A Multifunctional Polypeptide via Ugi Reaction for Compact and

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A Multifunctional Polypeptide via Ugi Reaction for Compact and Biocompatible Quantum Dots with Efficient Bioconjugation Hang Zhang, Jinlong Chen, Chunsheng Xiao, Youhua Tao, and Xianhong Wang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00072 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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Bioconjugate Chemistry

A Multifunctional Polypeptide via Ugi Reaction for Compact and Biocompatible Quantum Dots with Efficient Bioconjugation Hang Zhang,

†, ‡

†

†

†

Jinlong Chen, Chunsheng Xiao, 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. *Corresponding author: [email protected] and [email protected]

ABSTRACT The growing application of quantum dots (QDs) in biomedical research necessitates, in turn, continuous development of surface functionalizing ligands to optimize their performance for ever more challenging and diverse biological applications. Here, we demonstrate the novel multifunctional polypeptide ligands for compact and biocompatible QDs. The target ligand preparation exploits the efficient, activating agent-free Ugi reaction of four functional components to incorporate lipoic acid, pyridine, zwitterion motifs, and reactive functionalities in a one-pot procedure under mild conditions. Cap exchange with these multifunctional polypeptide ligands generates hydrophilic QD dispersions, which are colloidally stable for prolonged periods of time. The zwitterionic ligation delivers compact and small QDs, and the existence of reactive functionalities enables coupling of the QDs to biologics through bio-orthogonal coupling chemistry, such as ligation of azide-modified QDs to DNA. Therefore, this QD functionalization strategy via Ugi reaction is believed to be a viable approach for compact and biocompatible QDs with efficient bioconjugation.

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INTRODUCTION Fluorescent Quantum dots (QDs) that possess distinctive photo and physical properties have been intensively investigated as fluorophores in a wide range of biomedical applications, such as cellular labeling and biochemical sensing.1-9 These QDs are entirely dispersible in organic solvents (e.g., hexane and toluene).10-12 Therefore, to prepare hydrophilic and biocompatible QDs that are suitable for application in biology, an additional surfacefunctionalization step is needed. Thus far, the ligand exchange strategy offers an efficient way to obtain surface modification, and new ligands can modify solubility or introduce specific functionalities to the QDs.13-22 Despite of the approach employed, for the successful integrating of QDs into biology, the following three challenges need to be addressed. First, to improve the long-range colloidal stability of QDs, polymer ligands should feature multi-chelating groups.23-26 Compared to ligands that only bearing a few anchors, polymer ligands combining two different chelating groups could increase the overall ligand-to-QD coordination, thereby extraordinarily enhancing the colloidal stability of the nanocrystals. Second, the comparatively big size of the QDs restricts many proposed applications.14,27 For instance, big QDs may not enter the crowded neuronal synapse.15 To minimize the QD size without sacrificing colloidal stability, ligands must contain small volume motifs, such as zwitterion moieties.28-31 Third, to modulate interactions with cellular systems, preparing the QDs that allow further decorating with target biomolecules through covalent coupling has been vigorously pursued. 32-34 Addressing these issues, several studies have been conducted on the preparation of multicoordinating and multifunctional polymeric ligands in recent years. For example, Medintz et al. prepared poly(acrylic acid) ligands bearing pyridine and short PEG pendant groups via


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carbodiimide coupling chemistry.25 Mattoussi synthesized poly(isobutylene-alt-maleic anhydride) ligands with lipoic acid/imidazole anchoring groups, hydrophilic zwitterion moieties, and specific reactive groups using nucleophilic addition reaction toward maleic anhydride.29,32 Bawendi et al. reported well-controlled random copolymer ligands from RAFT polymerization of three types of monomers featuring imidazole, polyethylene glycol (PEG), and primary amines, respectively.35 In this contribution, we report a novel kind of multicoordinating and multifunctional polypeptide ligands which can present multiple chelating groups, small-sized zwitterion and tunable functionalities for compact and biocompatible QDs with efficient bioconjugation (Figure 1). Highly efficient Ugi reaction is used for the ligand synthesis, in which functional aldehyde and amine, oligo-ethylene-glycol (OEG) functionalized isocyanide, and low molecular weight poly(glutamic acid) , PGA (Mn = 5400 g/mol), react in a one-step procedure under room temperature. In contrast to previous ligand synthesis, the present chemistry is efficient, activating agent-free, and un-necessary for tedious purification.36-41 Coating of the QDs with these biocompatible polypeptides marries the advantages of thiol and pyridine affinity, and offers highly fluorescent nanocrystals which can present long-range colloidal stability. On the other hand, this approach delivers QDs with compact size via introduction of small-sized zwitterions motifs. We also demonstrate that containing azide groups in the polypeptide ligands allows conjugation of biological molecules to the QDs via copper-free azide-alkyne reaction. Different from commonly used coupling reactions (EDC/NHS chemistry), the aforementioned procedure do not need tedious purification. We anticipate that these results will not only allow the wide application of multidentate polypeptide for the QDs ligation, but more importantly improve the utility of QDs in the fields that require highly stable and compact fluorescent probes.


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Figure 1. (A) Synthesis approach to multifunctional polypeptides via Ugi Reaction. (B) Chemical structures of polypeptide ligands (PL1, PL2 and PL3). (C) Schematic illustration of the ligands exchange strategy.


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RESULTS AND DISCUSSION

Synthesis of Multifunctional Polypeptide Ligands. Polypeptide ligands were synthesized starting from biocompatible poly(L-glutamic acid) (PGA) precursor. PGA was synthesized from the ring-opening polymerization of γ-benzyl-L-glutamate N-carboxyanhydrides, followed by removal of protecting groups, it had a number average molecular weight of 5400 Da with a polydispersity index of 1.3, or contained approximately 42 -COOH groups. As shown in Figure 1, the polypeptide ligands were then synthesized through one-pot Ugi reaction between PGA with amine-functionalized components, namely zwitterions-amine 1a, lipoic acid (LA)-amine 1b, and azide-amine 1c, pyridine-aldehyde 2, and OEG-isocyanide 3a-3b. This approach has several distinctive characteristics. Activating agents were not necessary for this reaction, which can facilitate purification process. Ugi reaction enables the simultaneous incorporating of distinct and complementary components within the same ligand: lipoic acid and/or pyridine anchoring motifs for potent affinity to the nanocrystals, zwitterion motifs for minimal size, OEG motifs for water solubilization, and functional groups for targeted coupling to biologics. By using Ugi reaction, we have prepared three sets of polymer ligands: PL1, PL2, and PL3 (Figure 1B). Ugi reaction is usually performed in methanol as solvent.37 Having the preparation of polypeptide ligands in mind, this solvent seems unsuitable since PGA is not soluble in methanol. Therefore, other solvent or mixed solvent is needed. For this, alkaline H2O was employed for the synthesis of PL1, and mixed solvent of methanol and DMSO was tested for the synthesis of PL2 and PL3 (Table 1). The polypeptide ligands were first characterized by NMR spectra. Figure 2 showed 1

H NMR and

13

C NMR spectra of PL1 as a typical example. The pyridine units display two

broad peaks at 7.5, and 8.6 ppm, respectively. The repeating ethylene oxide units of OEG have an intense peak around 3.3-3.6 ppm, and the zwitterion groups appear at 2.0-3.4 ppm as broad


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peaks. Comparison of the peak integration ratios for each unit helped provide an estimate of the grafting percentages. Details about the NMR data of PL2 and PL3 are shown in the Supporting Information (Figure S2−S3). Moreover, Table 1 showed grafting percentages for the three polypeptide ligands indicating, that the Ugi reaction gave significantly higher grafting percentages in alkaline H2O (56 %) than in methanol/DMSO (28 % for PL2 and 38 % for PL3, respectively). Therefore, we have thus demonstrated that a simple Ugi reaction provides a relatively facile method toward multifunctional polypeptides for compact and biocompatible QDs. Table 1. Characterization of the polypeptide ligandsa

ligand

Reaction composition

Solvent

Grafting percentageb

Mn.NMR

Mn,SEC

(kDa)c

(kDa)d

Mw/Mnd

Isolated yielde (%)

(%) PL1

PGA+1a+2+3b

H 2O

56

16.7

13.0

1.3

60

PL2

PGA+1b+2+3a

CH3OH/DMSO (1/1)

28

13.6

10.9

1.2

31

PL3

PGA+1b+1c+2+3a

CH3OH/DMSO (1/1)

38

15.9

11.2

1.3

43

a

Ligands were synthesized at room temperature for 72h. bThe percentage was calculated from the integration ratio of 1H NMR peaks of the pyridine and methine group of the PGA units. c Mn,NMR=[(Mamine+Maldehyde+Misocyanide-MH2O) × DPPGA × Grafting Percentages] + MPGA. dMeasured by SEC with polystyrene as standard and H2O as eluent. e The isolated yield of polymer ligand after dialysis.


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Figure 2. Representative 1H , and 13C NMR spectra of PL1, measured in D2O. Ligand Exchange Using the Polypeptide Ligands. As shown in Figure 1C, to efficiently replace the native hydrophobic ligands on the QDs surface with the polypeptide ligands, we initially used a two-step ligands exchange method reported by Medintz et al.25, in which the native ligands were replaced with intermediate and lower affinity ligands, followed by ligands exchange with the final polypeptide ligands. Here, we chose 2-(2-aminoethoxy) ethanol as the intermediate ligands. And, this relatively weak ligands bound on the QDs surface can then be replaced with stronger binding ligands in a second step. Indeed, similar two-step ligands exchange methods have been successfully demonstrated to prepare QDs coated with thiol-based ligands.32 We also carried out direct one-step ligand exchanges of the as-prepared hydrophobic QDs with the polypeptide ligands. However, the one-step methods often resulted in incomplete ligands exchange or larger hydrodynamic sizes compared with the two-step methods (Figure S6A, B). For the PL2 and PL3, after the ligands exchange procedure, mild photoligation strategy was carried out to ensure the coordination of the LA groups. Photoligation can keep the integrity


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of reactive functionalities, such as azide, and biotin. The ability of the photoligation approach is due to the photochemical sensitivity of the strained dithiolane ring to UV excitation.42 Characterization of the Compact and Biocompatible QDs. The resultant compact and biocompatible QDs were evaluated by optical spectroscopy, 1H NMR and FT-IR spectra, and dynamic light scattering (DLS). Optical Characterization. The absorption and photoluminescence (PL) spectra of four sets of QDs (emission peak at 565 nm) before and after ligands exchange with PL1, PL2, and PL3 were recorded. As shown in Figure 3A, the photoluminescence properties of the QDs coated with various polypeptide ligands in water are nearly identical to that of hydrophobic QDs in chloroform, demonstrating the integrity of the QDs following phase transfer. Meanwhile, it was observed that there was a slight red shift (~5 nm) in emission peaks observed for QDs capped with polypeptide ligands. Similar results were also observed in the previously reported work.25 It is really difficult to explain red shift phenomenon of emission peak observed in the ligand exchanged QDs. Probably, this peak shift indicates that the surface polypeptide ligands have some effects on the electronic properties of the inorganic QD cores.43 Absorption spectra in Figure S4 also showed similar trends. We should also note that the PL2 or PL3 coated QDs exhibit a stronger fluorescence (Figure 3B and Figure S5), after 20 min of UV irradiation. Such enhancement in fluorescence could be attributed to two reasons: the synergistic effect that incorporated two kinds of anchoring groups beneficial for the colloidal stability, and UVpromoted photoannealing of QDs.44-48 The quantum yield (QY) of the QDs after ligation and phase-transfer from organic to aqueous media was decreased, compared to that of the hydrophobic ones dispersed in chloroform (0.42). The QYs of QDs after ligand exchange with PL1-PL3 were 0.30, 0.24, and 0.26, respectively. Such decrease in the fluorescence QY of QD


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samples after ligand exchange is a very common phenomenon, which arises primarily from incomplete surface passivation.25,49,50

Figure 3. (A) emission spectra of QDs with trioctylphosphine/trioctylphosphine oxide (TOP/TOPO) in chloroform and with PL1-PL3 in water. (B) Plot of the photoluminescence intensity for PL2 coated QDs before and after UV irradiation. (C, D, E) FT-IR spectra of the different ligands coated QDs, with TOP/TOPO (black line), with polypeptide ligands (blue line), and the ligands only (red line). The band at 2105 cm−1 is attributed to the azide group. Dynamic Light Scattering. We next examined the hydrodynamic diameters (HD) of the polypeptide-coated QDs using dynamic light scattering (DLS). As shown in Figure S6, the HD


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of the QDs coated with PL1-PL3 ranged from 11.4 to 12.5 nm. The diameter of the QD sample before ligand exchange was directly determined by transmission electron microscopy (TEM) to be 5 nm (Figure S7). These increases in diameter of polypeptide-coated QDs arises from the hydrodynamic interactions of the ligand layer. 1

H NMR and FT-IR spectra. The hydrophilic polypeptide-coated nanocrystals were

examined by the 1H NMR and FT-IR spectra to confirm that the native hydrophobic ligands were efficiently replaced by the multi-functionalized polypeptide ligands. As shown in Figure S8, chemical shifts of the spectrum of QD dispersions are in aggrement with the 1H NMR characteristics of the starting ligands. And the chemical shifts of the spectrum of original QD capped with TOP/TOPO are absent. These results clearly indicated that the native and the intermediate ligands have been entirely replaced by polypeptide in the exchange steps. FT-IR again verified that the ligands exchange process could preserve the integrity of the reactive functionalities on QDs. Specifically, the FT-IR spectra collected from the pure ligand PL1, PL2 and PL3 are identical with the QD capped with corresponding ligand (Figure 3C-3E). And in Figure 3E, a clearly peak at 2105 cm-1 belong to the azide absorption were observed in both FTIR spectra of PL3 and PL3 coated QD, which indicated ligand exchange process preserved the intergrity of azide groups.


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Figure 4. Colloidal stability testing. (A) Relative fluorescence intensities of 0.3 µM QDs coated with PL1. (B) Relative fluorescence intensities of 0.3 µM QDs coated with PL2.

Colloidal Stability. The colloidal stability of aqueous QDs ligated with multifunctional polypeptides was examined under various conditions, such as a wide pH range. Figure 4 showed the pH-dependent fluorescence intensities of 0.3 µM yellow-emitting QDs, in buffers at pH 3-13, and in deionized water (DI) water. All QD dispersions kept good colloidal stability at DI water as well as from neutral to basic pHs (pH 7~13) for at least 3 weeks, with no sign of aggregate build up or loss in fluorescence. And reduced stability including loss of fluorescence and sedimentation of the QDs was happed in the lower pHs, which can be explained by the protonation of pyridine anchoring group (pKa = 5.6 for p-alkyl substituted pyridine). The protonation of pyridine group reduced the coordination ability of the ligands onto the surface, which caused ligand desorption from the QD surface and subsequent aggregation of the QDs. Similar observations have been reported for other pyridine-based ligands at pH ≤ 5.25 As shown in Figure 4, the fluorescence intensity of PL2 capped QDs dispersions were higher than PL1 capped QDs dispersions after 45 days, which attributed to the synergistic effect of LA and pyridine anchoring groups in the PL2.


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Cellular Imaging. The uses of QDs for biological and biomedical applications have received considerable interest in recent years.51 The unique optical properties of QDs make them an exciting fluorescent tool for in vivo and in vitro imaging.52 To apply them in biological environment, it is essential to prepare hydrophilic and biocompatible QDs. The above Ugi chemistry can afford an array of hydrophilic and biocompatible polypeptides-based QDs for cellular imaging. Therefore, we tested the suitability of the resultant compact and biocompatible QDs for biological imaging by fluorescent microscopy. Specifically, HeLa cells were treated with 5 µM solution of QDs coated with PL1 and PL2. After incubation for 6 h at 37°C, cells were gently washed three times with PBS and fixed in PBS containing 4% paraformaldehyde for 2 h at room temperature. Then HeLa cells were washed with PBS for three times and visualized using Zeiss confocal microscopy (LSM 700, Carl Zeiss, Germany). As shown in Figure 5 and Figure S9, Both PL1 and PL2 coated QDs could enter HeLa cells and exhibited clear green fluorescence in the cells. Thus, these compact and biocompatible QDs coated with multifunctional polypeptides could be excellent candidates for cellular imaging.

Figure 5. Fluorescent microscopy images of HeLa cells treated with 500 nM QDs coated with PL1 for 6 h.

Bioconjugation to Nucleic Acids. The application of QDs in biology directly depends on their effectiveness to conjugate with target biologics. This is determined by the reactive groups


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and the ability of the coupling methods applied for assembling QD-bioconjugates. Additionally, conjugation of biomolecules to various inorganic nanocrystals has played an increasingly important role in biomaterials.53-55 Here, we have delivered polypeptides bearing functional groups and evaluated coupling of the QDs to biologics via copper-free click reaction. For click chemistry, we synthesized polypeptide ligand PL3 for which 16% of the repeating unit were terminated

with

azides,

that

were

conjugated

to

strained

cyclooctynes

such

as

dibenzylcyclooctyne (DBCO) under mild conditions in phosphate buffer (50 mM, pH = 8.0) (Figure 6A). As shown in Figure 6B, a mobility shifts in agarose gel electrophoresis was observed, indicated the successful conjugation of the DBCO-DNA with azide functionalized QD.

Figure 6. (A) Schematic depiction of QD-DNA coupling reactions via strain-promoted click chemistry. (B) Gel electrophoresis result for QD-DNA conjugations.

CONCLUSION In summary, we describe the preparation of a series of multifunctional polypeptides for compact and biocompatible QDs through Ugi reaction. Cap exchange with these polypeptides has delivered water-soluble QDs that possess outstanding colloidal stability over a broad pH range. The flexibility of incorporating functional groups into the ligands has simplified the synthesis of QD-bioconjugates through copper-free click reaction. The delivered QD-


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bioconjugates have been evaluated in several biological examples, such as cellular imaging. We anticipate that the chemical design described herein can also be used to deliver ligands that are optimally adapted for ligation a wide range of nanomaterials. Our design is also amenable to incorporating biologics, such as nucleic acid, into the ligand structures.

EXPERIMENTAL PROCEDURES Materials.

poly(γ-benzyl

glutamate)

synthesized

via

the

γ-benzyl-L-glutamate-N-

carboxyanhydride polymerization, using hexylamine as initiator at room temperature for three days. 4-Pyridinecarboxaldehyde, lipoic acid, oligo-ethylene-glycol 350 (OEG7.5), triethylene glycol monomethyl ether, were all purchased from Energy Chemical. Amine-modified LA and amino-zwitterion were prepared according to the previous works.31,56 Amine-azide was synthesized following the literature.14,15 The OEG3-isocyanide and OEG7.5-isocyanide were synthesized by the dehydration from their corresponding formamide precursors which were obtained from OEG3-NH2 and OEG7.5-NH2.57 Hydrophobic CdSe−ZnS Core−Shell QDs coated with trioctylphosphine/trioctylphosphine oxide (TOP/TOPO) were synthesized following the literature method. 58 Methods. DLS was performed on a Malvern Zeta-sizer Nano. The FT-IR spectra were carried out on a Bruker Tensor-27 spectrometer. UV-vis spectra were obtained by Shimadzu UV2450 spectrophotometer. Fluorescence measurements were performed on a PerkinElmer LS-55 phosphorescence spectrophotometer. 1H and

13

C NMR spectra were recorded using a Bruker

Avance II 600 MHz NMR, using D2O as the solvent. Absolute photo-luminescence quantum


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yield values as measured by the HAMAMATSU C9920-02 Absolute Quantum Yield Measurement systems. Synthesis of polypeptide ligand PL1. A NaOH solution (0.19 mmol in 0.5 ml H2O, pH = 13.6) of amino-zwitterion hydrochloride (47.8 mg, 0.19 mmol) and 4-Pyridinecarboxaldehyde (20 mg, 0.19 mmol) was stirred at room temperature for 1 h. PGA (20 mg, 0.16 mmol) and OEG3-isocyanide (32.2 mg, 0.19 mmol) were then added. And the mixture was reacted for 72 h at room temperature. The mixture was dialyzed against DI water for 2 days using the Dialysis Membrane (MW cutoff 5000). After lyophilization, a light yellow solid was obtained (72 mg, 60 %). Synthesis of polypeptide ligand PL2. A solution of amino-LA (60 mg, 0.24 mmol) and 4pyridinecarboxaldehyde (24.4 mg, 0.23 mmol) in 0.5 mL of CH3OH was stirred at room temperature for 1 h. PGA (25.8 mg, 0.2 mmol), OEG7.5-isocyanide(86 mg, 0.24 mmol) dissolved in DMSO (0.5 mL) were added. And the mixture was reacted for 72 h at room temperature. The mixture was dialyzed against deionized water for 2 days using the Dialysis Membrane (MW cutoff 5000). After lyophilization, a brown solid was obtained (61 mg, 31%). Synthesis of polypeptide ligand PL3.A solution of amino-LA (46 mg, 0.19 mmol), aminoazide (10.2 mg, 0.06 mmol) and 4-pyridinecarboxaldehyde (25 mg, 0.23 mmol) in 0.5 mL of CH3OH was stirred at room temperature for 1 h. PGA (25 mg, 0.19 mmol ), OEG7.5-isocyanide (84 mg, 0.23 mmol) dissolved in DMSO (0.5 ml) were added. And the mixture was reacted for 72 h at room temperature. The mixture was dialyzed against deionized water for 2 days using the Dialysis Membrane (MW cutoff 5000). After lyophilization, a brown solid was obtained (81 mg, 43 %).


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Ligand Exchange. The protocols of ligand exchange were carried out by a modified procedure as described earlier.25,44 It involved two steps for PL1, and three steps for PL2 and PL3. Typical procedures for PL1 ligation. The QDs (1 mg) dispersed in CHCl3 (0.2 mL), was mixed with ethanol (0.5 mL), and 2-(2-aminoethoxy) ethanol (0.15 mL) in nitrogen atmosphere. And the reaction was stirred at 60 oC for 1 h. The resultant QDs were precipitated by the addition of ethyl acetate and hexane, sonicated for 1 min, and centrifuged at 3800 rpm for 5 min, generating a pellet. Following dissolved in 0.3 mL of DMSO, PL1 (15.0 mg) in 0.80 mL of DMSO was added, and the mixture was reacted at 60 oC for another 3.5 h. The QDs were again precipitated with hexane and acetone, sonicated for 1 min, and centrifuged at 3800 rpm for 5 min. Then, the QDs were collected, and the procedure was repeated. The final precipitate was dried under vacuum for 5 min, and redispersed in DI water. Following sonication for 2 min, the dispersion of QD was filtered through a syringe filter (0.45 µm), and excess unbound polypeptides and other byproducts were removed with Amicon Ultra centrifugal filters (50 kDa MWCO). The resultant dispersion of QDs was preserved at 4 oC under dark. Typical procedures for PL2 and PL3 ligation. For the coordination of pyridine groups of PL2 and PL3 to the surface of QD with native ligands, the ligands exchange stratergy was carried out following the precedure of PL1. After that, the ligation of LA anchoring groups appended in PL2 and PL3 was implemented via a photomediated ligand exchange. Briefly, following precipitation of the QDs with hexane and acetone, sonication and centrifugation, the pellet of PL2/PL3 coated QDs were dried under vacuum, and dissolved in 1mL H2O. And then an aqueous solution of tetramethylammonium hydroxide (5 mM, 30 µL) was added, the reaction mixture was stirred under UV irradiation for 20 min. After filtration with 0.45µm syringe filter,


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and purification with Amicon Ultra centrifugal filters, the aqueous dispersion of QDs were preserved at 4 oC under dark. Cellular Imaging of QDs coated with polypeptide-based ligands. The cellular imaging experiment was performed according to our previous report.26 Specifically, HeLa cells were seeded in a 96-well plate at 7000 cells per well and incubated for 24 h at 37°C. Cells were then attached on culture plates. Next, HeLa cells were treated with 5 µM solution of QDs coated with PL1 and PL2. After incubation for 6 h at 37°C, cells were gently washed three times with PBS and fixed in PBS containing 4% paraformaldehyde for 2 h at room temperature. Then HeLa cells were washed with PBS for three times and visualized using Zeiss confocal microscopy (LSM 700, Carl Zeiss, Germany). Conjugation of PL3 coated QDs to DBCO-terminated DNA. Dibenzocyclooctyl terminated DNA (length 24 bp) was purchased from Sangon Biotech. The sequence was 5′-DBCO-TCC ATG ACG TTC CTG ACG TTT TTT-3′. The PL3 coated QD in phosphate buffer (50 mM, 100 µL, pH = 8.0) was reacted with equivalent amounts of DNA. The reaction was stirred at room temperature for 24 h. Conjugation was measured using electrophoresis in agarose gels (0.5% agarose). ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected].


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* E-mail: [email protected] 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 Nos. 20160414001GH and 20180201070GX), the Opening Foundation of State Key Laboratory of Medicinal Chemical Biology, Nankai University (Grant No. 2017016).

ABBREVIATION LA, lipoic acid; QDs, quantum dots; OEG, oligo-ethylene-glycol; PGA, poly(L-glutamic acid); SEC, size-exclusion chromatography;

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A Multifunctional Polypeptide via Ugi Reaction for Compact and

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