Article Cite This: Macromolecules XXXX, XXX, XXX−XXX
Polymerization of N‑Substituted Glycine N‑Thiocarboxyanhydride through Regioselective Initiation of Cysteamine: A Direct Way toward Thiol-Capped Polypeptoids Xinfeng Tao,†,‡ Botuo Zheng,† Tianwen Bai,† Min-Hui Li,†,‡,§ and Jun Ling*,† †
MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China ‡ PSL Université Paris, CNRS, Institut de Recherche de Chimie Paris, UMR8247, Chimie ParisTech, 11 rue Pierre et Marie Curie, 75005 Paris, France § Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology, 15 North Third Ring Road, Chaoyang District, 100029 Beijing, China S Supporting Information *
ABSTRACT: Because of the high reactivity, N-carboxyanhydride (NCA) can be initiated by the thiol group. On the contrary, N-thiocarboxyanhydride (NTA) is more stable and is able to tolerate it. Herein, we apply cysteamine as a regioselective initiator for ring-opening polymerization (ROP) of N-substituted glycine N-thiocarboxyanhydride (NNTA) to synthesize well-defined thiol-capped polypeptoids. ROPs of sarcosine NTA (Sar-NTA) and N-ethylglycine NTA (NEG-NTA) are well controlled when [M]/[I] ≤ 100 with high yields (>87.5%) producing polypeptoids with designable molecular weights and low polydispersity indices (87.5%) (samples 4−8 in Table 2). The structure of the product was determined by 1H NMR and 1 H−1H COSY analyses. As shown in 1H NMR spectra (Figure 3), PSar repeat units signals (Hc+c′ and Hd+d′) are assigned the same as our previous report,20 and the methyl and methylene proton signals of the chain end sarcosine unit are present at 2.49 ppm (Hf+f′) and 3.81 ppm (He+e′). The signals at 2.54 ppm (Ha) and 3.25 ppm (Hb) are assigned to methylene protons of cysteamine residue. The signals at 2.74 ppm (Ha′) and 3.45 ppm (Hb′) are attributed to the formation of disulfide linkage due to oxidation reaction between partial thiol groups (see the discussion below). The assignments of end-groups are also verified by treatment with trifluoroacetic anhydride. Ha, He, and Hf signals disappear after the addition of trifluoroacetic anhydride (Figure S6) in NMR tube with DMSO-d6 as solvent. The thiol group and amino group react with trifluoroacetic anhydride to form the thioester and amide group, respectively, and the corresponding adjacent protons signals of methylene (Ha’) and methyl (Hf’) shift to downfield at 2.58−2.66 ppm
Figure 2. (A) 1H NMR spectrum of sample 3. (B) SEC traces of PSar (sample 3) before and after treatment with cysteine and TCEP.
1.2−1.5 ppm were absent. Calculated by the intensities of Ha and Hd, the degrees of polymerization (DPs) of the products supported the single-site initiation of benzylamine, consistent with feed molar ratios and monomer conversions. Monomodal and symmetrical SEC trace (Figure 2B) showed a narrow PDI (Đ = 1.10), similar to our previous report.20 Although the BA+DT initiator is quite dilute in the polymerization solution, we still consider the possibility that thiol group is deprotonated by amine group through the hydrogen bonding. It may make thiol able to initiate the polymerization, while the thioester group is rearranged by amine group during the polymerization to form the more stable C
DOI: 10.1021/acs.macromol.8b00259 Macromolecules XXXX, XXX, XXX−XXX
Article
Macromolecules
formed at the beginning of the polymerization while rearranged by the amine group during the polymerization. To exclude the possibility, we studied the early stage (30 min) of cysteamineinitiated polymerization (sample S1 in Table S1). The signal of the thioester carbon around 190−200 ppm46−49 is not detectable in 13C NMR spectra of both early stage (sample S1, Figure S3D) or prolonged product (sample 4, Figure S3E). The characteristic stretching vibration of thioester17 around 1700−1750 cm−1 is also absent in FTIR spectra of early stage product (Figure S4D). All the above results indicate that the thiol group of cysteamine cannot initiate the polymerization of Sar-NTA, and only amine-initiated α-thiol-ω-aminotelechelic PSars are obtained. MALDI-ToF mass spectrum of sample 4 (Figure 4A−C) shows a symmetrical and monomodal distribution, different from bimodal distribution products initiated by 2-amino-1ethanol or 3-amino-1-propanol,31 indicating all the products are pure α-thiol-ω-aminotelechelic PSars without the existence of α,ω-diaminotelechelic PSars. A bimodal distribution SEC trace of sample 5 is observed (Figure S8), but the area ratio of the two peaks changes in repeating tests. We attribute it to the formation of disulfide bond products due to the oxidation reaction between the thiol end-groups. The eluent of SEC measurement contains 2‰ triethylamine. The alkaline condition and high temperature accelerate the oxidation of thiol groups. PSar chains with disulfide linkages are also observed in the MALDI-ToF MS spectrum of sample 5 after the solution was exposed in the open air for hours (Figure S9). Thus, we added dithiothreitol (DTT) to the sample 5 solution to prevent the oxidation of thiol groups during the SEC measurement. The SEC trace of sample 5 became monomodal and symmetrical (Figure S8), and the peak is exactly coincident with lower MW peak of the bimodal distribution SEC trace. All the above confirm that the bimodal distributed SEC trace are caused by formation of PSar containing disulfide group instead of α,ω-diaminotelechelic PSar from the simultaneous initiations by both thiol and amino groups. With the addition of DTT in the measurement, SEC traces of samples 4−8 are all
Table 2. Polymerization of Sar-NTA and NEG-NTA Initiated by Cysteaminea
The polymerizations were carried out at 60 °C for 24 h in acetonitrile for Sar-NTA or THF for NEG-NTA with [M] = 0.5 mol/L. bAs determined by 1H NMR. cAs determined by SEC measurement. dThe end-group integral in 1H NMR spectra is not accurate enough for the calculation. a
while the methylene (He’) signal overlaps with those of sarcosine methylene protons (3.9−4.5 ppm). 1H−1H COSY spectra (Figure S7) further confirm the assignments of endgroups. The methylene signal Ha of cysteamine residue is coupled with Hb (inset A in Figure S7). Likewise, the methylene signals Ha′ and Hb′ on disulfide product are also coupled (inset B in Figure S7). DPs and MWs determined by 1 H NMR are consistent with the feeding ratios of monomer to initiator when [M]/[I] ≤ 100, which certifies the single-site initiation of the amine group in well-controlled characteristics. What is more, if the thiol group initiated the polymerization of Sar-NTA, a thioester group would be formed (Scheme S4). However, the coupled signal of adjacent methylene to thioester (green box in Scheme S4) around 3.0 ppm46−49 is not observed in Figure S7. It is also possible that the thioester group is
Figure 3. 1H NMR spectrum of PSar (sample 4 in Table 2) in D2O. D
DOI: 10.1021/acs.macromol.8b00259 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules
Figure 4. MALDI-ToF mass spectra of PSar in sample 4 (A) and the corresponding product after thiol−ene reaction with styrene (D), with zoom-in views (B and E), and the corresponding chemical structures (C and F).
ToF profile (Figure S11) and SEC trace (Figure S12) confirmed that there was no MW decrease after the treatment. We also tried the NCL method30 to confirm the absence of the thioester group. Sample S1 (Table S1) was treated with cysteine and TCEP in aqueous solution (pH = 7.0) at room temperature for 16 h. The SEC traces (Figure S13) and MALDI-ToF mass spectra (Figure S14) of sample S1 kept the same before and after treatment of cysteine and TCEP, and no PSar with cysteine residue was observed in Figure S14D−F, indicating the absence of the thioester group and no breakage of polymer chains. Therefore, the thiol group on cysteamine remained intact during the polymerization of Sar-NTA. Finally, the free thiol content of the polymer was determined by using Ellman’s assay.57−59 After the cysteamine-initiated polymerization of Sar-NTA, the PSar was collected by remove the solvent under vacuum and dissolved in reaction buffer (0.1 M disodium hydrogen phosphate, 1 mM EDTA, pH 8.0) for test. The molar number of thiol group determined by Ellman’s assay is 7.323 × 10−5 mol (Table S2), which agrees well with the thiol value in feed (7.392 × 10−5 mol). More than 99% polymer chains contain the thiol end-group, which indicates that the thiol group of cysteamine cannot initiate the polymerization of NNTA and only amine-initiated α-thiol-ωaminotelechelic polypeptoids are obtained. Controlled polymerizations of NEG-NTA initiated by cysteamine were achieved in THF at 60 °C, producing PNEGs with high yields (>90.5%) and narrow MW distribution (Đ = 1.12−1.20) (samples 9−12 in Table 2). The thiol endgroup of the obtained PNEG was confirmed by 1H NMR (Figure 6), 13C NMR (Figure S3F), and 1H−1H COSY spectra (Figure S15). DPs of the products calculated from the 1H NMR spectra are consistent with the feed molar ratios when [M]/[I] ≤ 100, which demonstrates the controlled manner of the cysteamine-initiated polymerizations. As the monomodal SEC traces shown in Figure S16, MW of PNEG increases with the growth of [NEG-NTA]/[cysteamine]. The high-MW shoulders in some SEC traces are caused by the aggregation of polypeptoid chains in solvents.31,54−56 MALDI-ToF mass spectra of samples 9 and 10 (Figure 7 and Figure S17) reveal
monomodal with narrow distribution (Đ < 1.18) (Figure 5). The MWs increase gradually with the rise of feed molar ratios
Figure 5. SEC traces of samples 4−8 (excess DTT was added to the polymer solutions).
of Sar-NTA to cysteamine. The MWs determined by SEC are close to the results initiated by benzylamine at the same [M]/ [I].20 We attribute the high-MW shoulders of some SEC traces to the aggregation of polypeptoid chains in solvents.31,54−56 The MALDI-ToF mass spectrum of sample 6 (Figure S10) reveals monomodal distributions with no high-MW shoulders while its SEC trace (green line in Figure 5) has a slight shoulder, which supports pure α-thiol-ω-aminotelechelic PSars and their aggregations in SEC solvents but not the existence of α,ω-diaminotelechelic polypeptoids. To further confirm the absence of thioester group in the products, we incubated sample 4 with excess benzylamine in acetonitrile at 60 °C for 16 h. If thioester group had existed, PSar with the benzyl end-group would have been formed due to the aminolysis of the thioester group. In the MALDI-ToF spectrum shown in Figure S11, only PSars with thiol endgroups were observed after being treated with benzylamine without any benzyl-terminated polymer chains. The MALDIE
DOI: 10.1021/acs.macromol.8b00259 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules
Figure 6. 1H NMR spectrum of PNEG (sample 9 in Table 2) in D2O.
Figure 7. MALDI-ToF mass spectra of PNEG in sample 9 (A) and the corresponding product after thiol−ene reaction with styrene (D), with zoomin views (B and E), and the corresponding chemical structures (C and F).
monomodal distributions of exclusively α-thiol-ω-aminotelechelic products with no shoulders at the high-MW range while their SEC profiles in Figure S16 have shoulders, which demonstrates the shoulders are polymer aggregations in SEC solvents but not the existence of α,ω-diaminotelechelic polypeptoids. According to the 1H NMR spectrum of sample 10 after treatment with cysteine and TCEP (Figure S18), the methine signal and carboxylic signal of cysteine residue around 4.7 and 11.0 ppm, respectively, are not observed, which confirms the absence of the thioester group and noninitiation of the thiol group on cysteamine. We achieve thiol group-tolerated and regioselective polymerization of NNTA initiated by
cysteamine to prepare thiol-capped polypeptoids in a direct way. Thiol−Ene Reactions. We carried out radical-mediated thiol−ene reaction not only to reveal the convenience of thiol− ene click reaction but also to certify the integrality of the thiol end-group. Styrene was selected as the model compound. Thiol-capped PSar (sample 4) and PNEG (sample 9) reacted with excess AIBN and styrene at 70 °C for 24 h (Scheme 1). In the isolated products, 1H NMR spectra (Figures S19 and S20) presented the appearance of benzene proton signals at 6.75− 7.50 ppm. All the polypeptoid chains having an oligostyrene (DP = 1−5) end-group shown in MALDI-ToF spectra (Figures 4D−F and 7D−F) demonstrated the successful thiol−ene F
DOI: 10.1021/acs.macromol.8b00259 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules
(3) Shen, Y.; Fu, X. H.; Fu, W. X.; Li, Z. B. Biodegradable stimuliresponsive polypeptide materials prepared by ring opening polymerization. Chem. Soc. Rev. 2015, 44, 612−622. (4) Deming, T. J. Synthesis of side-chain modified polypeptides. Chem. Rev. 2016, 116, 786−808. (5) Song, Z. Y.; Han, Z. Y.; Lv, S. X.; Chen, C. Y.; Chen, L.; Yin, L. C.; Cheng, J. J. Synthetic polypeptides: from polymer design to supramolecular assembly and biomedical application. Chem. Soc. Rev. 2017, 46, 6570−6599. (6) Knight, A. S.; Zhou, E. Y.; Francis, M. B.; Zuckermann, R. N. Sequence programmable peptoid polymers for diverse materials applications. Adv. Mater. 2015, 27, 5665−5691. (7) Secker, C.; Brosnan, S. M.; Luxenhofer, R.; Schlaad, H. Poly(αpeptoid)s revisited: synthesis, properties, and use as biomaterial. Macromol. Biosci. 2015, 15, 881−891. (8) Gangloff, N.; Ulbricht, J.; Lorson, T.; Schlaad, H.; Luxenhofer, R. Peptoids and polypeptoids at the frontier of supra- and macromolecular engineering. Chem. Rev. 2016, 116, 1753−1802. (9) Chan, B. A.; Xuan, S. T.; Li, A.; Simpson, J. M.; Sternhagen, G. L.; Yu, T. Y.; Darvish, O. A.; Jiang, N. S.; Zhang, D. H. Polypeptoid polymers: synthesis, characterization, and properties. Biopolymers 2018, 109, e23070. (10) Birke, A.; Ling, J.; Barz, M. Polysarcosine-containing copolymers: synthesis, characterization, self-assembly, and applications. Prog. Polym. Sci. 2018, 81, 163−208. (11) Kricheldorf, H. R. Polypeptides and 100 years of chemistry of αamino acid N-carboxyanhydrides. Angew. Chem., Int. Ed. 2006, 45, 5752−5784. (12) Deming, T. J. Synthetic polypeptides for biomedical applications. Prog. Polym. Sci. 2007, 32, 858−875. (13) Hadjichristidis, N.; Iatrou, H.; Pitsikalis, M.; Sakellariou, G. Synthesis of well-defined polypeptide-based materials via the ringopening polymerization of α-amino acid N-carboxyanhydrides. Chem. Rev. 2009, 109, 5528−5578. (14) Zhang, D. H.; Lahasky, S. H.; Guo, L.; Lee, C. U.; Lavan, M. Polypeptoid materials: current status and future perspectives. Macromolecules 2012, 45, 5833−5841. (15) Luxenhofer, R.; Fetsch, C.; Grossmann, A. Polypeptoids: a perfect match for molecular definition and macromolecular engineering? J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 2731−2752. (16) Tao, X. F.; Deng, C.; Ling, J. PEG-amine-initiated polymerization of sarcosine N-thiocarboxyanhydrides toward novel doublehydrophilic PEG-b-polysarcosine diblock copolymers. Macromol. Rapid Commun. 2014, 35, 875−881. (17) Tao, X. F.; Deng, Y. W.; Shen, Z. Q.; Ling, J. Controlled polymerization of N-substituted glycine N-thiocarboxyanhydrides initiated by rare earth borohydrides toward hydrophilic and hydrophobic polypeptoids. Macromolecules 2014, 47, 6173−6180. (18) Deng, Y. W.; Zou, T.; Tao, X. F.; Semetey, V.; Trepout, S.; Marco, S.; Ling, J.; Li, M. H. Poly(ε-caprolactone)-block-polysarcosine by ring-opening polymerization of sarcosine N-thiocarboxyanhydride: synthesis and thermoresponsive self-assembly. Biomacromolecules 2015, 16, 3265−3274. (19) Tao, X. F.; Du, J. W.; Wang, Y. X.; Ling, J. Polypeptoids with tunable cloud point temperatures synthesized from N-substituted glycine N-thiocarboxyanhydrides. Polym. Chem. 2015, 6, 3164−3174. (20) Tao, X. F.; Zheng, B. T.; Kricheldorf, H. R.; Ling, J. Are Nsubstituted glycine N-thiocarboxyanhydride monomers really hard to polymerize. J. Polym. Sci., Part A: Polym. Chem. 2017, 55, 404−410. (21) Zheng, B. T.; Tao, X. F.; Ling, J. Water tolerated polymerization of N-substituted glycine N-thiocarboxyanhydride initiated by primary amines. Acta Polym. Sin. 2018, 72−79. (22) Kricheldorf, H. R.; Sell, M.; Schwarz, G. Primary amine-initiated polymerizations of α-amino acid N-thiocarbonic acid anhydrosulfide. J. Macromol. Sci., Part A: Pure Appl. Chem. 2008, 45, 425−430. (23) Cao, J. B.; Siefker, D.; Chan, B. A.; Yu, T. Y.; Lu, L.; Saputra, M. A.; Fronczek, F. R.; Xie, W. W.; Zhang, D. H. Interfacial ring-opening polymerization of amino-acid-derived N-thiocarboxyanhydrides toward well-defined polypeptides. ACS Macro Lett. 2017, 6, 836−840.
reaction between thiol end-group and styrene. After the thiol− ene reaction with styrene, the SEC traces of samples 4 and 9 are still monomodal with narrow distributions (Figures S21 and S22).
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CONCLUSION The free thiol group has no influence to the primary amineinitiated NNTA polymerization. We successfully carried out thiol group-tolerated NNTA polymerizations initiated by cysteamine to synthesize well-defined thiol-capped polypeptoids. Together with excellent regioselective between amino and thiol ends, the polymerizations show good controllability to produce thiol-capped polypeptoids with high yields (>87.5%) and low PDIs (
