422–425 nm and shoulders at445–450 nm, which are characteristic of polyfluor-enes.12 The emission maximum of PFP-G2 is blue-shifted to 416 nm,which also results from the lowerpolymerization degree. More interestingly, the flu-orescence quantum efficiencies (QEs) of PFP-G0-2

enhance as the dendritic generation grows.To obtain better understanding of QE as a func-

tion of aggregation, we examined the aggrega-tions of PFP-G0-2 in water with varying amountsof THF using fluorescence spectroscopy. PFP-G0-2

are amphiphilic macromolecules, their backbones

Figure 1. 1H-NMR and 13C-NMR spectra of Boc-PPF-G1 (a and b) in CDCl3 and 1H-NMR spectra ofPPF-G1 in DMSO-d6 (c) at room temperature.



and alkyl side chains are hydrophobic moieties,and the charged cationic amino groups are hydro-philic ones. Thus tight aggregates of PFP-G0-2 inwater are formed by interchain hydrophobic inter-actions to result in fluorescence self-quenching.3d

As shown in Figure 3, when THF is added the QEvalues of PFP-G0-2 increase gradually because ofthe breaks of their aggregates. However, the QEvalues begin to decrease when the THF content ishigher than 40% for PFP-G0, 50% for PFP-G1,and 60% for PFP-G2. These results show thatnew aggregations are formed which are domi-

nated by the electrostatic interactions of chargedamine groups and charge compensating chlorideions. These new aggregates lead to lower emissionintensity again due to p-p interactions.3d Notedthat the QE values of PFP-G2 have changed leastrelative to those of PFP-G0,1 with the addition ofTHF. It shows that the PFP-G2 can prevent theaggregation in some extent, which possiblyresults from the higher generation dendrons andthe lower polymerization degree.

Interactions of Dendritic-Conjugated Polymerswith DNA

The interactions between dsDNA and PFP-G0-2

were investigated by probing the changes of PFP-G0-2 fluorescence intensities. As shown in Figure4(a and b), there is a substantial decrease in theemission of PFP-G0 ([PFP-G0-2] ¼ 1.0 � 10�6 M)upon adding dsDNA ([dsDNA] ¼ 0 � 1.0 �10�7 M) in phosphate buffer (50 mM, pH ¼ 7.40).Because the maxima and shapes of the fluores-cence spectra do not change and no new peaksappear, possible mechanism for the quenching ofPFP-G0 is attributed to its aggregation nearthe negatively charged dsDNA, leading to

Table 1. Summary of Molecular Weights and Optical Properties of PFP-G0-2

Polymer Mna (Da) Mw

a (Da) PDI DP UV (kmax, nm) PL (kmax, nm) e (105 M�1 cm�1) QEb (%)

PFP-G0 9,000 17,100 1.90 12 379 425 0.46 31PFP-G1 10,400 20,400 1.96 8 375 422 0.30 49PFP-G2 8,600 11,400 1.32 4 345 416 0.21 57

aThe molecular weights were measured from Boc-protected dendritic conjugated polymers using GPC with polystyrenestandards, which were a little bit less than those calculated by 1H NMR analysis.

bQEs were measured in aqueous solution with quinine sulfate as standard.

Figure 2. Absorption (a) and fluorescence (b) spec-tra of PFP-G0-2 inaqueous solution. The excitation is370 nm for PFP-G0 and PFP-G1 and 345 nm forPFP-G2.

Figure 3. Fluorescence quantum efficiency of PFP-G0-2 as a function of THF content in water.



self-quenching.3d Similar results were obtainedfor PFP-G1 and PFP-G2.

The interactions between dsDNA and PFP-G0-2

were also investigated using cationic ethidiumbromide (EB) as fluorescent probe. EB is adsDNA-specific intercalator13 and is widely usedto study the binding properties of other cationicspecies to dsDNA through competition studies.14

The stability of the dendrimer-DNA complexes isenhanced when the generation of dendrimersincreases.8,15 We can imagine that the electro-static bindings of PFP-G0-2 with dsDNA/EB com-plex would result in displacement of intercalatedEB from DNA double helix to the solutionbecause of the electrostatic repulsion accompany-ing by a quenching of EB fluorescence. Thus onecan estimate the stability of the dendrimer/DNA

complex by probing the fluorescence change ofEB. In these experiments, the dsDNA and EBwere premixed in phosphate buffer solution(50 mM, pH ¼ 7.40) ([dsDNA] ¼ 1.0 � 10�7 M,[EB] ¼ 2.0 � 10�6 M), and the PFP-G0-2 was suc-cessively added ([PFP-G0-2] ¼ 0 to 1.0 � 10�6 M)to the solution followed by fluorescence measure-ments with excitation wavelength of 510 nm. Asshown in Figure 5(a), the fluorescent intensity ofEB decreases gradually as the concentration ofPFP-G2 or molar ratio of amine (on PFP-G0-2) tophosphate (on dsDNA) increases. Similar resultswere observed for PFP-G0 and PFP-G1. It isnoted that the intensity of dsDNA/EB emissiondecreases greatly on adding PFP-G2 than addingPFP-G0 and PFP-G1 [Fig. 5(b, c)].

The electrophoresis analysis supported theresults of fluorescence experiments. As shown inFigure 6, the dsDNA itself moves as a clear singleband noted as line 4. Similar band can be seen inlane 3 for the PFP-G0/dsDNA complex, just theband intensity is weaker relative to the lane 4.For PFP-G1/dsDNA complex (lane 2), a weakband can been seen. For PFP-G2/dsDNA complex,no band was clearly observed in lane 1. The exper-imental results indicate that the charge densitiesof PFP-G0-2 can control the stabilization ofthe dsDNA/PFP-G0-2 complexes by electrostaticinteractions. The PFP-G2 with highest genera-tion of dendritic side chains possesses a highestcharge density and can form most stable complexwith dsDNA.

CONCLUSIONS

In summary, a new series of cationic water-solu-ble dendritic-conjugated polyfluorenes (PFP-G0-2)were synthesized and characterized. The PFP-G0-2

exhibit good QEs in water media. More interest-ingly, the QEs of PFP-G0-2 enhance as the dendri-tic generation grows because higher generationdendrons prevent the formation of p-stackingaggregates between conjugated polymer back-bones. Studies on the interactions betweendsDNA and PFP-G0-2 using ethidium bromide asfluorescent probe show that the electrostatic bind-ings of PFP-G0-2 with dsDNA/EB complex resultin displacement of EB from DNA double helix tothe solution. The PFP-G2 with highest generationof dendritic side chains forms most stable complexwith dsDNA. These water-soluble dendritic-conju-gated polyfluorenes may prove useful in biosensorapplication or probing gene delivery.

Figure 4. (a) Emission spectra of PFP-G0 in thepresence of dsDNA. (b) Normalized emission intensityof PFP-G0 as a function of dsDNA concentration. kex¼ 345 nm, [PFP-G0] ¼ 1.0 � 10�6 M, [dsDNA] ¼ 0 to1.0 � 10�7 M. Measurements are in phosphate buffersolution (50 mM, pH ¼ 7.40).



The authors are grateful for the financial support fromthe ‘‘100 Talents’’ program of Chinese Academy of Scien-ces, the National Natural Science Foundation of China(20725308, 20721061, 20574073) and the NationalHigh-Tech R andDProgram (No. 2006AA02Z130).

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Figure 5. (a) Emission spectra of dsDNA/EB as afunction of PFP-G2 concentration. (b) Normalizedemission intensity of dsDNA/EB as functions of PFP-G0-2 concentrations. [dsDNA] ¼ 1.0 � 10�7M, [EB] ¼2.0 � 10�6 M, [PFP-G0-2] ¼ 0 to 1.0 � 10�6 M. (c)Normalized emission intensity of dsDNA/EB as func-tions of molar ratio of amine (on PFP-G0-2) to phos-phate (on dsDNA). Measurements were performed inpotassium phosphate-sodium hydroxide buffer solu-tion (50 mM, pH ¼ 7.40). The excitation wavelengthis 510 nm.



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Documents

Water-soluble dendritic-conjugated polyfluorenes: Synthesis, characterization, and interactions with DNA