Letter pubs.acs.org/macroletters
Ring-Opening Polymerization of N‑Carboxyanhydrides Initiated by a Hydroxyl Group Špela Gradišar, Ema Ž agar, and David Pahovnik* National Institute of Chemistry, Department of Polymer Chemistry and Technology, Hajdrihova 19, 1000 Ljubljana, Slovenia S Supporting Information *
ABSTRACT: We report on a method for preparation of welldefined synthetic polypeptides by ring-opening polymerization (ROP) of α-amino acid N-carboxyanhydrides (NCA) initiated by a hydroxyl group. To overcome the issue of slow initiation by hydroxyl group, an acid catalyst was used in the initiation step to catalyze opening of the NCA ring by the hydroxyl group and to simultaneously suppress further chain propagation by protonation of the formed amine group. In this way, we have separated slow initiation from the fast chain propagation, since such a combination leads to poorly defined products, and instead performed them in a successive manner. Only after completion of the initiation, the propagation was started by the addition of a base to deprotonate the ammonium group. This method was successfully applied for the synthesis of homopolypeptides by using alcohol as an initiator as well as polypeptide-based block copolymers by using poly(ethylene glycol) or poly(styrene) macroinitiator terminated with the hydroxyl group. This approach not only expands the pool of possible initiators, but also significantly facilities the preparation of polypeptide-based hybrid polymers.
S
tions10−12 or macroinitiator is synthesized from the multifunctional initiator bearing the amine protected group,13−18 making commercially available amine-functionalized macroinitiators, like frequently used amine-terminated poly(ethylene glycol) (PEG),2 significantly more expensive than their hydroxylfunctionalized analogues. Recently, several attempts have been made to initiate ROP of NCA by the hydroxyl group. For example, Hadjichristidis et al.19 used various aminoalcohols bearing secondary or tertiary amine group as the initiators of ROP of the NCA with hydrogen-bonding thiourea organocatalyst. While the initiation did proceed from the hydroxyl group, the benzyl alcohol and the aminoalcohol with the Boc-protected amine group did not initiate the ROP, indicating a limitation of this procedure due to the necessity of the presence of the amine group on the initiator. On the other hand, Zhang et al.20 successfully applied alcohols and hydroxyl-terminated macroinitiators for the preparation of polypeptoids by ROP of the N-substituted NCA using organic superbase 1,1,3,3-tetramethylguanidine (TMG) as a catalyst.21 However, TMG is most probably not an appropriate catalyst for the polypeptide synthesis since it can interact with the slightly acidic proton at the 3-N position of the unsubstituted NCA, making an undesired activated monomer polymerization mechanism highly likely.7
ynthetic polypeptides are, together with polypeptide-based hybrid polymers, interesting biomaterials for various applications in the biomedical field.1−6 Well-defined synthetic polypeptides are prepared by ring-opening polymerization (ROP) of N-carboxyanhydride (NCA) monomers derived from α-amino acids, which enables good control over the molecular weight characteristics. While several different initiating/catalytic systems have been developed for ROP of NCA, primary amines are the most commonly used initiators, especially for the preparation of hybrid block copolymers as well as polypeptides of complex macromolecular architectures by the so-called normal amine polymerization mechanism.7,8 In the initiation step of ROP via normal amine mechanism, the NCA ring is opened by nucleophilic attack of the primary amine initiator, resulting in formation of a carbamic acid that via decarboxylation transforms into an amine group, which is then the active species for chain propagation. Rate of initiation with the primary amine group is usually fast, and therefore, all the polymer chains start to grow simultaneously. On the contrary, initiation of ROP of NCA by the hydroxyl group proceeds significantly slower than the chain propagation, leading to uncontrolled polymerization and, consequently, to poorly defined products.9 This is in contrast to ROP of several other heterocyclic monomers, like epoxides, lactones/lactides, and cyclic carbonates, where the alkoxide/hydroxyl end-groups are the active species. Due to this reason, the preparation of polypeptide-based hybrid block copolymers demands transformation of the end-hydroxyl group of polyether, polyester, or polycarbonate macroinitiator to the amine group, which is usually accomplished either through the multistep reac© XXXX American Chemical Society
Received: May 23, 2017 Accepted: June 8, 2017
637
DOI: 10.1021/acsmacrolett.7b00379 ACS Macro Lett. 2017, 6, 637−640
Letter
ACS Macro Letters In this work, we present a new and efficient synthetic approach in which alcohols or hydroxyl-terminated polymers were used as the (macro)initiators for controlled ROP of NCA to prepare well-defined polypeptides as well as hybrid block copolymers. To overcome the limitation of slow initiation by the hydroxyl group while still performing initiation and propagation steps of polymerization in one-pot manner, the initiation step was separated from the propagation step by using an acid catalyst during initiation. Acid has to play a double role, that is, to efficiently catalyze the opening of the NCA ring by the hydroxyl group and to simultaneously suppress further chain propagation by protonating the amine group formed after decarboxylation of the carbamic acid. Only after the initiation had been completed, the chain propagation was started by addition of a base that deprotonated the ammonium groups (Scheme 1). Scheme 1. Reaction Pathway of ROP of NCA Using Hydroxyl Group as an Initiator
Figure 1. (A) Enlarged 1H NMR spectra of the reaction aliquots withdrawn at different time of initiation of BLG NCA with PPA, showing the transformation of R-CH2-OH groups (3.44 ppm) into RCH2-OCO-R ester bonds (4.17 ppm) (sample B in Table 1). (B) MALDI-TOF mass spectra of the reaction aliquots withdrawn at different time of initiation of BLA NCA with hydroxyl-terminated PEG macroinitiator (sample E in Table 1). Measured monoisotopic signals are denoted in the enlarged regions of mass spectra together with the calculated exact masses ionized with sodium ion (in parentheses) for the proposed structures.
For the initiation, we have tested several acids. Among them, methansulfonic acid (MSA) proved to be the most efficient one, whereas other acids, like HCl/Et2O, diphenyl phosphate, and trifluoroacetic acid, resulted in lower conversion of alcohol to ester in reaction time comparable to MSA. On the other hand, trifluoromethanesulfonic acid was a very efficient catalyst for the initiation since it was finished in a couple of hours; however, in this case, a much higher extent of the benzyl protection group cleavage as a side reaction was also observed. A ratio between the acid and the initiator is extremely important for controlled polymerization since acid is consumed during initiation for protonation of the formed amine groups. Therefore, a mole equivalent of acid with respect to initiator was not sufficient for full conversion of the hydroxyl groups into the ester bonds. The most efficient ratio was found to be three mole equivalents of the acid with respect to the initiator. Higher amounts of added acid did not significantly increase the initiation rate, but led to a higher extent of side reactions. Solvent also plays a very important role in the initiation step. In DMF, usually used for polypeptide synthesis, as well as in THF, the initiation was not successful, most likely due to strong interactions of the solvent with the acid catalyst. On the other hand, chlorinated solvents that have also been used for NCA polymerizations17−19 proved to be much better choice, since full conversion of hydroxyl groups to ester bonds was achieved in chloroform at 40 °C in 24 h, as revealed by 1H NMR for the initiation of γ-benzyl-L-glutamate (BLG) NCA with 3-phenyl-1propanol (PPA) (Figure 1A). If no acid was used under otherwise the same reaction conditions, almost no initiation by alcohol was observed after 24 h, as indicated by the absence of an ester group in NMR spectra. In dichloromethane as a solvent, the initiation proceeds as well; however, the rate of initiation was significantly slower as compared to that in chloroform. Similar experimental conditions (MSA/macroinitiator ratio of 3/1, CHCl3, 40 °C) were found to be optimal also for the preparation of polypeptide-based hybrid block
copolymers by using monohydroxyl-terminated PEG or poly(styrene) (PS) as a macroinitiator (samples E and F, Table 1). In the case of PEG-initiated ROP of β-benzyl-Laspartate (BLA) NCA, we were able to follow the initiation by MALDI-TOF MS (Figure 1B). After an initiation time of 8 h, the residual unreacted PEG was still visible, whereas after 48 h it was completely consumed. The mass spectrum recorded after 48 h initiation time shows besides the expected distribution of peaks due to the PEG chains bearing one BLA unit, also an additional distribution of peaks of very low intensity that belongs to the PEG chains bearing two BLA repeating units. These results confirm successful suppression of chain propagation by MSA in CHCl3 at 40 °C, which was also observed for reaction of NCA with hydrochloride salts of amines, hydroxylamines and O-alkylhydroxylamines, where the reaction stops after the first acetylation step.22,23 Similar selfregulating initiation was observed when carboxylic acids were used as the initiators in combination with phosphazene superbase for ROP of an epoxide.24 On the other hand, hydrochloride salts of primary amines are well-established initiators for controlled ROP of NCA in DMF at elevated temperatures at which the salt dissociates. In this way, the activated monomer mechanism has been suppressed.25,26 Propagation was started only after the completion of initiation by addition of the base, N-ethyldiisopropylamine, that deprotonates the ammonium groups similar to that reported by Vacogne and Schlaad27 on pausing and resuming ROP of NCA by addition of HCl and triethylamine, 638
DOI: 10.1021/acsmacrolett.7b00379 ACS Macro Lett. 2017, 6, 637−640
Letter
ACS Macro Letters Table 1. Experimental Parameters for ROP of NCA and Molecular Weight Characteristics of Reaction Products M
a
I
[M]0/[I]0
Mtheora
Mexpb
Mw/Mnb
Y (%)
4900 (4000) 5300 (5100) 7200 (6500) 6900 (6600) 7650 8500
1.17
90
1.38
75
1.07
80
1.57
85
1.15 1.08
80 85
A
BLA
PPA
25:1
5300
B
BLG
PPA
25:1
5600
C
BLA
PPA
40:1
8300
D
BLG
PPA
40:1
8900
E F
BLA BLA
PEG-OH PS-OH
40:1 40:1
10200 10400
Calculated from the monomer to initiator ratio. bMw determined by SEC-MALS and in parentheses Mp determined by MALDI-TOF MS.
by intramolecular cyclization via attack of the amine end-group on the benzyl ester group. This side reaction is commonly observed when the polymerization of BLG NCA is performed at higher temperatures in DMF.31 In our case, cooling down the reaction mixture to 0 °C before addition of the base did not completely suppress this side reaction, indicating that intramolecular cyclization to pyroglutamate was facilitated under the experimental conditions used. Since formation of pyroglutamate prevents further chain propagation, the molecular weight distributions of PBLG homopolypeptides (samples B and D in Table 1) are broader as compared to those observed for the PBLA analogues (samples A and C in Table 1). SEC-MALS analysis of the PEG-b-PBLA and PS-b-PBLA block copolymers revealed monomodal SEC traces that clearly shifted to lower elution volume as compared to the SEC traces of the corresponding macroinitiators, indicating an increase in molecular weight of the hybride block copolymers. Thus, SECMALS together with 1H NMR results (Figures S10 and S11), which confirm the presence of both blocks in the copolymers, prove effectiveness of our approach for the preparation of polypeptide-based hybrid block copolymers by using the hydroxyl-terminated macroinitiators (Figure 3).
respectively. The consumption of NCA monomer during propagation was followed by 1H NMR. Rate of propagation was found to be dependent on the acid to base mole ratio, with the ideal initiator/acid/base ratio of 1/3/2.5. When a less amount of the base had been added to the reaction mixture, a significant decrease in polymerization rate was observed, while the addition of larger amounts of base resulted in uncontrolled polymerization and, consequently, in poorly defined products. MALDI-TOF mass spectrum of poly(β-benzyl-L-aspartate) (PBLA; Figure 2) shows that the main product of BLA NCA
Figure 2. Top: MALDI-TOF mass spectrum and its enlarged region with denoted measured monoisotopic signals of the PBLA homopolypeptide (sample A in Table 1) prepared by ROP of BLA NCA initiated with PPA. Bottom: The proposed PBLA structures and their calculated exact masses ionized with sodium ion [M + Na]+. Figure 3. SEC-MALS chromatograms of PEG-b-PBLA (A) and PS-bPBLA (B) block copolymers (black curves; samples E and F in Table 1) together with the corresponding PEG-OH and PS-OH macroinitiators (red curves). Solid curves: refractive index detector responses; dotted curves: light-scattering detector responses at 90° angle; squares: molar mass as a function of elution volume.
polymerization using PPA initiator is the desired polypeptide bearing the initiator bound via the ester bond to one chain end and the primary amine group on the other chain end, which thus reveals the controlled ROP. Additional distributions of peaks of very low intensities indicate the initiation by water and the cleavage of initiator at the ester bond as well as the cleavage of one benzyl protection group to release a benzyl alcohol that can also act as an initiator. These results confirm the importance to carry out polymerizations in dry conditions, which are typically needed for controlled ROP of NCA.28−31 MALDI-TOF mass spectra of poly(γ-benzyl-L-glutamate) (PBLG) homopolypeptides initiated by PPA (Figures S7 and S8) show two major peak populations. Both of them were initiated by the alcohol, however, one of them is terminated with the desired amine group, while the other one is terminated with a pyroglutamate unit. Pyroglutamate end group is formed
In summary, this work demonstrates that the hydroxyl group in combination with the acid catalyst can be successfully applied for the initiation and controlled ROP of NCA. This methodology narrows the differences between the ROP of NCA and the ROP of a large group of other heterocyclic monomers like epoxides, lactones and carbonates, which contrary to the NCA propagate through the hydroxyl group. As a consequence, this new synthetic methodology substantially facilitates the preparation of well-defined polypeptide-based hybrid polymers by using hydroxyl-terminated macroinitiators. In this way, we can avoid time-consuming end-group 639
DOI: 10.1021/acsmacrolett.7b00379 ACS Macro Lett. 2017, 6, 637−640
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ACS Macro Letters
(18) Le Hellaye, M.; Fortin, N.; Guilloteau, J.; Soum, A.; Lecommandoux, S.; Guillaume, S. M. Biomacromolecules 2008, 9, 1924−1933. (19) Zhao, W.; Gnanou, Y.; Hadjichristidis, N. Polym. Chem. 2015, 6, 6193−6201. (20) Chan, B. A.; Xuan, S.; Horton, M.; Zhang, D. Macromolecules 2016, 49, 2002−2012. (21) Ishikawa, T. Guanidines in Organic Synthesis. In Superbases for Organic Synthesis: Guanidines, Amidines, Phosphazenes and Related Organocatalysts; Ishikawa, T., Ed.; John Wiley & Sons, Ltd.: United States, 2009; pp 93−143. (22) Knobler, Y.; Bittner, S.; Frankel, M. J. Chem. Soc. 1964, 0, 3941− 3951. (23) Knobler, Y.; Bittner, S.; Virov, D.; Frankel, M. J. Chem. Soc. C 1969, 0, 1821−1824. (24) Zhao, J.; Pahovnik, D.; Gnanou, Y.; Hadjichristidis, N. Macromolecules 2014, 47, 1693−1698. (25) Dimitrov, I.; Schlaad, H. Chem. Commun. 2003, 2944−2945. (26) Lutz, J.-F.; Schütt, D.; Kubowicz, S. Macromol. Rapid Commun. 2005, 26, 23−28. (27) Vacogne, C. D.; Schlaad, H. Chem. Commun. 2015, 51, 15645− 15648. (28) Aliferis, T.; Iatrou, H.; Hadjichristidis, N. Biomacromolecules 2004, 5, 1653−1656. (29) Zou, J.; Fan, J.; He, X.; Zhang, S.; Wang, H.; Wooley, K. L. Macromolecules 2013, 46, 4223−4226. (30) Kramer, J. R.; Deming, T. J. Biomacromolecules 2010, 11, 3668− 3672. (31) Habraken, G. J. M.; Peeters, M.; Dietz, C. H. J. T.; Koning, C. E.; Heise, A. Polym. Chem. 2010, 1, 514−524.
functionalization of the hydroxyl-terminated macroinitiator or protection/deprotection reactions in the case of using multifunctional initiators. Moreover, this synthetic methodology opens up new avenues for preparation of polypeptide-based hybrid polymers, such as one-pot sequential polymerization of different heterocyclic monomers and NCA.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.7b00379. Experimental procedures, 1H NMR and MALDI-TOF MS spectra, and SEC-MALS chromatograms (PDF).
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
David Pahovnik: 0000-0001-8024-8871 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge the financial support from the Slovenian Research Agency (Research Core Funding No. P20145 and Project No. Z2-6755) and the Centre of Excellence, Polymer Materials and Technologies, for access to the MALDITOF mass spectrometer. Š.G. is a Ph.D. student at the Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia.
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DOI: 10.1021/acsmacrolett.7b00379 ACS Macro Lett. 2017, 6, 637−640
