Article pubs.acs.org/Biomac
Fast and Living Ring-Opening Polymerization of α‑Amino Acid N‑Carboxyanhydrides Triggered by an “Alliance” of Primary and Secondary Amines at Room Temperature Wei Zhao,† Yves Gnanou,‡ and Nikos Hadjichristidis*,† King Abdullah University of Science and Technology (KAUST), †Physical Sciences and Engineering Division, KAUST Catalysis Center, Polymer Synthesis Laboratory, and ‡Physical Sciences and Engineering Division, Thuwal 23955, Kingdom of Saudi Arabia S Supporting Information *
ABSTRACT: A novel highly efficient strategy, based on an “alliance” of primary and secondary amine initiators, was successfully developed allowing the fast and living ring-opening polymerization (ROP) of α-amino acid N-carboxyanhydrides (NCAs) at room temperature.
■
INTRODUCTION Polypeptides, an important class of biopolymers, have found extensive applications, including drug delivery, tissue engineering, sensing, and catalysis.1 The most efficient method to produce polypeptides is by ring-opening polymerization (ROP) of α-amino acid N-carboxyanhydrides (NCAs) triggered by different nucleophiles.2 Although the polymerization of NCAs has been extensively studied since the early 1950s,3 their controlled polymerization was first achieved in 1997 by Deming who used transition metal complexes as initiators.4 In the following years, a few other controlled NCA polymerizations were reported using as initiators primary amine hydrochlorides or trifluoroboranes,5 organosilicon derivatives,6a rare earth metal complexes,6b,c or primary amine under high vacuum, low temperature, or nitrogen flow.7 Readily available primary, secondary, and tertiary amines can be used as initiators to produce polypeptides on a large scale without metal residuals. However, in the case of secondary and tertiary amines, two polymerization mechanisms, the so-called “normal amine” (NAM) and the “activated monomer” (AMM), occur simultaneously, yielding polypeptide samples with uncontrolled molecular weight and broad or bimodal molecular weight distribution.8 Only in the case of primary amines does polymerization exhibit the features of a “living” process, the propagation occurring by the sole normal amine mechanism (NAM) and at a slow rate (NAM and AMM, see Supporting Information). In a recent contribution,9 we unveiled that in an initiator like triethylaminetriamine (TREN) both its core tertiary amine and the three growing primary amines act cooperatively and in synergy to bring about a fast and yet “living” polymerization of NCAs through the so-called accelerated amine mechanism through monomer activation (AAMMA) (Scheme 1). As an extension of the latter work, we show in the following paper © XXXX American Chemical Society
Scheme 1. Accelerated Amine Mechanism through Monomer Activation (AAMMA) for the Polymerization of NCAs Initiated by TREN
that primary and secondary amines can also act cooperatively and in synergy to trigger the “living” polymerization of NCAs.
■
EXPERIMENTAL SECTION
General Methods. All reactions were carried out under a dry and oxygen-free argon atmosphere by using Schlenk techniques or under an argon atmosphere in a MBraun glovebox. Solvents were purified using the MBraun SPS system. Anhydrous dimethylformamide (DMF) was further dried by passing through an activated alumina column. All liquids were dried over activated 4 Å molecular sieves for a week and distilled before use, and all solid materials were used as received. All purified anhydrous reagents were stored over 4 Å molecular sieves in the glovebox. H-Glu(OBn)-OH and H-Lys(Z)-OH were purchased from Sigma-Aldrich and used as received. Glu-NCA and Lys-NCA were prepared and recrystallized four times following published procedures.6 The average water content in DMF and monomers (GluNCA and Lys-NCA) was 47 and 30 ppm, respectively, as measured using the C30 Compact Karl Fischer Coulometer from Mettler Toledo. Instruments and Measurements. 1H, 13C, and 15N NMR spectra were recorded on a Bruker AV600 spectrometer. NMR assignments were confirmed by 1H−1H (COSY), 1H−13C (HMQC), and 13C NMR (DEPT) experiments when necessary. Operando IR study of NCA polymerization was carried out by using ReactIR 45m Received: January 30, 2015 Revised: March 10, 2015
A
DOI: 10.1021/acs.biomac.5b00134 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules Table 1. Homo- and Sequential Block Copolymerization of Glu-NCA and Lys-NCA Initiated by Various Amines
entrya
initiator
[M]0/[I]0
time (min)
conv.b (%)
Mn,calcd ×10−4c
Mn,NMR ×10−4d
Mn,SEC‑LS ×10−4e
PDIe
1 2 3f 4g 5 6 7 8 9 10 11 12 13 14 15 16h 17
TETA TETA TETA TETA TETA TETA TETA TETA TETA DETA TEPA PEHA HMDA PEA PEA DMEDA HMDA/DMEDA
10/1 25/1 (25 + 25)/1 (25 + 25)/1 50/1 75/1 100/1 120/1 200/1 75/1 75/1 75/1 75/1 75/1 75/2 75/1 75/(1 + 1)
40 40 40 + 40 40 + 120 60 60 120 120 180 60 60 60 60 60 60 60 60
>99 >99 >99 >99 >99 >99 >99 >99 >99 93 95 97 69 45 72 88 90
0.22 0.55 1.10 1.20 1.10 1.64 2.19 2.60 4.38 1.53 1.56 1.60 1.13 0.74 0.59 1.45 1.48
0.23 0.60 1.20 1.26 1.17 1.64 2.32 2.69 4.51 1.60 1.63 1.64 1.12 0.85 0.71 2.83 2.31
0.24 0.63 1.27 1.33 1.18 1.80 2.34 2.77 4.61 1.59 1.60 1.61 1.18 0.80 0.68 1.69 1.48
1.04 1.08 1.13 1.10 1.11 1.18 1.18 1.17 1.29 1.14 1.16 1.14 1.17 1.17 1.18 1.36 1.12
a The polymerizations were carried out in DMF at 25 °C with [Glu-NCA]0 = 0.19 M. bFT-IR was used to monitor the conversion of NCA by determining the intensity of the NCA anhydride absorption band at 1787 cm−1. cCalculated from [Glu-NCA]/[I] × 219.24 × conv. d1H NMR spectroscopy. eSEC-MALS, 0.1 M LiBr in DMF at 60 °C. fSequential ROP of two portions of Glu-NCA. gSequential ROP of γ-benzyl-L-glutamate NCA (Glu-NCA) and ε-Cbz-L-lysine (Lys-NCA). hThe SEC trace was bimodal.
Figure 1. (A) Mn,SEC‑LS and PDI vs conversion (polymerization time in parentheses) for the ROP of Glu-NCA; [Glu-NCA]/[TETA] = 100, [GluNCA]0 = 0.19 M; DMF at 25 °C. (B) SEC profiles (RI signals) of representative samples, entries 1, 2, 5, 6, 7, and 9 in Table 1. (C) SEC profiles (RI signals) of the polymerization experiment: peak A, after prepolymerization of Glu-NCA (25 equiv to TETA, 40 min), Mn = 0.63 × 104, PDI = 1.08; peak B (entry 3) after polymerization of 25 equiv more Glu-NCA (40 min), Mn = 1.27 × 104, PDI = 1.13. (D) SEC profiles (RI signals) of copolymerization of Glu-NCA and Lys-NCA: peak A, after prepolymerization of Glu-NCA (25 equiv to TETA, 40 min), Mn = 0.63 × 104, PDI = 1.08; peak C (entry 4), after block copolymerization of Glu-NCA and Lys-NCA ([Glu-NCA]0/[ Lys-NCA]0/[I]0 = 25/25/1, 120 min), Mn = 1.33 × 104, PDI = 1.10.
B
DOI: 10.1021/acs.biomac.5b00134 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules
Figure 2. (A) Kinetics of the ROP of Glu-NCA initiated by different initiators (the automatic sampling interval is 5 s). (B) Mark−Houwink− Sakurada plots of PBLGs obtained from the ROP of Glu-NCA initiated by two different amines. with MCT Detector from Mettler Toledo AutoChem. DiComp (Diamond) probe was connected via AgX 9.5 mm × 1.5 m fiber (Silver Halide). The area from 2800 to 650 cm−1 was used for analysis (8 wavenumber resolution, automatic sampling interval: 5 s). The realtime concentration of NCA was quantified by measuring the intensity of NCA’s anhydride peak at 1787 cm−1 by FT-IR. The conversion of NCA was determined by comparing the NCA concentration in the polymerization solution to the NCA concentration at t = 0. The polymer characterizations were carried out by combining an Agilent 1260 infinity GPC instrument with a multiangle laser light scattering (MALLS) detector at 60 °C. The system was equipped with one guard and three columns (Styragel HT 2 DMF, Styragel HT 3 DMF, and Styragel HT 4 DMF), a 1200 HPLC pump, an Optilab T-rEX RI detector, a ViscoStar-II viscomety detector, and a DAWN HELEOS-II multiangle laser-light scattering (MALLS) detector (wavelength: 690 nm; from Wyatt Technology). HPLC-grade DMF (containing 0.1 M LiBr) was used as the mobile phase at a flow rate of 1.0 mL/min and 60 °C. Concentrations between 5.0 and 10.0 mg/mL were injected into the columns at an injection volume of 200 μL. ASTRA software from Wyatt Technology was used to collect and analyze the data. Typical Polymerization Procedure. A typical procedure for polymerization of NCA was performed in a 10 mL ampule in a Braun Labmaster glovebox. Taking the polymerization of entry 5 in Table 1 as an example, to a vigorously stirred solution of TETA in 2 mL of DMF (2.2 mg, 0.015 mmol) was added NCA monomer (0.2 g, 0.759 mmol) in 2 mL of DMF. The reaction mixture was stirred for a specific time at room temperature. After the measured time interval, a small amount of aliquot (several drops) was taken from the reaction mixture via a syringe for the determination of monomer conversion via FT-IR. At the same time, 0.2 mL of the reaction mixture was taken out from the system and diluted to 10 mg (PBLG)/mL using DMF (containing 0.1 M LiBr). The solution was then analyzed by SEC to determine the molecular weight and PDI of PBLG. The remaining final reaction mixture was precipitated with methanol, sonicated and centrifuged to remove the solvent. The obtained PBLG was collected and dried under vacuum overnight after two repetitions of sonicationcentrifugation procedure.
sity index (PDI) of the synthesized poly(γ-benzyl-L-glutamate) (PBLG). All polymerizations were accomplished with practically complete conversion in 3 h at room temperature and exhibited all characteristic features of “livingness”: (a) complete consumption of the monomer; (b) linearity of Mn with conversion; (c) excellent control of molecular weight assuming initiation occurs exclusively by primary amines; (d) narrow molecular weight distributions; and (e) synthesis of block copolypeptides by sequential monomer addition.10 Indeed GluNCAs could be polymerized in the two sequences without any chain breaking reactions and well-defined block copolymers of PBLG-b-PZLL could be prepared by sequential addition of Glu-NCA and ε-Cbz-L-lysine NCA (Lys-NCA) (Table 1, entries 3−4 and Figure 1C,D). The intriguing feat that AMM was totally suppressed in the latter cases in spite of the presence of secondary amines prompted us to better probe the respective roles of primary and secondary amines in TETA. The excellent match between targeted molecular weights calculated assuming that only primary amines initiate polymerization and experimentally measured ones indicates that secondary amines in such a system are not responsible for creating chains unlike primary amines. Yet the polymerization with such an initiating system does not follow the regular normal amine mechanism as evidenced by the fast polymerization observed. Kinetics of ROP of Glu-NCA Initiated by Different Amines. Indeed the kinetics of ROP of Glu-NCA studied in DMF at 25 °C using four concentrations of TETA (3.80, 2.53, 1.90, and 1.58 mM) indicates that it is much faster than that measured for polymerizations carried out in the presence of HMDA alone. The plots of ln([NCA]0/[NCA]t) versus the polymerization time are straight lines with zero intercepts for all [TETA]0 (Figure S1), confirming that the rate of polymerization −d[NCA]/dt = kapp[NCA], where kapp = kp[TETA]x, is first-order in [NCA] and that no termination reaction occurred. The polymerization rates (kapp) calculated from the slopes of the straight lines are 0.0578, 0.0415, 0.0324, and 0.0292 min−1 respectively. The order (x) of [TETA] determined from the slope of ln kapp versus ln[TETA]0 is found to be 0.80 and the rate constant, kp, determined from the intercept is equal to 0.0199 mM−1 min−1 (Figure S2). The observed fractional dependency of 0.80 (close to one) on the initiator concentration suggests that nearly all active species are monomeric (no aggregation) and that the polymer chains propagate through one only type of active center.11 Very similar results to those generated with TETA were obtained with a series of initiators containing both secondary and primary amines: DETA, TEPA, and PEHA. The respective
■
RESULTS AND DISCUSSION Living Polymerization of NCA Initiated by Triethylenetetramine (TETA). Triethylenetetramine (TETA) is an example of such an initiator that contains both primary and secondary amines. After our preliminary experiments showed that TETA not only exhibits a very high activity but also allows a remarkable control over the polymerization at room temperature, we then run a systematic investigation of NCA polymerization initiated by TETA. As shown in Table 1 (entries: 1−9) and Figure 1A,B, TETA is an efficient initiator for the ROP of Glu-NCA affording excellent control over molecular weight (MW) and polydisperC
DOI: 10.1021/acs.biomac.5b00134 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules
Figure 3. 1H NMR and 15N NMR spectra of TETA and Glu-NCA (CDCl3, 298 K).
Scheme 2. Proposed Mechanism for the ROP of NCA Initiated by TETA
polymerization preventing the formation of star and ill-defined structures. Proposed Explanation of the High Activity of “Allied Amines” (TETA, DETA, TEPA, or PEHA). How to explain that propagation is much faster with TETA, DETA, TEPA, or PEHA than with HMDA, a regular diamine initiator? We then turned to the NMR characterization of these initiators and compare it with that of the monomer. As shown in Figure 3, the nitrogen of monomer is “electron-poor” and its amido proton is more “acid” than those of the initiator, whereas the secondary
rate constants corresponding to the different initiators could be derived and an order established kp,DETA/kp,TETA/kp,TEPA/kp,PEHA = 0.83:1:1.03:1.17, PEHA with its four secondary amines turning to bring about the fastest polymerization (Figure 2A). A confirmation of the formation of linear polypeptides when using either TETA, DETA, TEPA, or PEHA as initiators is brought by the MHS plots which show unambiguously that the variation of intrinsic viscosity is that typical of linear samples (Figure 2B); an additional proof is thus provided that −NH− groups in either TETA, DETA, TEPA, or PEHA do not initiate D
DOI: 10.1021/acs.biomac.5b00134 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules amine nitrogens of TETA are “electron-rich” and more “basic” than that carried by NCA. In the presence of monomer, the electron-rich nitrogens of TETA are bound to interact with the acidic protons of monomer, facilitating and boosting the attack of the latter by the two primary amines in TETA (Scheme 2). The typical reaction that occurs in the presence of secondary amines alone in AMM and results in the slow abstraction of the monomer amido proton is thus prevented. Secondary amines in TETA, DETA, TEPA, or PEHA are thus not passive and silent but activate the monomer to the point that growing primary amines consume the latter much faster. To further substantiate the proposed mechanism, we considered a mixture of individual primary and secondary amines as initiators for NCA polymerization. A mixture of HMDA (primary amine) and N, N′-dimethyl-1,2-ethanediamine (DMEDA; secondary amine) was thus employed to initiate the polymerization of Glu-NCA (Table 1, entry 17). This bimolecular system produced well-defined samples. When an equivalent DMEDA (vs HMDA) was added to HMDA, the rate of polymerization was comparable to that of the polymerization initiated by the sole DMEDA but the propagation exhibited all features of “livingness”, unlike the DMEDA case: the obtained polypeptides indeed possess symmetrical and unimodal SEC traces and very low PDIs that dramatically differ from the polymers prepared from DMEDA (Figure S3). In the case of polymerization of GluNCA mediated by DMEDA, ln([NCA]0/[NCA]t) was plotted versus the polymerization time. No first order dependence on monomer concentration was observed (Figure 1B, green line) and in addition ill-defined polypeptide was resulted, indicating that the polymerization was not following the “normal amine” mechanism. The kapp value (6.140 × 10−4s−1) of the polymerization initiated by HMDA/DMEDA (1:1) was 3/5 of that (9.644 × 10−4s−1) obtained for the polymerization by TETA, but it was twice as much as that of the polymerization initiated by HMDA alone (3.236 × 10−4 s−1; Figure 2A). However, when we increase the ratio of DMEDA/HMDA to 2 which corresponds to the same molar ratio of secondary amines to primary amines as in PEHA, a bimodal GPC trace of the resulting polymer is observed, indicating that AMM prevails when DMEDA is used in excess. Unlike the case of PEHA which can activate only one monomer after another one due to steric reasons, a ratio of 2:1 in the DMEDA/HMDA system cannot prevent the excess of secondary amines present from interacting with more than one monomer at a time, allowing this excess to activate and abstract the monomer amido protons: this reaction thus occurs because the concentration of growing primary amines is too low to totally subdue proton abstraction. Also, the secondary amine nitrogens of DMEDA are “electron-richer” than those PEHA and can thus more easily abstract the amido protons of monomer (see 15N MNR spectra in Figure 4).
Figure 4. 15N NMR spectra of initiators bearing secondary amines (DETA, TETA, TEPA, and PEHA), HMDA, N,N′-DMEDA, and HMDA/N,N′-DMEDA (1:1) mixture (CDCl3, 298 K).
for NCA polymerization. Future work in our laboratory will seek to leverage this novel polymerization mechanism and to extend the scope of this strategy for synthesis of well-defined polypeptides with different architectures.
■
ASSOCIATED CONTENT
S Supporting Information *
Additional kinetic data, SEC traces, and proposed mechanism are presented. This material is available free of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*Tel.: + 966-(0)12-8080789. E-mail: nikolaos.hadjichristidis@ kaust.edu.sa. Notes
The authors declare no competing financial interest.
■ ■
ACKNOWLEDGMENTS Research reported in this publication was supported by the King Abdullah University of Science and Technology REFERENCES
(1) (a) Deming, T. J. Adv. Drug Delivery Rev. 2002, 54, 1145−1155. (b) Wang, X. Y.; Kim, H. J.; Wong, C.; Vepari, C.; Matsumoto, A.; Kaplan, D. L. Mater. Today 2006, 9, 44−53. (c) Dos Santos, S.; Chandravarkar, A.; Mandal, B.; Mimna, R.; Murat, K.; Saucede, L.; Tella, P.; Tuchscherer, G.; Mutter, M. J. Am. Chem. Soc. 2005, 127, 11888−11889. (d) Mart, R. J.; Osborne, R. D.; Stevens, M. M.; Ulijn, R. V. Soft Matter 2006, 2, 822−835. (e) Deng, C.; Wu, J.; Cheng, R.; Meng, F.; Klok, H.; Zhong, Z. Prog. Polym. Sci. 2014, 39, 330−364. (f) Lu, H.; Wang, J.; Song, Z.; Yin, L.; Zhang, Y.; Tang, H.; Tu, C.; Lin, Y.; Cheng, J. Chem. Commun. 2014, 50, 139−155. (2) Deming, T. J. Adv. Polym. Sci. 2006, 202, 1−18. (3) Kricheldorf, H. R. Angew. Chem., Int. Ed. 2006, 45, 5752−5784. (4) (a) Deming, T. J. Nature 1997, 390, 386−389. (b) Deming, T. J. J. Am. Chem. Soc. 1998, 120, 4240−4241. (5) (a) Dimitrov, I.; Schlaad, H. Chem. Commun. 2003, 2944−2945. (b) Conejos-Sánchez, I.; Duro-Castano, A.; Birke, A.; Barz, M.; Vicent, M. Polym. Chem. 2013, 4, 3182−3186. (c) Birke, A.; Huesmann, D.;
■
CONCLUSION In summary, we found that primary amines in “alliance” with secondary amines could function as excellent initiators for the fast and living ROP of α-amino acid N-carboxyanhydrides. In contrast to conventional amine-mediated NCA polymerizations, these “allied” amines do not require low reaction temperatures to prevent side reactions from occurring, and they can afford well-defined polypeptides at room temperature. This unexpected finding and its associated AAMMA mechanism provides a new strategy to develop effective metal-free initiators E
DOI: 10.1021/acs.biomac.5b00134 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules Kelsch, A.; Weilbächer, M.; Xie, J.; Bros, M.; Bopp, T.; Becker, C.; Landfester, K.; Barz, M. Biomacromolecules 2014, 15, 548−557. (6) (a) Lu, H.; Cheng, J. J. Am. Chem. Soc. 2007, 129, 14114−14115. (b) Peng, H.; Ling, J.; Shen, Z. J. Polym. Sci., Part A: Polym. Chem. 2012, 50, 1076−1085. (c) Peng, H.; Ling, J.; Zhu, Y.; You, L.; Shen, Z. J. Polym. Sci., Part A: Polym. Chem. 2012, 50, 3016−3029. (7) (a) Aliferis, T.; Iatrou, H.; Hadjichristidis, N. Biomacromolecules 2004, 5, 1653−1656. (b) Vayaboury, W.; Giani, O.; Cottet, H.; Deratani, A.; Schué, F. Macromol. Rapid Commun. 2004, 25, 1221− 1224. (c) Pickel, D. L.; Politakos, N.; Avgeropoulos, A.; Messman, J. M. Macromolecules 2009, 42, 7781−7788. (d) Habraken, G. J. M.; Wilsens, K. H. R. M.; Koning, C. E.; Heise, A. Polym. Chem. 2011, 2, 1322−1330. (e) Zou, J.; Fan, J.; He, X.; Zhang, S.; Wang, H.; Wooley, K. L. Macromolecules 2013, 46, 4223−4226. (8) Hadjichristidis, N.; Iatrou, H.; Pitsikalis, M.; Sakellariou, G. Chem. Rev. 2009, 109, 5528−5578. (9) Zhao, W.; Gnanou, Y.; Hadjichristidis, N. Chem. Commun. 2015, 51, 3663−3666. (10) Fetters, L. Encyclopedia of Polymer Science and Engineering, 2nd ed.; Wiley-Interscience: New York, 1987; Vol. 10, pp 19−25. (11) (a) Penczek, S.; Duda, A. Makromol. Chem., Macromol. Symp. 1991, 47, 127−140. (b) Duda, A.; Penczek, S. Macromol. Rapid Commun. 1994, 15, 559−566.
F
DOI: 10.1021/acs.biomac.5b00134 Biomacromolecules XXXX, XXX, XXX−XXX
