Synthetic protocols toward polypeptide conjugates via chain end functionalization after RAFT polymerization

Abstract

We report a new protocol to synthesize conjugates of polypeptides with vinyl polymers prepared by reversible addition–fragmentation chain transfer (RAFT) polymerization. Well-defined polystyrene-b-poly(γ-benzyl L-glutamate) (PS-b-PBLG) diblock copolymers are synthesized as a typical example of such conjugates. Polystyrene is prepared by RAFT polymerization using conventional and commonly used 2-cyano-2-propyl dodecyl trithiocarbonate as a RAFT chain transfer agent bearing an inert R group. Amine functionalization is carried out by a one-pot process combining aminolysis with the in situ thiol capping reaction by acrylate or methanethiosulfonate containing a tert-butoxy carbonyl (Boc) protected amine group. After deprotection of the Boc group, the released amino group initiates ring-opening polymerization of γ-benzyl L-glutamate N-carboxyanhydride (BLG NCA) to generate the desired block copolymers. Hydrolysis of the benzyl group affords the corresponding polystyrene-b-poly(L-glutamic acid) (PS-b-PGlu) amphiphilic block copolymers which self-assemble into spherical micelles in aqueous media. Circular dichroism (CD) spectra show secondary structural changes of PGlu in different pH conditions. When methanethiosulfonate is used as the capping agent, polystyrene and PBLG segments are conjugated by a reduction-responsive disulfide linkage, which is confirmed by size-exclusion chromatography and destruction of the micelle after treating with dithiothreitol (DTT). This protocol is a simple way to synthesize diblock copolymers of amino acids with vinyl monomers without specific design of RAFT CTA, which is practical for various polymer conjugates with tunable properties.

---

Introduction

Polypeptide conjugates with synthetic polymers show significant application potential in biomedicine and biotechnology fields, due to the combined advantages of both blocks, which not only makes it possible to mimic the biological activity and secondary structure arrangement of proteins, but also incorporates new elements to meet the functional requirement for special applications.^1–3

Novel biomedical materials for controlled drug release, gene delivery, tissue engineering and regenerative medicine call for multi-stimuli responsive features. Vinyl polymers have enjoyed fast development in the last decade due to the discovery of controlled radical polymerizations (CRPs) which allow precise design of both polymer architecture and composition. It is exciting and challenging to introduce them into polypeptide conjugates to obtain multifunctional materials programmable for both structural versatility and functionality.^4,5

Disulfide bond is important cleavable linkage in organisms which contributes to protein folding and stabilization of protein structures. Moreover, its ability to cleave in reductive environment frequently encountered in biological systems makes synthetic polymers with disulfide bond attract more and more attention.⁶

Reversible addition–fragmentation chain transfer (RAFT) approach is attracting increasing attention due to advantages including metal-free synthesis, tolerance toward many functional groups, and a wide range of polymerizable monomers. It has been widely used in the synthesis of biomedical materials.^7–9 Polypeptides can be easily obtained by a mature method of amine-initiated ring opening polymerization (ROP) of amino acid N-carboxyanhydride (NCA).^10,11 Combining RAFT with NCA ROP is a promising way to prepare synthetic “smart” polypeptides in the case that stimuli-responsive segments are involved such as poly(N-isopropylacrylamide) (PNIPAM) with thermo-responsive feature.

There are two strategies to conjugate polypeptide segments to RAFT-prepared polymers via either polymeric click reactions¹² or macroinitiator approaches.¹³ Polymeric click reactions can be very efficient in connecting polymer chains, but always contain complicated post-treatment to remove catalysts or unreacted homopolymers. Moreover, copper catalyst is not favored in biological applications.

Macroinitiator approaches are cleaner ways by designing appropriate amine-group capped macroinitiators for NCA polymerizations. It is an issue to avoid the well-known nucleophilic attack of amine group to trithiocarbonate (TTC) moiety in RAFT chain transfer agent (CTA) leading to high polydispersity of final products. A complicated design of CTA is always required, which limits its application. Only a few combinations of RAFT and NCA ROP have been reported.^13–15 Very recently, Jacobs et al. used a special RAFT CTA of phthalimidomethyl trithiocarbonate with a protected amino group at R-side to obtain macroinitiator for NCA ROP after removal of TTC group and subsequent deprotection of phthalimide.¹⁶

In this work, we report a versatile approach to synthesize amino and disulfide-linked amino functionalized vinyl polymer macroinitiator from precursor obtained by RAFT polymerization using commonly available CTAs. Instead of complicated design of amine-containing R group CTAs, a one-pot aminolysis and thiol capping strategy is applied to convert TTC moiety to a tert-butoxy carbonyl (Boc) protected amino end group. This approach generates amino group at ω-terminus and shows more convenience than most utilized conventional R group method to prepare amino functional vinyl polymers.^17–19 The following deprotection releases amine to initiate NCA ROP producing the target polypeptide conjugates. Well-defined polystyrene-b-poly(γ-benzyl L-glutamate) (PS-b-PBLG) block copolymers with or without disulfide linkage are synthesized as models to demonstrate the feasibility of our method. After deprotection, the corresponding amphiphilic polystyrene-b-poly(L-glutamic acid) (PS-b-PGlu) self-assembles to micelles in aqueous solutions. Our strategy allows synthetic polypeptide conjugation to any vinyl polymers readily produced by RAFT polymerization.

Experimental

Materials

Butylamine (J&K), acryloyl chloride (Shanghai Jingchun), trifluoroacetic acid (TFA, Acros) hydrogen bromide (HBr, 33 wt%, Acros), and DL-dithiothereitol (DTT) (Adamas) were used as received. 2,2′-Azobis(isobutyronitrile) (AIBN, Adamas) was recrystallized from methanol. Dioxane and triethylamine (TEA) were distilled, and styrene and N,N-dimethylformamide (DMF) were distilled under reduced pressure prior to use. γ-Benzyl L-glutamate N-carboxyanhydride (BLG NCA),^20–22 2-(tert-butoxycarbonylamino)ethanol,²³ sodium methanethiosulfonate,²⁴ N-tert-butoxycarbonyl-2-bromoethylamine²⁵ and 2-cyano-2-propyl dodecyl trithiocarbonate (CDT)²⁶ were synthesized according to literatures.

Characterization

Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance DMX 500 spectrometer (¹H: 500 MHz) using CDCl₃ as solvent and tetramethylsilane as internal reference. Molecular weights (MWs) and molecular weight distributions (MWDs) were determined by size-exclusion chromatography (SEC) on a Waters-150C apparatus equipped with Waters Styragel HR3 and HR4 columns and a Waters 2414 refractive index detector. Two solvents were used as eluents. THF was used with a flow rate of 1.0 mL min⁻¹ at 40 °C and commercial polystyrenes were used as calibration standards. DMF containing 0.05 mol L⁻¹ LiBr was used with a flow rate of 1.0 mL min⁻¹ at 60 °C with poly(methyl methacrylate) standards. The morphologies of micelles were observed on JEOL JEM-1230 (HR) transmission electron microscopy (TEM) instrument with the accelerating voltage of 80 kV. A drop of 0.5 mg mL⁻¹ micellar solution was put onto the surface of carbon-film coated copper grids. Excess solvent was quickly removed with a filter paper. Circular dichroism (CD) spectra were measured at 25 °C in a 2 mm path length quartz cell using a Biologic MOS-450 spectrometer (France).

Synthesis of 2-(tert-butoxycarbonylamino)ethyl acrylate (1)

To a solution of 2-(tert-butoxycarbonylamino)ethanol (8.96 g, 55.6 mmol) and triethylamine (8.5 mL, 61.1 mmol) in 90 mL CH₂Cl₂, acryloyl chloride (5.0 mL, 61.1 mmol) in 15 mL CH₂Cl₂ was added dropwise at 0 °C. The mixture was stirred for 12 h at room temperature. The solution was washed with 1 mol L⁻¹ HCl, saturated brine and water, successively. The organic layer was dried over MgSO₄ and evaporated to afford the product as a white solid (9.72 g, 81.3%). ¹H NMR (500 MHz, CDCl₃, δ in ppm): 6.44 (dd, 1H), 6.15 (dd, 1H), 5.86 (dd, 1H), 4.83 (br, 1H), 4.23 (t, 2H), 3.44 (t, 2H), 1.45 (s, 9H).

Synthesis of 2-(tert-butoxycarbonylamino)ethyl methanethiosulfonate (2)

A solution of sodium methanethiosulfonate (2.53 g, 18.9 mmol) and N-tert-butoxycarbonyl-2-bromoethylamine (4.06 g, 18.1 mmol) in DMF was stirred at 70 °C for 4 h. The precipitate was filtered out and the solvent was evaporated in vacuum. The residue was dissolved in CH₂Cl₂ and washed with water. After dried over MgSO₄, a crude product was obtained after evaporation and yielded white crystals (2.78 g, 60.1%) by recrystallization in hexane. ¹H NMR (500 MHz, CDCl₃, δ in ppm): 4.96 (br, 1H), 3.49 (t, 2H), 3.37 (s, 3H), 3.30 (t, 2H), 1.45 (s, 9H).

Synthesis of polystyrene (PS) precursor

In a typical polymerization, styrene (6.25 g, 60 mmol), AIBN (28.2 mg, 0.17 mmol) and CDT (0.27 g, 0.78 mmol) were mixed in 4 mL dioxane, deoxygenated by three freeze–pump–thaw cycles, sealed, and polymerized at 70 °C for 24 h. The product was precipitated in methanol, filtered, and dried under high vacuum for 24 h to become yellow powder (3.08 g, 45.0%).

Aminolysis of end-group in the presence of capping agent 1

PS₃₅–TTC (2.0 g, 0.5 mmol) and 1 (0.54 g, 2.5 mmol) were dissolved in DMF (15 mL) to be a yellow solution. After degassed, a solution of n-butylamine (0.15 mL, 1.5 mmol) and triethylamine (0.21 mL, 1.5 mmol) in 3 mL DMF was added by a syringe. The reaction mixture was stirred at room temperature for 15 h and became a colorless solution. The product was white powder after precipitation in methanol (1.7 g, 85.0%).

Aminolysis of end-group in the presence of capping agent 2

The procedure was the same as the above with PS₅₇–TTC (1.7 g, 0.27 mmol), 2 (0.34 g, 1.35 mmol) and n-butylamine (0.63 mL, 6.25 mmol), giving similar yield (1.4 g, 82.3%).

Deprotection of t-Boc group

A solution of PS₃₅–NHBoc (1.0 g) in CH₂Cl₂ (5 mL) was treated with excess amount of trifluoroacetic acid (1 mL) at 0 °C for 2 h. 45 mL CH₂Cl₂ was added to the reaction mixture, and the mixture was then washed with saturated NaHCO₃ aqueous solution and water. The CH₂Cl₂ phase was dried over MgSO₄, concentrated, and precipitated in methanol. PS₃₅–NH₂ was obtained as white powder (0.65 g, 65%).

Synthesis of PS-b-PBLG block copolymers

All polymerizations were performed in Schlenk tubes under argon atmosphere. As a typical example, BLG NCA (0.25 g, 0.95 mmol) and PS₃₅–NH₂ initiator (88 mg, 0.023 mmol) were dissolved in 4.5 mL DMF. The solution was stirred for 5 days at room temperature. The product was isolated by precipitation from diethyl ether and dried in vacuum (0.228 g, 80%).

Deprotection of PS-b-PBLG block copolymer

In a typical experiment, PS–PBLG1 (0.15 g, 8.2 μmol) was dissolved in 1 mL TFA. After the addition of HBr–acetic acid (33 wt%, 0.2 mL), the mixture was stirred at 0 °C for 4 h. The polymer was precipitated from diethyl ether, filtered, and washed with distilled water. Final product was obtained after dry in vacuum for 48 h (69 mg, 69%).

Deprotection of PS-SS-b-PBLG block copolymer

A 2 mL aqueous solution of NaOH (0.14 g, 3.5 mmol) was added into a DMF (5 mL) solution containing PS–PBLG5 (0.223 g, 1.1 μmol) at room temperature. After 10 h, the reaction mixture was dialyzed against deionized water. The final mixture was lyophilized to give PS–PGlu5 as white powder (0.13 g, 75%).

Preparation of polymeric micelles

In a typical example, PS–PGlu1 (2.5 mg) was dissolved in 1.2 mL DMSO. 1.2 mL of NaOH solution (0.5 mol L⁻¹) was added dropwise into the polymer solution under vigorous stir. After 2 h, the solution was dialyzed against deionized water to remove DMSO and the excess NaOH. Final volume was 5 mL to make a micelle solution of 0.5 mg mL⁻¹.

Results and discussion

Postmodification through one-pot aminolysis and thiol capping reaction of RAFT-prepared polymers has been proved to be an efficient way to convert CTA chain ends to designed functional groups.^27–30 This strategy shows great versatility and atom-efficiency in preparing end functionalized vinyl polymers. Moreover, quantitative end group transformation is guaranteed by efficient thiol chemistry such as thiol-ene click reaction³¹ and thiol–methanethiolsulfonate reaction.³² Because of the incompatibility of amine and TTC groups, we use two Boc-protected amine containing compounds, acrylate 1 and methanethiosulfonate 2, as two thiol capping agents to introduce amine end group.

Scheme 1 illustrates the whole picture of our new synthetic protocol. PS precursor PS–TTC is prepared by RAFT polymerization. Although CDT is taken as an example, it is feasible to use any trithiocarbonate or dithioester CTAs. Styrene is also open to be changed into most vinyl monomers with demanded properties. It is noteworthy that monomers containing nucleophilic groups, for instance hydroxyl and amino groups, need protection since the following NCA ROP does not tolerate them.

Scheme 1 Synthetic pathways of PS-b-PGlu block copolymers with and without cleavable disulfide linkage.

Herein, well-defined polystyrene PS₃₅–TTC and PS₅₇–TTC are synthesized through CDT mediated RAFT polymerization (Table 1). The two polymers, precursors for polypeptide conjugation, possess a TTC moiety at the ω-chain end and an inert cyano group at the α-chain end and represent commonly used vinyl polymers prepared by RAFT polymerization. Different compositions of these two PS polymers are primarily due to the different monomer concentrations used for the polymerization which resulted in different yields.

Table 1 PS obtained by CDT mediated RAFT polymerization and corresponding protected amine-functionalized PS

Polymer	[M]/[CTA]/[I]	Yield (%)	Mn,theo^c (kDa)	Mn,SEC^d (kDa)	Mn,NMR^e (kDa)	MWD^d
a Polymerization condition: 70 °C, 24 h, [M]a = 6 mol L^−1, [M]b = 7.7 mol L^−1.b Polymerization condition: 70 °C, 24 h, [M]a = 6 mol L^−1, [M]b = 7.7 mol L^−1.c Calculated by Mn,theo = [M]/[CTA] × yield × 104.d Obtained from SEC in THF with PS calibration.e Calculated by ^1H NMR in Fig. 2A according to Mn,NMR = I(H^d)/[I(H^c)/2.5 × 104.
PS35–TTC^a	77/1/0.2	45	3.6	3.7	4.0	1.06
PS35–NHBoc	—	—		3.7	—	1.05
PS57–TTC^b	80/1/0.2	67	5.5	5.3	6.3	1.08
PS57–SS–NHBoc	—	—		5.2	—	1.07

---

TTC group is converted to Boc-protected amine group with different linkage by aminolysis in the presence of capping agent 1 or 2 (5 equivalents to TTC group). PS–NH₂ and PS–SS–NH₂ macroinitiators are obtained by deprotection of Boc group and further used in ROP of BLG NCA to produce PS-b-PBLG and PS-SS-b-PBLG conjugates, respectively. One-pot aminolysis of PS₃₅–TTC precursor followed by in situ Michael addition with acrylate 1 generates PS₃₅–NHBoc quantitatively. PS₃₅–NH₂ is obtained after deprotection of Boc group. Their SEC analyses show unimodal profiles with narrow MWDs and unchanged MWs (Table 1). Moreover, no shoulder with high MW is observed, which indicates the absence of polymeric disulfide oxidized by mercapto groups (Table 1 and Fig. S1 in ESI† and Fig. 1A and B). Structures of three species are confirmed by ¹H NMR. The dodecylthiocarbonothioylthio moiety at the chain end of PS₃₅–TTC shows characteristic signals of methyl protons H^a, dodecyl methylene protons H^b and H^c at 0.88, 1.25 and 3.25 ppm, respectively (Fig. 2A). Quantitative end group transformation after aminolysis and Michael addition is evidenced by the complete disappearance of dodecyl proton signals and the emerged Boc methyl at 1.44 ppm. Compared with capping agent 1 (data in Experimental section), no vinyl proton is remaining in PS₃₅–NHBoc, while signals of methylene protons neighboring sulfur atom (H^p and H^q) are found at 2.17–2.50 ppm. In addition, methylene protons Hⁱ and H^j shift upfield to 3.30 and 4.03 ppm (Fig. 2B) from those in compound 1 (3.44 and 4.23 ppm) after thiol-ene addition. All the above evidence a successful reaction generating PS polymer bearing a Boc protected amine chain end. The functionality is determined as 96% according to the intensity ratio of I(H^j)/I(H^c) using I(H^d) as internal standard in Fig. 2A and B. After the removal of TTC moiety, yellow PS₃₅–TTC changes to white powder of PS₃₅–NHBoc.

Fig. 1 SEC traces of PS₃₅–TTC (A), PS₃₅–NH₂ (B), PS–PBLG1 (C), PS–PBLG2 (D) and PS–PBLG3 (E) in DMF.

Fig. 2 ¹H NMR spectra of PS₃₅–TTC (A), PS₃₅–NHBoc (B) and PS₃₅–NH₂ (C) in CDCl₃ (*: H₂O).

Deprotection of PS₃₅–NHBoc releases PS₃₅–NH₂ via treatment with excess TFA in CH₂Cl₂ and neutralization. ¹H NMR analysis confirms a significant decrease of H^h at 1.44 ppm (Boc group) and a signal shift from 3.30 (Hⁱ) to 2.92 ppm (H^k) contributing to methylene protons adjacent to amine group (Fig. 2C). Under the experimental conditions, PS₃₅–NH₂ is obtained with 50% yield, along with original PS–NHBoc and unidentified species. Incomplete amine end group deprotection from Boc group was also reported using similar experimental procedures.^33–35

Incomplete deprotection of Boc group is not a fatal problem to our protocol because some reports have succeeded in preparing pure amine-terminated macroinitiator including poly(ε-caprolactone)³⁶ and poly(L-lactide)³⁷ by TFA similarly, considering the ester bonds in poly(ε-caprolactone) and poly(L-lactide) are less stable than vinyl polymer structures, we believe complete deprotection can be achieved by improving experimental conditions, however our trials according to their method still show unpromising result.

Since we are aiming at proving the feasibility of the protocol, so the detailed deprotection conditions are not further investigated in our work. Fortunately, both the undeprotected polymers and side products are not able to initiate ROP of NCA and remain as polystyrene in the copolymers. They are easy to be removed from PS-b-PBLG copolymers by precipitation in diethyl ether which is a good solvent for PS but a poor one for the copolymer. Therefore, the crude PS₃₅–NH₂ is directly used to initiate ROP of BLG NCA without further purification.

Chain extension with BLG NCA is conducted in DMF for 5 days at room temperature to guarantee complete monomer consumption. PS-b-PBLG block copolymers with various PBLG lengths are synthesized by simply tuning monomer feed ratio, showing good controllability of PS–NH₂ initiator over ROP of NCA without removal of unidentified PS species (Table 2). All of them show monomodal SEC traces, obvious MW increases, narrow MWDs and absence of remaining PS homopolymers (Fig. 1), indicating successful synthesis of block copolymers.

Table 2 ROP of BLG NCA using PS macroinitiators at room temperature in DMF

Polymer	Macroinitiator	[M]/[I]	DPtheory^a	DPexp^b	MWD
a Concerning 50% amine functionality for PS35–NH2 initiator and 60% for PS57–SS–NH2.b Calculated by ^1H NMR in Fig. 3 according to DPPBLG = DPPS{2.5 × I(H^h)/[I(H^c) + I(H^i) − 2.5 × I(H^h)]}.
PS–PBLG1	PS35–NH2	20	40	67	1.23
PS–PBLG2	PS35–NH2	40	80	88	1.20
PS–PBLG3	PS35–NH2	60	120	120	1.19
PS–PBLG4	PS57–SS–NH2	20	33	37	1.20
PS–PBLG5	PS57–SS–NH2	40	67	65	1.23
PS–PBLG6	PS57–SS–NH2	60	100	99	1.24

---

Fig. 3A shows a typical ¹H NMR spectrum of a block copolymer (PS–PBLG1). Signals related to both PS and PBLG segments are fully identified. Because the product was obtained by precipitation from ether where PS homopolymer should be well dissolved, the signals of styrene units in Fig. 3 reveal that PS segments must link to PBLG segments. Segmental lengths and compositions of the products in Table 2 are calculated from the intensities of methylene H^h at 4.99 ppm in benzyl group of BLG and aromatic protons from both styrene and BLG units.

Fig. 3 ¹H NMR spectra of PS–PBLG1 (A) and PS–PGlu1 (B) in DMSO-d₆ (*: DMF, ×: DMSO and H₂O).

Amphiphilic polystyrene-b-poly(glutamic acid) (PS-b-PGlu) block copolymers are finally obtained by the removal of benzyl protection in BLG units in TFA and HBr–glacial acetic acid mixture. They are confirmed by the absence of benzyl protons H^h and Hⁱ in ¹H NMR spectra, while a new signal corresponding to carboxyl proton emerged at 12.15 ppm (Fig. 3B). The process is mature in literature.³⁸

PS–PGlu1 (hydrolyzed from PS–PBLG1) micelles are prepared by dialysis (see Experimental section). Its average hydrodynamic diameter is measured as 63.3 nm by DLS measurement at 25 °C and pH of 5.5. TEM image shows spherical particles with diameter of ca. 50 nm in good agreement with the DLS result (Fig. S2†). The micelle is quite stable in aqueous solution without detectable aggregates after storage for weeks.

The micelle exhibits pH-responsive properties. The carboxyl groups of glutamic acid units are already partially deprotonated at pH 5.5. A small increase of hydrodynamic diameter to 80 nm at pH 9 according to DLS measurement suggests complete negative charged PGlu segments and fully extended chain conformations (Fig. 4A) rather than aggregations. While being acidated to pH of 2, PGlu segments are fully protonated with dramatic decreased hydrophilicity, and thus the micelles aggregate to large nanoparticles with diameters around 1000 nm. The micelle is reversible to the size of 80 nm by disaggregation of nanoparticles when pH value returns to 9 again (Fig. 4A and Table 3).

Fig. 4 DLS size distribution (A) and CD spectrum (B) of 0.5 mg mL⁻¹ PS–PGlu1 in aqueous solution at pH of 9, 5.5 and 2.

Table 3 DLS data of PS–PGlu1 and PS–PGlu5 micelles measured at 25 °C

Sample	pH	Diameter (nm)	PDI
PS–PGlu1	5.5	63.3	0.165
	9	80.2	0.135
	2	1036	0.295
	Back to 9	80.7	0.152
PS–PGlu5	5.5	366.7	0.083
DTT treated PS–PGlu5	5.5	—	—

---

Because of the pH-sensitive secondary conformation of PGlu segments, PS–PGlu1 assemblies are monitored by CD spectra (Fig. 4B). At pH 9, a typical CD signature of extended random coil conformation is observed with a minimum at 199 nm and a maximum at 217 nm, while at pH 2, two minima at 208 and 222 nm in the CD curve suggest α-helix structure.¹⁴ The CD spectra of the copolymers at pH of 5.5 show a pattern between those at pH of 2 and 9.

Besides the versatility of vinyl monomers and CTAs in RAFT polymerization, our modification on TTC group has a very straightforward advantage of the capability to introduce cleavable disulfide bond between polypeptide and vinyl polymer segments. Although a similar disulfide linkage can be incorporated through R-group approach of CTA,^39,40 its application is limited by the complicated synthesis of CTA and incomplete functionality (<90%).

A methanethiosulfonate 2 containing Boc-protected amine is synthesized for the first time in this work. It is used as capping agent to prepare disulfide-carrying initiator during the aminolysis of TTC group. The end group transformation strategy utilizing methanethiosulfonate is derived from Roth's quantitative functionalization of poly(methyl methacrylate) chain end with methyl and alkynyl groups.^41,42

As a result, PS₅₇–TTC precursor (M_n = 6300, MWD = 1.08) is successfully converted to disulfide-containing PS₅₇–SS–NHBoc. A complete transformation (>94% based on ¹H NMR) is achieved using 5 equiv. of compound 2 instead of 20 equiv. in Roth's report.⁴¹ The unimodal and symmetrical SEC profiles of PS₅₇–SS–NHBoc (Fig. S3B† and Table 1) and PS₅₇–SS–NH₂ (Fig. S5A†) after deprotection of Boc groups indicate that the backbone of PS is stable in the reactions. A small hump with doubled MW is observed in SEC trace of PS₅₇–SS–NH₂, which is probably due to coupled polymeric disulfide, i.e. PS–SS–PS, during the deprotection of Boc groups using TFA. Fortunately, it does not have any amine end group to initiate NCA ROP and is easily removed by precipitation in the next step. Therefore, PS₅₇–SS–NH₂ is also used directly to initiate ROP of BLG NCA to produce PS-SS-b-PBLG block copolymers (Table 2), all of which show narrow distributions in SEC and are fully characterized by NMR analyses (Fig. S3–S6†).

To demonstrate the reduction-responsive property of PS-SS-b-PBLG, SEC curves of PS–PBLG6 are compared in Fig. 5 before and after the treatment of excess dithiothreitol (DTT) which is a well-known reduction agent. The reduced product shows two peaks (Fig. 5, line C). One appears at the same retention time of PS₅₇–SS–NH₂ (Fig. 5, line A), and the other is PBLG homopolymer with lower MW than PS–PBLG6 (Fig. 5, line B). All the original block copolymer molecules are proved to contain a disulfide linkage between PS and PBLG segments.

Fig. 5 SEC traces of PS₅₇–SS–NH₂ (A), PS–PBLG6 (B) and after reduction by DTT for 48 h (C) in DMF.

Sample PS–PBLG5 has a similar PBLG length and longer PS compared with PS–PBLG1 (Table 2). The corresponding hydrolyzed product PS–PGlu5 has a larger hydrophobic PS content than PS–PGlu1. Given the fact that the difference of linkage does not influence the self-assembly behavior, we expect larger micelles of PS–PGlu5 than PS–PGlu1 and a reduction-responsive property.

In the first attempt, we use conventional HBr/HAc method to deprotect benzyl group of PS–PBLG5. However, it causes cleavage of disulfide bond with the precipitation of PGlu homopolymer in diethyl ether according to ¹H NMR analysis. When we turn to an alternative deprotection under basic condition using NaOH similar to Dong's report⁴³ (Experimental section), PS–PGlu5 is obtained as a sodium salt after the lyophilization. Disappearance of benzyl methylene signal in ¹H NMR spectrum at 5.0 ppm confirms the successful deprotection (Fig. S6B†). PS–PGlu5 micelle is prepared by a similar dialysis. Both DLS and TEM support larger spherical micellar size of PS–PGlu5 (366.7 nm) than PS–PGlu1 (63.3 nm) (Table 3 and Fig. S7†). When the micelle solution is treated with 20 mM of DTT under argon atmosphere, the disulfide linkage between PS and PGlu blocks breaks. After incubation for 48 h, suspending sediments decrease the transmittance compared with the original micelle solution (Fig. 6A), indicating that hydrophobic PS segments precipitate after the detachment of hydrophilic PGlu segments. After filtration of both two solutions in Fig. 6A, DTT-treated solution does not have Tyndall phenomenon to scatter light as the untreated micelles do (Fig. 6B). DLS analysis also shows absence of assembly structure in DTT-treated solution, which suggests the destruction of micellar structure in reductive environment and further proves the feasibility of introducing reduction-responsive feature to the block copolymer. With our post-modification protocol, disulfide bond can be easily incorporated to enable the synthesis of dual or multi stimuli-responsive polypeptide conjugates for bio-related applications.

Fig. 6 Photos of PS–PGlu5 micelle solutions (A) before (left) and after (right) treatment with DTT, and Tyndall phenomenon (B) of PS–PGlu5 micelle solution (left) and DTT-treated sample after filtration (right) by a laser with a wavelength of 650 nm.

Conclusion

We report a versatile protocol to synthesize polypeptide conjugates with common RAFT-prepared polymers. Key step includes one-pot aminolysis and thiol capping which remove TTC group and incorporate amine or disulfide-carrying amine functionality simultaneously. Well-defined PS-b-PBLG and corresponding amphiphilic PS-b-PGlu block copolymers with or without disulfide linkage are successfully synthesized. The pH-responsiveness of PS-b-PGlu micelles originating from hydrophilic–hydrophobic transformation of PGlu segments in aqueous solution is monitored by DLS and CD. Reduction-responsive behavior of PS–SS–PGlu micelles is observed by cleavage of disulfide linkage and micelle destruction in reductive environment, verifying a robust and simple way to incorporate disulfide linkage quantitatively between two blocks.

The convenient synthesis protocol produces block copolymers of polypeptide and vinyl polymer. Various polymers are accessible from NCA and vinyl monomers in this way.

Acknowledgements

This work was supported by National Natural Science Foundation of China (21174122 and 21374093) and Special Funds for Major Basic Research Projects (G2011CB606001).

References

1. J. Huang and A. Heise, Chem. Soc. Rev., 2013, 42, 7373–7390.
2. H. Lu, J. Wang, Z. Song, L. Yin, Y. Zhang, H. Tang, C. Tu, Y. Lin and J. Cheng, Chem. Commun., 2013, 50, 139–155.
3. T. J. Deming, in Peptide Hybrid Polymers, ed. H. A. Klok and H. Schlaad, 2006, vol. 202, pp. 1–18.
4. C. Deng, J. Wu, R. Cheng, F. Meng, H.-A. Klok and Z. Zhong, Prog. Polym. Sci., 2014, 39, 330–364.
5. T. J. Deming, Prog. Polym. Sci., 2007, 32, 858–875.
6. M. D. Rikkou and C. S. Patrickios, Prog. Polym. Sci., 2011, 36, 1079–1097.
7. C. Boyer, V. Bulmus, T. P. Davis, V. Ladmiral, J. Liu and S. Perrier, Chem. Rev., 2009, 109, 5402–5436.
8. A. E. Smith, X. Xu and C. L. McCormick, Prog. Polym. Sci., 2010, 35, 45–93.
9. A. Gregory and M. H. Stenzel, Prog. Polym. Sci., 2012, 37, 38–105.
10. N. Hadjichristidis, H. Iatrou, M. Pitsikalis and G. Sakellariou, Chem. Rev., 2009, 109, 5528–5578.
11. H. R. Kricheldorf, Angew. Chem., Int. Ed., 2006, 45, 5752–5784.
12. R. Fu and G.-D. Fu, Polym. Chem., 2011, 2, 465–475.
13. X. Zhang, J. Li, W. Li and A. Zhang, Biomacromolecules, 2007, 8, 3557–3567.
14. L. Deng, K. Shi, Y. Zhang, H. Wang, J. Zeng, X. Guo, Z. Du and B. Zhang, J. Colloid Interface Sci., 2008, 323, 169–175.
15. X. Zhang, M. Oddon, O. Giani, S. Monge and J.-J. Robin, Macromolecules, 2010, 43, 2654–2656.
16. J. Jacobs, N. Gathergood and A. Heise, Macromol. Rapid Commun., 2013, 34, 1325–1329.
17. V. Vazquez-Dorbatt, Z. P. Tolstyka and H. D. Maynard, Macromolecules, 2009, 42, 7650–7656.
18. A. Postma, T. P. Davis, R. A. Evans, G. Li, G. Moad and M. S. O'Shea, Macromolecules, 2006, 39, 5293–5306.
19. A. W. Jackson and D. A. Fulton, Macromolecules, 2010, 43, 1069–1075.
20. H. Peng, J. Ling and Z. Shen, J. Polym. Sci., Part A: Polym. Chem., 2012, 50, 1076–1085.
21. H. Peng, J. Ling, Y. Zhu, L. You and Z. Shen, J. Polym. Sci., Part A: Polym. Chem., 2012, 50, 3016–3029.
22. J. Ling, H. Peng and Z. Shen, J. Polym. Sci., Part A: Polym. Chem., 2012, 50, 3743–3749.
23. M. T. Reetz, M. Rentzsch, A. Pletsch, A. Taglieber, F. Hollmann, R. J. G. Mondiere, N. Dickmann, B. Hoecker, S. Cerrone, M. C. Haeger and R. Sterner, ChemBioChem, 2008, 9, 552–564.
24. Y.-J. Zhao, Y.-q. Zhai, Z.-G. Su and G.-H. Ma, Polym. Adv. Technol., 2010, 21, 867–873.
25. H. Li, M.-a. Hao, L. Wang, W. Liang and K. Chen, Org. Prep. Proced. Int., 2009, 41, 301–307.
26. Y. K. Chong, G. Moad, E. Rizzardo and S. H. Thang, Macromolecules, 2007, 40, 4446–4455.
27. C. Boyer, V. Bulmus and T. P. Davis, Macromol. Rapid Commun., 2009, 30, 493–497.
28. C. Boyer, A. Granville, T. P. Davis and V. Bulmus, J. Polym. Sci., Part A: Polym. Chem., 2009, 47, 3773–3794.
29. J. R. McKee, V. Ladmiral, J. Niskanen, H. Tenhu and S. P. Armes, Macromolecules, 2011, 44, 7692–7703.
30. X.-P. Qiu and F. M. Winnik, Macromol. Rapid Commun., 2006, 27, 1648–1653.
31. C. E. Hoyle and C. N. Bowman, Angew. Chem., Int. Ed., 2010, 49, 1540–1573.
32. D. J. Smith, E. T. Maggio and G. L. Kenyon, Biochemistry, 1975, 14, 766–771.
33. C. Deng, G. Z. Rong, H. Y. Tian, Z. H. Tang, X. S. Chen and X. B. Jing, Polymer, 2005, 46, 653–659.
34. C. Deng, H. Y. Tian, P. B. Zhang, J. Sun, X. S. Chen and X. B. Jing, Biomacromolecules, 2006, 7, 590–596.
35. M. Le Hellaye, N. Fortin, J. Guilloteau, A. Soum, S. Lecommandoux and S. M. Guillaume, Biomacromolecules, 2008, 9, 1924–1933.
36. M. Schappacher, A. Soum and S. M. Guillaume, Biomacromolecules, 2006, 7, 1373–1379.
37. Y. J. Fan, G. P. Chen, J. Tanaka and T. Tateishi, Biomacromolecules, 2005, 6, 3051–3056.
38. J. Babin, D. Taton, M. Brinkmann and S. Lecommandoux, Macromolecules, 2008, 41, 1384–1392.
39. J. Liu, V. Bulmus, C. Barner-Kowollik, M. H. Stenzel and T. P. Davis, Macromol. Rapid Commun., 2007, 28, 305–314.
40. C. Boyer, J. Liu, L. Wong, M. Tippett, V. Bulmus and T. P. Davis, J. Polym. Sci., Part A: Polym. Chem., 2008, 46, 7207–7224.
41. P. J. Roth, D. Kessler, R. Zentel and P. Theato, J. Polym. Sci., Part A: Polym. Chem., 2009, 47, 3118–3130.
42. P. J. Roth, D. Kessler, R. Zentel and P. Theato, Macromolecules, 2008, 41, 8316–8319.
43. W.-F. Dong, A. Kishimura, Y. Anraku, S. Chuanoi and K. Kataoka, J. Am. Chem. Soc., 2009, 131, 3804–3805.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra17094f

---