Multidentate Comb-Shaped Polypeptides Bearing ... - ACS Publications

Subscriber access provided by University of Colorado Boulder

Article

Multidentate Comb-shape Polypeptides Bearing Trithiocarbonate Functionality: Synthesis and Application for Water-Soluble Quantum Dots Hang Zhang, Jinlong Chen, Xiaojie Zhang, Chunsheng Xiao, Xuesi Chen, Youhua Tao, and Xianhong Wang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01760 • Publication Date (Web): 31 Jan 2017 Downloaded from http://pubs.acs.org on February 1, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Multidentate

Comb-shape

Polypeptides

Bearing

Trithiocarbonate Functionality: Synthesis and Application for Water-Soluble Quantum Dots Hang Zhang,†,# Jinlong Chen,† Xiaojie Zhang,† Chunsheng Xiao,† Xuesi Chen,† Youhua Tao,*,† Xianhong Wang*,† †

Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Renmin Street 5625, Changchun 130022, People’s Republic of China. #

University of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China.

ABSTRACT We describe here the synthesis of multidentate comb-shape polypeptides bearing trithiocarbonate functionality and their application in the preparation of water-soluble quantum dots (QDs). A new L-lysine-based N-carboxyanhydride monomer containing trithiocarbonate functionality was designed and synthesized. Ring-opening polymerization of the resulting monomer initiated by hexamethyldisilazane, afford polypeptides bearing pendent trithiocarbonate groups (P(TTCLys)) with controllable molecular weights. P(TTCLys) was then applied to mediate the RAFT polymerization of oligo(ethylene glycol)acrylate for the metal-free preparation of hydrophilic comb-shape polypeptides. Simple reduction of trithiocarbonate functionality enabled the introduction of multiple thiol anchoring groups to the above-mentioned comb-shape polypeptides. Finally, the obtained multidentate polypeptide-based ligands were successfully applied in the ligand exchange procedures to generate water-soluble QDs. The fluorescent microscopic images suggested that the resultant water-soluble QDs could be effectively internalized into HeLa cells to realize bright cellular imaging. Therefore, our work can result in a new kind of valuable polypeptide-based QDs with hydrophilic character and biocompatibility for cellular imaging.

ACS Paragon Plus Environment

1

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 24

INTRODUCTION Polypeptides from the ring-opening polymerization (ROP) of α-amino acid Ncarboxyanhydrides (NCAs) are important biomaterials for their special properties, e.g., stimuliresponsiveness, defined secondary structures, and so on.1-10 Among which, side-chain functionalized polypeptides are particularly interesting, since they can further conjugate with bioactive molecules, leading to structural and functional mimics of natural peptides and facilitating their applications in biomedical research.11-14 Side-chain functionalized polypeptides have emerged as one of the most popular topics in the polypeptides field in recent years. Hammond et al. developed the first example of the NCA monomer having a “clickable” propargyl group.15 This monomer can undergo the efficient click reaction to introduce PEG, sugar16-18 or polyhedral oligomeric silsesquioxane (POSS) groups19 into side-chain. Deming prepared polypeptides with azide pendants by living polymerization of new azide-containing NCA monomers.20 Zhang reported the synthesis of polypeptides bearing chloro- or azido- functional groups that were amendable to click chemistry.21 Cheng and Schlaad synthesized polypeptides with allyl-22-25 or vinyl benzyl-26groups and these side-chains can be further derivatized by thiol–ene addition reactions. And Cheng also prepared brush-like polymers bearing polypeptides as the side chains through combination of ROMP and NCA.27 Li developed polypeptides bearing ATRP initiator functionality and demonstrated efficient preparation of bottlebrushes with a poly(α-lysine) backbone via combination of NCA and ATRP.28 Despite these remarkable efforts, probably due to the inherent side reactions, the synthesis of polypeptides bearing trithiocarbonate functionality still remains a challenge and further exploration on the preparation of comb-shape polypeptides through NCA polymerization and RAFT is also highly desired. Indeed, marriage of NCA polymerization and RAFT offers a


2

Page 3 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

feasible route for the metal-free preparation of functional polypeptides, such process is considered to be crucial for biomedical applications of polypeptides because the removal of toxic metal residues is unnecessary. Quantum dots (QDs) have attracted a great deal of attention because of their promising applications in the biomedical fields as fluorescence imaging reagents.29-32 For biomedical applications, it is important to prepare QDs with water-solubility and cell compatibility. However, hydrophobic compounds (i.e., trioctylphosphine oxide) are usually added to generate the outer layer during the QDs synthesis process. Therefore, the resultant QDs possess poor water dispersibility.33,34 To solve this problem, many approaches have been adopted to improve the hydrophilic character and biocompatibility of QDs. Ligand replacement with hydrophilic ligands, is one of the most extensive phase transfer techniques for water-soluble QDs.35-40 For example, a series of multidentate hydrophilic polymer ligands containing multiple thiol groups as terminal groups were synthesized for water-soluble QDs.41 Mattoussi reported on the synthesis of dihydrolipoic acid derivatives appended with poly(ethylene glycols) (PEG) of various lengths to generate hydrophilic and biocompatible QDs.42 Ishihara reported the synthesis of watersoluble phospholipid polymer for modifying QDs with outstanding cytocompatibility.43 Recently, Zentel and Barz synthesized heterotelechelic polysarcosine with di- or trilipoic acid-based anchoring groups and demonstrated their use as multidentate ligands in the preparation of stable water-soluble QDs.44 In this contribution, we report the synthesis of trithiocarbonate functionalized L-lysine Ncarboxyanhydride

(TTC-LysNCA)

and

its

controlled

ROP

to

produce

P(TTCLys)

homopolypeptides (Scheme 1). P(TTCLys) was then applied to mediate the RAFT polymerization of OEGA for the metal-free preparation of hydrophilic trithiocarbonate


3

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 24

functionalized comb-shape polypeptides (P(TTCLys)-g-POEGA). Moreover, we envisioned that simple reduction of trithiocarbonate functionality would lead to hydrophilic comb-shape polypeptides with multiple thiol anchoring groups that were susceptible to ligand exchange with hydrophobic QDs, and thereby water-soluble QDs were obtained. The biocompatible nature of the comb-shape polypeptides and their composition can be manipulated by NCA polymerization and RAFT, providing a great opportunity for tuning QDs functions. To the best of our knowledge, this is the first report of multidentate comb-shape polypeptides for application of water-soluble and cytocompatible QDs. Scheme 1. Metal-free Synthesis of P(TTCLys) and P(TTCLys)-g-POEGA through marriage of NCA Polymerization and RAFT

EXPERIMENTAL SECTION Materials and Methods. The following chemicals were used as received without further purification unless otherwise stated. Tetrahydrofuran (THF) was refluxed with sodium and distilled under N2. Ethyl acetate (EtOAc), hexane, dimethyl formamide (DMF), methanol, ethanol (EtOH), chloroform (CHCl3), dichloromethane (DCM) and petroleum ether (PE) were dried

with

molecular

sieve.

1-Dodecanethiol

(98%,

Hydroxysuccinimide(98%,Chemlin),N-alpha-(tert-Butoxycarbonyl)-L-lysine


Aladdin),N(98%,

Energy 4

Page 5 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Chemical), Trifluoroacetic acid (TFA, 99%, Aladdin), Triphosgene (98%, Energy Chemical), Hexamethyldisilazane (HMDS, 99.9%, Sigma-Aldrich), Triethylene glycol monomethyl ether (96%, Energy Chemical) and Acrylyl chloride (98%, Energy Chemical) were used as received. The hydrodynamic diameter of the water-dispersion QDs were determined by the dynamic light scattering (Malvern Zeta-sizer Nano). Circular dichroism spectra were measured on a JASCO J-820 spectrometer. The FT-IR spectra were performed on a Bruker Tensor-27 spectrometer.

UV-Vis

absorption

spectra

were

conducted

on

Shimadzu

UV-2450

spectrophotometer. Fluorescence emission spectra were measured on a PerkinElmer LS-55 phosphorescence spectrophotometer. Absolute photo-luminescence quantum yield values as measured by the HAMAMATSU C9920-02 Absolute Quantum Yield Measurement systems. The Ring-opening polymerization of TTC-LysNCA In a glove box, TTC-LysNCA monomer (86 mg) was dissolved in anhydrous THF (1 mL). Initiator HMDS in anhydrous THF in appropriate amount were added to the NCA solution at room temperature. The reaction was monitored by FTIR and, upon complete consumption of the monomer. The product was precipitated using excess petroleum ether and collected via centrifugation. P(TTCLys) as a yellow solid was obtained in ~80% yield. 1H NMR (400M, CDCl3/CF3COOD 5/1, ppm): δ 4.45 (NHCHC(=O)), 3.27 (C11H23CH2SC(=S)S), 3.19 (CHCH2CH2CH2CH2NH),

1.57-2.04

(CHCH2CH2CH2CH2NH,

C(=O)C(CH3)2SC(=S)S,

C10H21CH2CH2S), 1.44-1.57 (CHCH2CH2CH2CH2NH), 1.19-1.44 (CHCH2CH2CH2CH2NH, SCH2CH2C9H18CH3), 0.88 (SCH2CH2C9H18CH3). The RAFT polymerization of OEGA OEGA (in appropriate amount), CTA of PTTCLys (44mg, 0.091mmol of the repeating unit), AIBN (1.5mg, 0.0091mmol) and THF were placed in a dry glass Schlenk flask. The solution was


5

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 24

degassed by three times freeze-pump-thaw cycles and the flask was immersed into preheated oil bath at 65°C for 24 h. The flask was then cooled in liquid N2 and exposure to air. The polymer was precipitated using excess diethyl ether and collected via centrifugation. P(TTCLys)-gPOEGA as a pale yellow solid was obtained. (For P(TTCLys)20-g-POEGA11, P(TTCLys)20-gPOEGA26, P(TTCLys)20-g-POEGA34 and P(TTCLys)60-g-POEGA9, the yields were 68%, 60%, 49% and 46%, respectively ). 1H NMR (400M, CDCl3, ppm): δ 4.04-4.37 (C(=O)OCH2CH2O), 3.89

(NHCHC(=O)),

3.51-3.76

(C(=O)OCH2CH2O(CH2CH2O)2OCH3), SC(=S)S),

2.24-2.49

CHCH2CH2CH2CH2NH), CHCH2CH2CH2CH2NH),

(C(=O)OCH2CH2O(CH2CH2O)2OCH3), 2.91-3.22

(CHCH2CH2CH2CH2NH,

(CH2CH(C=O)CH2), 1.61-1.86 1.02-1.61

1.86-2.04

3.39

C11H23CH2

(CH2CH(C=O)CH2,

(C(=O)C(CH3)2SC(=S)S),C10H21CH2CH2S,

(CHCH2CH2CH2CH2NH,SCH2CH2C9H18CH3),

0.896

(SCH2CH2C9H18CH3). Reduction of Trithiocarbonate Functionalized Polypeptides for Water-soluble QDs. To the solution of the P(TTCLys)20-g-POEGA34 (295 mg) in methanol (50 mL) was added slowly NaBH4 methanol solution (2.4 mL, 1M) under the nitrogen atmosphere. And reaction was stirred for 72 h at 25 oC. The polymer solution was dialyzed in H2O for 3 days to afford thio functionalized polymer (240 mg, 81 % yield). 1H NMR (400M, CDCl3, ppm): δ 3.99-4.40 (C(=O)OCH2CH2O), 3.78 (NHCHC(=O)), 3.48-3.73 (C(=O)OCH2CH2O(CH2CH2O)2OCH3), 3.39 (C(=O)OCH2CH2O(CH2CH2O)2OCH3), 3.16-3.26 (CHCH2CH2CH2CH2NH,), 2.23-2.48 (CH2CH(C=O)CH2), 1.78-2.04 (CHCH2CH2CH2CH2NH), 1.41-1.78 (CH2CH(C=O)CH2), 1.261.41 (NHC(=O)C(CH3)2CH2CH, CHCH2CH2CH2CH2NH).


6

Page 7 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

RESULTS AND DISCUSSION

Monomer Synthesis. As shown in Scheme 2, TTC-LysNCA was synthesized within four steps. Briefly, RAFT agent 1 was used as starting material, as similar compound was already synthesized in the literature.45 1 was activated with N-Hydroxysuccinimide (NHS) to generate 2, which underwent amidation reaction with Nα-Boc-L-lysine at 0 oC to afford 3. Direct cyclization of 3 using triphosgene failed to yield the corresponding NCA. Therefore, the Boc group was removed with trifluoroacetic acid (TFA) to afford the corresponding 4. To keep the integrity of trithiocarbonate groups, it is critical to undergo deprotection reaction at 0 oC for 1h. Crude TTCLysNCA monomer was then obtained by cyclization of 4 with triphosgene in THF. TTC-LysNCA was purified by a modified procedure reported by Deming et al.46 The monomer was fully characterized by 1H and 13C NMR (Figure S4), and mass spectrometry (Figure S5). Scheme 2. Synthesis of the trithiocarbonate functionalized L-lysine N-carboxyanhydride (TTCLysNCA) monomera

a

Reagents and conditions: (i) EDCI, NHS, DCM, 0 °C to rt, overnight; (ii) Nα-Boc-L-lysine, NaHCO3, THF/H2O , 0 °C, 24 h; (iii) TFA, CH2Cl2, 0 °C, 1h; (iv) C3Cl6O3, THF, 50 °C, 3 h. NCA Polymerization. Due to the susceptibility of trithiocarbonate group to free amine groups, it is unsuitable to use primary and secondary amines as initiators for ROP of TTC-


7

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 24

LysNCA. As a consequence of the potential bio-application of our resultant polypeptides, we employed hexamethyldisilazane (HMDS) 47 to mediate the ROP of TTC-LysNCA at 25 oC in THF at different monomer-to-initiator (M/I) ratios. As shown in Table 1, the polymerization all reached over 98% NCA conversion, as determined by FT-IR. Upon increasing M/I from 20/1 to 80/1, we obtained homopolypeptide P(TTCLys) with linearly increased Mn and molecularweight distributions (Ð) ranging from 1.4 to 1.5, as determined by SEC using PS as standards (Figure 1 and Table 1). Meanwhile, the obtained Mn of P(TTCLys) was close to the calculated Mn. These results indicated that the ROP of TTC-LysNCA initiated by HDMS were executed in a controlled manner. Figure 2A showed the corresponding 1H NMR spectrum of the P(TTCLys) homopolypeptides. The protons of the methine group of the polypeptide main chain displayed resonance at 4.45 ppm. The signals at 3.27 ppm (CH2CH2SC(=S)S) in the 1H NMR spectrum corroborated the existence of trithiocarbonate pendants on the homopolypeptides. We have thus demonstrated that ROP of TTC-LysNCA with HDMS provided an effective approach toward polypeptides with pendent trithiocarbonate groups. Table 1. Ring-opening polymerizations of TTC-LysNCAa Entry

Polymers

[M]/[I]

Mn,SEC (kg/mol)d

Ðd

(%)b

Mn,calc. (kg/mol)c

Conv.

1

P(TTCLys)20

20

>98

9.7

9.8

1.4

2

P(TTCLys)40

40

>98

19.6

16.7

1.5

3

P(TTCLys)60

60

>98

28.4

25.6

1.4

4

P(TTCLys)80

80

>98

37.9

42.0

1.5

a

Polymerizations were performed in THF at room temperature for 72h. bDetermined by FT-IR. Mn,calc.=M(monomer) × [M]0/([initiator]) × Conversion (monomer). dDetermined by SEC in THF, PS as standard.

c


8

Page 9 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 1. Plots of Mn and Mw/Mn of P(TTCLys) as a function of M/I in the HDMS mediated polymerization Synthesis of the Trithiocarbonate Functionalized Comb-shape Polypeptides. Due to the incompatibility of amino groups applied for NCA initiation and trithiocarbonate groups for RAFT polymerization, there is minimal work reported marriage of NCA polymerization and RAFT. Here, P(TTCLys) bearing trithiocarbonate side chains was employed to mediate the RAFT polymerization of OEGA to synthesize water-soluble comb-shape polypeptides (Scheme 1).


9

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 24

Figure 2. 1H NMR spectra of P(TTCLys) in CF3COOD + CDCl3 (5/1, v/v) (A), and P(TTCLys)g-POEGA in CDCl3 (B). The polymerization was carried out in THF at 65 oC, using AIBN as the initiator. The P(TTCLys)20 and P(TTCLys)60 were applied as macro-transfer agents. The polymerization results are summarized in Table 2. Figure 2B displayed the typical 1H NMR spectrum of P(TTCLys)-gPOEGA. The grafting of OEGA to P(TTCLys) was successful, with a new resonance (δ = 3.39 ppm) appearing, attributed to methyl groups of OEGA side chain. Due to the insolubility of the homopolypeptides in DMF, the Mn and Mw/Mn of the obtained homopolypeptides and combshape polypeptides was measured with different SEC systems (THF for P(TTCLys), DMF for P(TTCLys)-g-POEGA). SEC trace of P(TTCLys)20-g-POEGA34 showed a symmetric single peak (Figure S6), suggesting the successful synthesis of the comb-shape polypeptides with moderate control (Ð ≤ 1.7) through marriage of NCA polymerization and RAFT. In addition, though


10

Page 11 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

P(TTCLys) mediated RAFT polymerization of OEGA produced comb-shape polypeptides with moderate control, there still existed dead side chains due to the increasing local concentration of radical species, which may be responsible for the lower conversion with higher [M]/[CTA] ratio (Table 2). Table 2. Synthesis of P(TTCLys)-g-POEGAa Entry

Polymers

[M]0/[CTA]/

Conv.

Mn,NMR.

Mn,SEC

[AIBN]

(%)b

(kg/mol)c

(kg/mol)d

Ðd

1

P(TTCLys)20-g-POEGA11

15:1:0.1

73

59

55

1.5

2


40:1:0.1

66

128

77

1.7

3


60:1:0.1

57

164

102

1.4

4


15:1:0.1

60

129

98

1.7

a

Polymerizations are performed in THF at 65oC for 24h. b Determined by 1H NMR analysis. Mn,NMR = [M(monomer) × DPP(TTCLys) ×3I4.04-4.37/2I0.90] + M(macro-transfer agent). d Determined by SEC in DMF (0.01M LiBr), polystyrene as standard. c

The secondary structure of P(TTCLys), and P(TTCLys)-g-POEGA were measured by circular dichroism (CD) and FI-IR. P(TTCLys) homopolypeptides showed strong Cotton effect with two negative peaks at 222 and 207 nm (Figure 3A), a signature for the α-helix conformation of peptide. The a-helix content was about 84% in THF. Meanwhile, the CD spectrum of P(TTCLys)-g-POEGA exhibited two negative dichroic bands at 222 and 207 nm, which were signals of α-helical conformation. For P(TTCLys)20-g-POEGA34, the helicity was estimated to be 37% in water. We also used FT-IR to study the conformation of P(TTCLys) and P(TTCLys)-g-POEGA (Figure 3B). α-helical conformation was confirmed, with two absorptions (1653 and 1547 cm − 1, respectively) appearing, attributed to amide I and the N–H amide II.


11

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 24

Figure 3. (A) CD spectra of P(TTCLys)20 in THF (0.1 mg/mL) and P(TTCLys)20-g-POEGA34 in water (1 mg/mL). (B) FTIR spectra of P(TTCLys)20 and P(TTCLys)20-g-POEGA34. Reduction of Trithiocarbonate Functionalized Polypeptides for Water-soluble QDs. A notable feature of the multidentate comb-shape polypeptides that are obtained through marriage of NCA polymerization and RAFT is the presence of trithiocarbonate groups in the side chains. Simple reduction of this functionality should lead to polypeptides with multiple thiol anchoring groups, which are available for hydrophilic ligand for water-soluble QDs (Figure 4). We investigated the reduction reaction of P(TTCLys)-g-POEGA by NaBH4. The reactions were conducted in methanol under nitrogen atmosphere. The complete consumption of the characteristic trithiocarbonate absorbance peak at 316 nm was observed within 72 h (Figure S8).


12

Page 13 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Hydrophobic CdSe−ZnS Core−Shell QDs coated with trioctylphosphine oxide (TOPO) were prepared following a literature method.48 The ligand replacement technology was then carried out to obtain water-soluble QDs. P(TTCLys)20-g-POEGA34 polypeptide-based ligands (20 mg) and QDs (1 mg) were suspended in EtOH (1.5 mL) and stirred for 15 h at 65 oC. Afterward, EtOH evaporated, and the residue was suspended in hexane/CHCl3/EtOH (10:1:1, v/v/v, 30 mL). The supernatant was removed after centrifugation. The residue was then dispersed

Figure 4. Illustration on the reduction of trithiocarbonate functionalized polypeptides and preparation of water-soluble QDs. The inset showed the fluorescence image of the QDs capped with P(TTCLys)-g-POEGA in aqueous solution. in H2O, and the excess polypeptide-based ligands were removed by repeated centrifugation with Amicon Ultra centrifugal filters (50 kDa MWCO). FI-IR spectra (Figure S9) showed that characteristic bands of TOPO were disappeared, while new bands at 1650 cm−1 (attributed to amide I) and at 1541 cm−1 (attributed to amide II) were clearly observed, suggesting that thiol groups of the polypeptide-based ligands can successfully replace the hydrophobic outer layer of the QDs. The water-dispersion QDs retained the optical properties of the original QDs (Figure 5). We then determined the stability of water-soluble QDs via turbidity buildup and changes in the fluorescent intensity by collecting the emission spectra.40,49 And results showed that QDs


13

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 24

coated with P(TTCLys)20-g-POEGA34 ligands were stable in NaCl solutions (1 M) as well as in aqueous solution with pH ranging from 4.5-9.0. The photoluminescence quantum yield of hydrophilic QDs in water was decreased to 0.22, compared to 0.42 of the original QDs with TOPO ligands in chloroform. Similar phenomenon was also observed in the previously reported work.40,44,50 In addition, the hydrodynamic diameter of the water-dispersion QDs was about 56 nm, as determined by the dynamic light scattering measurements (Figure S11).

Figure 5. Absorption (0.5μM) and emission (0.5μM) spectra of initial hydrophobic QDs in chloroform, and the corresponding QDs coating with comb-shape polypeptide ligands in water.


14

Page 15 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 6. (A) Viability of HeLa cells by the MTT assay following treatment with the QDs coated with polypeptides for 48 h. The experiment was repeated for three times. (B) Fluorescent microscopy images of HeLa cells treated with 0.1 mg mL−1 QDs for 48 h. Cytotoxicity and Cellular Imaging. With an eye towards biological applications, we tested whether water-soluble QDs coated with polypeptide-based ligands showed any potentially cytotoxic effects using HeLa cells. As shown in Figure 6A, No obvious toxicity was obtained from QDs treated HeLa cells at concentrations up to 750 µg mL−1 for 48 h incubation, indicating the QDs coated with comb-shape polypeptides have good cytocompatibility and bear the potential for applications in biological imaging. Next, we examined the suitability of the QDs for biological imaging by fluorescent microscopy. The fluorescent microscopic images shown in Figure 6B indicated that water-soluble QDs could enter HeLa cells and exhibited clear green fluorescence in the cells. Thus, this class of water-soluble QDs coated with multidentate combshape polypeptides could be excellent candidates for cellular imaging.


15

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 24

CONCLUSION In conclusion, we have reported the metal-free synthesis of trithiocarbonate functionalized comb-shape polypeptides with controllable molecular weights through marriage of NCA polymerization and RAFT. The key element of our approach is to prepare trithiocarbonate functionalized L-lysine NCA monomer. The synthetic methodology developed here is very important toward biomedical applications of polypeptides. Moreover, the trithiocarbonate groups were reducted to introduce multiple thiol anchoring groups to the comb-shape polypeptides. The obtained hydrophilic polypeptide-based ligands are successfully applied in the ligand exchange procedures to generate water-soluble QDs. The fluorescent microscopic images suggested that the above-mentioned water-soluble QDs could be effectively internalized in HeLa cells and could be excellent candidates for cellular imaging. Indeed, this work not only offers a feasible route for the metal-free synthesis of multidentate comb-shape polypeptides bearing trithiocarbonate functionality but also afford us novel polypeptide-based ligands able to impose hydrophilic character and biocompatibility on QDs. ASSOCIATED CONTENT Supporting Information. Experimental details and characterization data. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. * E-mail: [email protected]


16

Page 17 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work is supported by “The Hundred Talents Program” from the Chinese Academy of Sciences, the National Natural Science Foundation of China (Grant Nos. 21474101 and 51673192), Jilin Science and Technology Bureau (Grant No. 20160414001GH). REFERENCES (1) Huang, J.; Heise, A. Stimuli responsive synthetic polypeptides derived from Ncarboxyanhydride (NCA) polymerisation. Chem. Soc. Rev. 2013, 42, 7373-7390. (2) Tao, Y.-h. New Polymerization Methodology of Amino Acid Based on Lactam Polymerization. Acta Polymerica Sinica 2016, 1151-1159. (3) Wang, K.; Liu, Y.; Li, C.; Cheng, S.-X.; Zhuo, R.-X.; Zhang, X.-Z. Cyclodextrin-Responsive Micelles Based on Poly(ethylene glycol)–Polypeptide Hybrid Copolymers as Drug Carriers. ACS Macro Lett. 2013, 2, 201-205. (4) Liu, G.; Dong, C.-M. Photoresponsive Poly(S-(o-nitrobenzyl)-l-cysteine)-b-PEO from a lCysteine N-Carboxyanhydride Monomer: Synthesis, Self-Assembly, and Phototriggered Drug Release. Biomacromolecules 2012, 13, 1573-1583. (5) Lai, H.; Chen, X.; Lu, Q.; Bian, Z.; Tao, Y.; Wang, X. A new strategy to synthesize bottlebrushes with a helical polyglutamate backbone via N-carboxyanhydride polymerization and RAFT. Chem Commun (Camb) 2014, 50, 14183-14186.


17

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 24

(6) Yuan, J.; Sun, Y.; Wang, J.; Lu, H. Phenyl Trimethylsilyl Sulfide-Mediated Controlled RingOpening Polymerization of α-Amino Acid N-Carboxyanhydrides. Biomacromolecules 2016, 17, 891-896. (7) Zhao, W.; Gnanou, Y.; Hadjichristidis, N. Well-defined (co)polypeptides bearing pendant alkyne groups. Polymer Chemistry 2016, 7, 3487-3491. (8) Deming, T. J. Synthesis of Side-Chain Modified Polypeptides. Chem. Rev. 2016, 116, 786808. (9) Yan, J.; Liu, K.; Li, W.; Shi, H.; Zhang, A. Thermoresponsive Dendronized Polypeptides Showing Switchable Recognition to Catechols. Macromolecules 2016, 49, 510-517. (10) Wang, J.; Gibson, M. I.; Barbey, R.; Xiao, S. J.; Klok, H. A. Nonfouling Polypeptide Brushes via Surface-initiated Polymerization of N(epsilon) -oligo(ethylene glycol)succinate-Llysine N-carboxyanhydride. Macromol Rapid Commun 2009, 30, 845-850. (11) Jiang, Y.; Wang, S.; Zhang, X.; Tao, Y.; Wang, X. Controlled metal-free polymerization toward well-defined thermoresponsive polypeptides by polymerization at low temperature. Journal of Polymer Science Part A: Polymer Chemistry 2016, 54, 2618-2624. (12) Tao, Y.; Chen, X.; Jia, F.; Wang, S.; Xiao, C.; Cui, F.; Li, Y.; Bian, Z.; Chen, X.; Wang, X. New chemosynthetic route to linear ε-poly-lysine. Chemical Science 2015, 6, 6385-6391. (13) Zhang, X.; Wang, S.; Liu, J.; Xie, Z.; Luan, S.; Xiao, C.; Tao, Y.; Wang, X. Ugi Reaction of Natural Amino Acids: A General Route toward Facile Synthesis of Polypeptoids for Bioapplications. ACS Macro Letters 2016, 5, 1049-1054.


18

Page 19 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(14)

Li, M.; Cui, F.; Li, Y.; Tao, Y.; Wang, X. Crystalline Regio-/Stereoregular Glycine-

Bearing Polymers from ROMP: Effect of Microstructures on Materials Performances. Macromolecules 2016, 49, 9415-9424. (15) Engler, A. C.; Lee, H. I.; Hammond, P. T. Highly efficient "grafting onto" a polypeptide backbone using click chemistry. Angew. Chem. Int. Ed. 2009, 48, 9334-9338. (16) Xiao, C.; Zhao, C.; He, P.; Tang, Z.; Chen, X.; Jing, X. Facile Synthesis of Glycopolypeptides by Combination of Ring-Opening Polymerization of an Alkyne-Substituted N-carboxyanhydride and Click “Glycosylation”. Macromolecular Rapid Communications 2010, 31, 991-997. (17) Krannig, K.-S.; Huang, J.; Heise, A.; Schlaad, H. Photochemical thiol-yne functionalization of polypeptide scaffolds. Polymer Chemistry 2013, 4, 3981-3986. (18) Bonduelle, C.; Huang, J.; Ibarboure, E.; Heise, A.; Lecommandoux, S. Synthesis and selfassembly of "tree-like" amphiphilic glycopolypeptides. Chem. Commun. 2012, 48, 8353-8355. (19) Lin, Y.-C.; Kuo, S.-W. Hierarchical self-assembly and secondary structures of linear polypeptides graft onto POSS in the side chain through click chemistry. Polymer Chemistry 2012, 3, 162-171. (20) Rhodes, A. J.; Deming, T. J. Soluble, Clickable Polypeptides from Azide-ContainingNCarboxyanhydride Monomers. ACS Macro Lett. 2013, 2, 351-354. (21) Tang, H.; Zhang, D. General Route toward Side-Chain-Functionalized α-Helical Polypeptides. Biomacromolecules 2010, 11, 1585-1592.


19

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 24

(22) Sun, J.; Schlaad, H. Thiol−Ene Clickable Polypeptides. Macromolecules 2010, 43, 44454448. (23) Krannig, K.-S.; Doriti, A.; Schlaad, H. Facilitated Synthesis of Heterofunctional Glycopolypeptides. Macromolecules 2014, 47, 2536-2539. (24) Krannig, K.-S.; Schlaad, H. pH-Responsive Bioactive Glycopolypeptides with Enhanced Helicity and Solubility in Aqueous Solution. J. Am. Chem. Soc. 2012, 134, 18542-18545. (25) Robinson, J. W.; Schlaad, H. A versatile polypeptoid platform based on N-allyl glycine. Chem. Commun. 2012, 48, 7835-7837. (26) Lu, H.; Bai, Y.; Wang, J.; Gabrielson, N. P.; Wang, F.; Lin, Y.; Cheng, J. Ring-Opening Polymerization of gamma-(4-Vinylbenzyl)-(L)-Glutamate N-Carboxyanhydride for the Synthesis of Functional Polypeptides. Macromolecules 2011, 44, 6237-6240. (27) Lu, H.; Wang, J.; Lin, Y.; Cheng, J. One-Pot Synthesis of Brush-Like Polymers via Integrated Ring-Opening Metathesis Polymerization and Polymerization of Amino Acid NCarboxyanhydrides. J. Am. Chem. Soc. 2009, 131, 13582-13583. (28) Liu, Y.; Chen, P.; Li, Z. Molecular bottlebrushes with polypeptide backbone prepared via ring-opening polymerization of NCA and ATRP. Macromol Rapid Commun 2012, 33, 287-295. (29) Zhou, J.; Yang, Y.; Zhang, C. Y. Toward Biocompatible Semiconductor Quantum Dots: From Biosynthesis and Bioconjugation to Biomedical Application. Chem. Rev. 2015, 115, 11669-11717.


20

Page 21 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(30) Palui, G.; Aldeek, F.; Wang, W.; Mattoussi, H. Strategies for interfacing inorganic nanocrystals with biological systems based on polymer-coating. Chem. Soc. Rev. 2015, 44, 193227. (31) Liu, W.; Howarth, M.; Greytak, A. B.; Zheng, Y.; Nocera, D. G.; Ting, A. Y.; Bawendi, M. G. Compact Biocompatible Quantum Dots Functionalized for Cellular Imaging. J. Am. Chem. Soc. 2008, 130, 1274-1284. (32) Liu, W.; Choi, H. S.; Zimmer, J. P.; Tanaka, E.; Frangioni, J. V.; Bawendi, M. Compact Cysteine-Coated CdSe(ZnCdS) Quantum Dots for in Vivo Applications. J. Am. Chem. Soc. 2007, 129, 14530-14531. (33) Murray, C. B.; Norris, D. J.; Bawendi, M. G. Synthesis and characterization of nearly monodisperse CdE (E = sulfur, selenium, tellurium) semiconductor nanocrystallites. J. Am. Chem. Soc. 1993, 115, 8706-8715. (34) Peng, Z. A.; Peng, X. Formation of High-Quality CdTe, CdSe, and CdS Nanocrystals Using CdO as Precursor. J. Am. Chem. Soc. 2001, 123, 183-184. (35) Susumu, K.; Uyeda, H. T.; Medintz, I. L.; Pons, T.; Delehanty, J. B.; Mattoussi, H. Enhancing the Stability and Biological Functionalities of Quantum Dots via Compact Multifunctional Ligands. J. Am. Chem. Soc. 2007, 129, 13987-13996. (36) Liu, W.; Greytak, A. B.; Lee, J.; Wong, C. R.; Park, J.; Marshall, L. F.; Jiang, W.; Curtin, P. N.; Ting, A. Y.; Nocera, D. G.; Fukumura, D.; Jain, R. K.; Bawendi, M. G. Compact Biocompatible Quantum Dots via RAFT-Mediated Synthesis of Imidazole-Based Random Copolymer Ligand. J. Am. Chem. Soc. 2010, 132, 472-483.


21

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 24

(37) Palui, G.; Na, H. B.; Mattoussi, H. Poly(ethylene glycol)-based multidentate oligomers for biocompatible semiconductor and gold nanocrystals. Langmuir 2012, 28, 2761-2772. (38) Yildiz, I.; Deniz, E.; McCaughan, B.; Cruickshank, S. F.; Callan, J. F.; Raymo, F. M. Hydrophilic CdSe−ZnS Core−Shell Quantum Dots with Reactive Functional Groups on Their Surface. Langmuir 2010, 26, 11503-11511. (39) Giovanelli, E.; Muro, E.; Sitbon, G.; Hanafi, M.; Pons, T.; Dubertret, B.; Lequeux, N. Highly Enhanced Affinity of Multidentate versus Bidentate Zwitterionic Ligands for Long-Term Quantum Dot Bioimaging. Langmuir 2012, 28, 15177-15184. (40) Zhan, N.; Palui, G.; Safi, M.; Ji, X.; Mattoussi, H. Multidentate zwitterionic ligands provide compact and highly biocompatible quantum dots. J. Am. Chem. Soc. 2013, 135, 13786-13795. (41) Yildiz, I.; McCaughan, B.; Cruickshank, S. F.; Callan, J. F.; Raymo, F. M. Biocompatible CdSe-ZnS core-shell quantum dots coated with hydrophilic polythiols. Langmuir 2009, 25, 7090-7096. (42) Uyeda, H. T.; Medintz, I. L.; Jaiswal, J. K.; Simon, S. M.; Mattoussi, H. Synthesis of Compact Multidentate Ligands to Prepare Stable Hydrophilic Quantum Dot Fluorophores. J. Am. Chem. Soc. 2005, 127, 3870-3878. (43) Liu, Y.; Oda, H.; Inoue, Y.; Ishihara, K. Movement of a quantum dot covered with cytocompatible and pH-responsible phospholipid polymer chains under cellular environment. Biomacromolecules 2016, 17, 3986-3994.


22

Page 23 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(44) Fokina, A.; Klinker, K.; Braun, L.; Jeong, B. G.; Bae, W. K.; Barz, M.; Zentel, R. Multidentate Polysarcosine-Based Ligands for Water-Soluble Quantum Dots. Macromolecules 2016, 49, 3663-3671. (45) Lai, J. T.; Filla, D.; Shea, R. Functional Polymers from Novel Carboxyl-Terminated Trithiocarbonates as Highly Efficient RAFT Agents. Macromolecules 2002, 35, 6754-6756. (46) Kramer, J. R.; Deming, T. J. General Method for Purification of α-Amino acid-Ncarboxyanhydrides Using Flash Chromatography. Biomacromolecules 2010, 11, 3668-3672. (47) Lu, H.; Cheng, J. Hexamethyldisilazane-Mediated Controlled Polymerization of α-Amino Acid N-Carboxyanhydrides. J. Am. Chem. Soc. 2007, 129, 14114-14115. (48) Jung, J.; Lin, C. H.; Yoon, Y. J.; Malak, S. T.; Zhai, Y.; Thomas, E. L.; Vardeny, V.; Tsukruk, V. V.; Lin, Z. Crafting Core/Graded Shell-Shell Quantum Dots with Suppressed Reabsorption and Tunable Stokes Shift as High Optical Gain Materials. Angew. Chem. Int. Ed. 2016, 55, 5071-5075. (49) Wang, W.; Ji, X.; Kapur, A.; Zhang, C.; Mattoussi, H. A multifunctional polymer combining the imidazole and zwitterion motifs as a biocompatible compact coating for quantum dots. J. Am. Chem. Soc. 2015, 137, 14158-14172. (50) Zhan, N.; Palui, G.; Grise, H.; Tang, H.; Alabugin, I.; Mattoussi, H. Combining ligand design with photoligation to provide compact, colloidally stable, and easy to conjugate quantum dots. ACS Appl. Mater. Interfaces 2013, 5, 2861-2869.


23

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 24

For Table of Contents Use Only


24

Multidentate Comb-Shaped Polypeptides Bearing ... - ACS Publications

Recommend Documents