Shin-nosuke
Nishimura
,
Nobuyuki
Higashi
* and
Tomoyuki
Koga
*
Department of Molecular Chemistry & Biochemistry, Faculty of Science & Engineering, Doshisha University, Kyotanabe, Kyoto, 610-0321, Japan. E-mail: tkoga@mail.doshisha.ac.jp; nhigashi@mail.doshisha.ac.jp
First published on 9th November 2018
Significant efforts have been dedicated toward designing conjugated sequence-controlled peptides and synthetic vinyl polymers as a new class of polymeric materials with ordered structures and specific biofunctions. The creation of novel synthetic strategies that enables the precise incorporation of multiple tailored peptides along a vinyl polymer backbone remains highly challenging. Herein, we report a useful method for preparing multiblock architectures composed of alternately aligned sequential peptides and various vinyl polymers through nitroxide-mediated polymerization (NMP). A new cyclic oligopeptide was developed containing a 2,2,5-trimethyl-4-phenyl-3-azahexane-3-nitroxide (TIPNO)-derived alkoxyamine bond in the framework. Controlled radical polymerization in a homogeneous liquid phase using this peptide initiator successfully provided a well-defined multiblock hybrid polymer in one step, in which chain extension and multimerization proceeded simultaneously. This method was applied to the polymerizations of a wide range of vinyl monomers, including styrene, acrylamides, acrylates and acrylonitrile. Expanding the compatibility of monomers with this approach is valuable for constructing complex multiblock architectures with structural and functional diversity.
One successful approach to constructing multiblock architectures involves a combination of click chemistry and living radical polymerizations.30,31 This approach, however, requires precise transformations of the chain ends on each block to provide appropriate reactive groups (i.e., azide/alkyne). Chromatographic purification is needed for isolating the objective polymer after the click reaction. Recently, we reported that the nitroxide-mediated polymerization (NMP) of styrene and its derivatives by cyclic peptide initiators having a 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO)-derived alkoxyamine bond in the framework successfully yielded multiblock hybrids composed of alternately aligned peptides and well-defined polystyrenes with low dispersities in one step.32 Significantly, this method permitted easy manipulation of the peptide sequence in the multiblock hybrids,32 although it was unfortunately only applicable to aromatic vinyl monomers due to the nature of the TEMPO group, which featured a relatively low dissociation rate constant (kd) of the C–ON bond.33 A new cyclic peptide initiator that provides more flexibility for polymer design is strongly required to produce functional multiblock architectures with structural diversity.
To overcome the limitations of monomer compatibility, we designed a novel hetero-bifunctional 2,2,5-trimethyl-4-phenyl-3-azahexane-3-nitroxide (TIPNO)-derived alkoxyamine (Fmoc-NH-TIPNO-COOH) for the synthesis of a versatile peptide initiator. A significant structural feature of TIPNO was the presence of a hydrogen atom attached to one of the α-carbons bonded to the nitrogen. This proton changed the kd value of the alkoxyamine bond and consequently increased the range of vinyl monomers that could be polymerized in a well-controlled living manner.33,34
Herein, we describe the preparation of a novel TIPNO-based cyclic peptide and its usefulness as an NMP initiator for the one-step production of a wide variety of multiblock copolymers composed of alternately aligned sequential peptides and various vinyl polymers such as polyacrylamides, polyacrylates, polyacrylonitrile and polystyrene.
We next prepared a new TIPNO-based cyclic peptide as an NMP initiator. In this system, the thermally promoted homolysis of the alkoxyamine bond generates a reactive carbon radical and a nitroxide radical simultaneously at the C and N termini of the peptide, respectively. In the presence of vinyl monomers, chain polymerization proceeds only from the carbon radical at the peptide terminus, not from the stable nitroxide radical; however, the nitroxide radical can trap the intermediate growing polymer radical reversibly, which provides a multiblock architecture during polymerization.
First, the peptide segment was constructed on an Fmoc-NH-SAL MBHA resin by SPPS using Fmoc-derivatives. We selected a tetraleucine sequence as an example because of its structural simplicity and easy characterization. TIPNO-derived alkoxyamine was next introduced at the N-terminus of the peptide using 7. The allyl group of the Asp residue and Fmoc group were removed by treating with palladium(0) and piperidine, respectively. Subsequently, intramolecular cyclization of the peptide was carried out on the resin through condensation between the N-terminus and the Asp side chain. Finally, the resin was treated with TFA to give the objective TIPNO-based cyclic peptide initiator. The molecular structure was verified by MALDI-TOF MS and 1H NMR analyses (Fig. 1). The observed molecular weight ([M + H]+obsd. = 1233.68) agreed well with the theoretical value ([M + H]+calcd. = 1233.57). 1H NMR analysis also gave satisfactory results.
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Fig. 1 A. MALDI-TOF MS spectrum of the TIPNO-based cyclic peptide initiator. Matrix: DHBA. B. 1H NMR spectrum of the peptide in DMSO-d6 at 25 °C. |
The versatility of this new type of peptide initiator was demonstrated by conducting the polymerization using a wide range of vinyl monomers, including acrylamides, acrylates, acrylonitrile, and styrene (Scheme 2). Initially, NMPs of the acrylamides were tested. Polymerization of N-isopropylacrylamide (NIPAM) was performed in DMF at 120 °C, and the molecular weights and polydispersity indexes (Đ) were evaluated by size-exclusion chromatography (SEC). Table 1 summarizes the resulting products obtained after polymerization for 1 (P1), 3 (P2), 6 (P3), 12 (P4), and 24 h (P5). The SEC traces of all polymers were unimodal and showed a clear shift toward high molecular weights with conversion (Fig. 2); however, the number average molecular weights (Mn) of these polymers deviated remarkably from the calculated theoretical values (Mn,Theor) based on the monomer conversion, assuming one peptide segment per chain (for P1 (Mn = 7000 g mol−1, Mn,Theor = 2820 g mol−1), P2 (Mn = 11200 g mol−1, Mn,Theor = 4850 g mol−1), P3 (Mn = 19
700 g mol−1, Mn,Theor = 6400 g mol−1), P4 (Mn = 24
500 g mol−1, Mn,Theor = 9100 g mol−1), P5 (Mn = 38
600 g mol−1, Mn,Theor = 11
400 g mol−1)). The polydispersities were relatively broad (Đ ≈ 1.8). These polymers were soluble in water and showed lower critical solution temperature (LCST) behaviors, due to the PNIPAM block. On the other hand, circular dichroism (CD) analysis clearly indicated the presence of a peptide segment in the polymer chain (e.g., for P2; Fig. 3B (blue line)). The CD spectrum showed a mixed pattern of β-sheet and random coil structures with a negative maximum at 208 nm and a shoulder at 218 nm. Moreover, the 1H NMR spectrum (Fig. 3C, blue line) supported the presence of both peptide and PNIPAM segments. These results indicated the production of multiblock structures through the ring-opening of the peptide initiator and subsequent NMP. Note that the observed LCST for the multiblock hybrid was distinctly shifted to the lower temperature side compared with that for the PNIPAM homopolymer even in the case of the hybrid with a long PNIPAM block length (Fig. S9†). This can be attributed to the introduction of hydrophobic tetraleucine blocks.
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Scheme 2 Schematic illustration of the one-step synthesis of versatile multiblock peptide–polymer hybrids via NMP utilizing the TIPNO-based cyclic peptide initiator. |
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Fig. 2 SEC traces (THF, 40 °C) of the multiblcok copolymers (P1–P5) obtained after polymerization for 1 (blue), 3 (red), 6 (green), 12 (pink) and 24 h (yellow). |
Polymer | Monomer | Polymn. time (h) | Polymn. temp. (°C) | Conv. (%) |
M
n, Theor![]() |
Before fragmentation | After fragmentation | ||||
---|---|---|---|---|---|---|---|---|---|---|---|
M n (g mol−1) |
M
p![]() |
Đ (Mw/Mn) | M n (g mol−1) |
M
p![]() |
Đ (Mw/Mn) | ||||||
a Theoretical number-average molecular weight of the fragmented polymer calculated by using the following equation: Mn,Theor. = [monomer]/[initiator] × conversion × molecular weight of monomer + molecular weight of the initiator. b Molecular weight at the top of the peak in the SEC curve. c Number-average molecular weight calculated by SEC analysis (PSt standard) in THF at 40 °C. d Number-average molecular weight calculated by SEC analysis (PMMA standard) in THF at 40 °C. e Number-average molecular weight calculated by SEC analysis (PMMA standard) in DMF (containing 10 mM LiBr) at 40 °C. | |||||||||||
P1 | NIPAM | 1 | 120 | 14.0 | 2820 | 7000d | 10![]() |
1.77 | 2400d | 3400 | 1.20 |
P2 | NIPAM | 3 | 120 | 32.1 | 4850 | 11![]() |
17![]() |
1.67 | 3800d | 4800 | 1.25 |
P3 | NIPAM | 6 | 120 | 45.6 | 6400 | 19![]() |
30![]() |
1.87 | 7500d | 9600 | 1.19 |
P4 | NIPAM | 12 | 120 | 69.8 | 9100 | 24![]() |
36![]() |
1.67 | 8300d | 12![]() |
1.21 |
P5 | NIPAM | 24 | 120 | 89.9 | 11![]() |
38![]() |
64![]() |
2.12 | 12![]() |
21![]() |
1.28 |
P6 | NAAMe | 18 | 120 | 87.6 | 15![]() |
32![]() |
53![]() |
1.46 | 12![]() |
19![]() |
1.23 |
P7 | tBuA | 17 | 120 | 79.4 | 11![]() |
26![]() |
42![]() |
1.89 | 9500d | 13![]() |
1.18 |
P8 | EA | 25 | 120 | 42.6 | 5500 | 9500d | 18![]() |
1.63 | 3700d | 6200 | 1.22 |
P9 | AN | 16 | 120 | 97.7 | 6400 | 34![]() |
43![]() |
1.54 | 13![]() |
15![]() |
1.38 |
P10 | St | 72 | 110 | 76.5 | 9200 | 38![]() |
55![]() |
1.77 | 8400c | 9100 | 1.10 |
These structures were characterized by conducting fragmentation experiments using a radical crossover reaction.37 Assuming a multiblock structure, the obtained polymer must include thermally labile alkoxyamine bonds in the main chain at multiple points, reflective of the block number. Fragmentation reactions of P1–P5 were performed in the presence of a large excess of TIPNO (>70 equiv.) in N-methyl-2-pyrrolidone at 120 °C over 12 h, and the resultant polymers were isolated using a reprecipitation method. As a representative example, SEC analysis of P2 (Fig. 3A) showed a marked decrease in both the molecular weight and polydispersity index after treatment, from Mn = 11200 g mol−1 (Đ = 1.67) to Mn = 3800 g mol−1 (Đ = 1.25). Other polymers showed similar fragmentation behaviors (Fig. S11A–S14A†). On the other hand, the 1H NMR (Fig. 3C, S11C–S14C†) and FTIR spectra before and after fragmentation (Fig. S10, S11B–S14B†) did not change significantly, demonstrating cleavage of the polymer chain without altering the compositions of the PNIPAM/peptide blocks, namely, fragmentation from a multi- to a diblock structure. Note that fragmentation caused a conformational change in the peptide block. The CD spectrum of the fragmented P2 (diblock form) showed a pattern typical of a random coil peptide, with a negative maximum at 200 nm (Fig. 3B, red line). Thus, the unique multiblock structure with alternately aligned peptides and PNIPAM blocks increased the local peptide concentration. As a result, the hydrophobic tetraleucine blocks self-organized into β-sheets in water. In fact, increasing the chain lengths of the PNIPAM blocks tended to suppress high-order structuring of the peptide blocks (Fig. S11D–14D†).
The most important feature of this method is that the PNIPAM block length was precisely controlled during conversion. In other words, a specific spatial arrangement of peptide blocks could be designed in a single polymer chain. The SEC charts of the fragmented diblock polymers obtained from P1–P5 were symmetrically unimodal and showed a clear shift toward high molecular weights as the polymerization time increased (Fig. 4A). Fig. 4B plots Mn and Đ of the fragmented polymers as a function of the monomer conversion. In this figure, the calculated values of the theoretical Mn,Theor are included as a solid line. The Đ values fell below 1.3 throughout the polymerization reaction, indicating that nearly monodisperse polymers were formed. The linearity of the Mnversus conversion plot, which was comparable to the Mn,Theor plot, clearly showed that NMP proceeded in a well-controlled living manner. These results also indicated that the initiation efficiency of this cyclic peptide initiator was sufficiently high. The ratio of the molecular weight of the original polymer to the corresponding fragmented one (Table 1) suggested that the multiblock copolymers [(Leu)4-b-PNIPAM]m contained approximately three repeats (m ≈ 3) of the (Leu)4-b-PNIPAM diblock unit (i.e. total hexablock). This TIPNO-based cyclic peptide initiator could be applied to other acrylamides derived from the amino acid, N-acryloyl-L-alanine O-methyl ester (NAAMe).
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Fig. 4 A. SEC traces (THF, 40 °C) of the fragmented block copolymers obtained from P1–P5, demonstrating excellent controlled polymerization. B. Plots of Mn (blue circles) and Đ (red triangles) as a function of the conversion rate. The solid line represents the theoretical Mn (see the footnote (a) in Table 1). |
Amino acid-based vinyl polymers have enormous potential as smart biomaterials because they show tunable thermo-responsive behaviors in water and excellent biocompatibilities.38–41 For example, we reported that the LCST/UCST behaviors of amino acid-based vinyl polymers depended on the type of amino acid and the chemical modification of the pendant group.39 The objective multiblock hybrid (P6) was successfully obtained by NMP in one step and was characterized by SEC, 1H NMR, and FTIR analyses in the same manner as described above (Table 1, Fig. S15†). This polymer was soluble in water (below the LCST of the PNAAMe block, 18 °C (ref. 39)) and cleavable by a thermal treatment (under excess TIPNO). The fragmented P6 showed an Mn value close to the Mn,Theor as well as a relatively low dispersity (Đ = 1.23). The hybrid P6 assumed an unprecedented and unique multiblock architecture composed of sequential peptides and amino acid-based polymers; therefore, it is highly attractive as both a stimuli-responsive and protein-mimetic biomaterial.
We next polymerized two acrylates: tert-butyl acrylate (tBuA) and ethyl acrylate (EA). Acrylate polymers are useful as general-purpose plastics over large areas. The one-step syntheses of the multiblock hybrids using TIPNO-based cyclic peptides were successful in both cases (P7 (Mn ≈ 26300) and P8 (Mn ≈ 9500)) (Table 1, Fig. S16–S18†). The fragmentation experiments showed that the dispersities of the PtBuA and PEA blocks remained satisfactorily low (Đ < 1.25), and both polymers featured three repeats (m ≈ 3) of the peptide-b-polyacrylate diblock unit. It should be noted that the PtBuA blocks in P7 could be converted easily into poly(acrylic acid) (PAA) blocks by removing the tBu groups (Fig. S17†). The resultant fully deprotected hybrid was soluble in water and exhibited pH-responsiveness.
In a previous study,32 we reported that the copolymerization of acrylonitrile (AN) and styrene (St) proceeded via a TEMPO-based cyclic peptide initiator only when St was the major component; however, the TIPNO-based cyclic peptide initiator allowed homopolymerization of AN to proceed in DMF at 120 °C and yielded a multiblock peptide/PAN hybrid of Mn ≈ 34200 (P9) (Table 1, Fig. S19†). FTIR and 1H NMR analyses verified the formation of PAN blocks (C
N stretching bond at ν = 2242 cm−1 and 1H signal at δ ≈ 3.2 ppm (–CH(C
N)–), although the observed dispersity (Đ = 1.38) was slightly larger than that of the other vinyl monomers. Of course, this new cyclic initiator, as well as the previously reported TEMPO-based initiator, is applicable to the polymerization of aromatic St and gives well-regulated multiblock hybrids (P10, Table 1, Fig. S20†). Notably, an enhanced polymerization rate was observed for the TIPNO-based cyclic peptide system (conversion 77% at 72 h) compared to the rate obtained using the TEMPO-based initiator (34% at 70 h)32 under the same reaction conditions, reflecting the difference between the dissociation rate constants of the two alkoxyamines.
Footnote |
† Electronic supplementary information (ESI) available: Experimental details and supplemental figures. See DOI: 10.1039/c8py01330f |
This journal is © The Royal Society of Chemistry 2019 |