Letter pubs.acs.org/macroletters
Stability of Trithiocarbonate RAFT Agents Containing Both a Cyano and a Carboxylic Acid Functional Group Adrian V. Fuchs†,‡ and Kristofer J. Thurecht*,†,‡ †
Australian Institute of Bioengineering and Nanotechnology and Centre for Advanced Imaging, University of Queensland, Brisbane 4072, Australia ‡ ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash University, Parkville 3052, Victoria, Australia S Supporting Information *
ABSTRACT: The hydrolytic degradation of widely used cyano-containing, acid-bearing trithiocarbonate reversible addition−fragmentation chain-transfer (RAFT) agents has been identified and shown to effect the RAFT polymerization and end-group fidelity of PMMA polymers. The hydrolysis occurred when the RAFT agents were stored under the recommended conditions. Degradation was identified in both commercially available and popular synthetic RAFT agents. 1H and 13C NMR as well as mass spectroscopy show that the cyano functionality hydrolyzes to the amide adduct. Doping of this amide degradation product into RAFT polymerizations of MMA results in increased dispersities and changes in expected end-group fidelities. The ability to identify this degradation product and remove it from the RAFT agent before use will allow better control over polymer properties and postmodification processes commonly used in complex polymer systems, nanomedicines, and bioconjugates.
T
popular radical-induced azo-initiator decomposition of a bis(thioacyl) disulfide.14,15 One such azo-initiator is the 4,4′azobis(4-cyanopentanoic acid) (V-501) reagent where the resultant RAFT agent contains a tertiary, cyano-containing R group suitable for use in the polymerization of many acrylates, methacrylates, styrenics, and derivatives thereof.16 Furthermore, the carboxylic acid functionality of the resultant polymer endgroups can be additionally modified to impart higher-order functionality through common ligation and coupling chemistries. These acid-functionalized RAFT agents are now commercially available as summarized by Keddie et al.17 which includes the popular 4-cyano-4[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid. Of great importance to any living radical polymerization process, including RAFT, is the integrity and purity of the chain transfer agent. The living characteristics of a RAFT polymerization are largely dictated by the addition and fragmentation rates of the RAFT agent. RAFT agent degradation and the resultant descendant species will likely cause variations in the addition and fragmentation rates and therefore produce illdefined polymers. Such effects are observed in the monomer conversion rates, polymer dispersities, and end-group functionalities. Herein we show that commonly utilized cyano-containing trithiocarbonate RAFT agents undergo cyano-hydrolysis
he advent of reversible addition−fragmentation chaintransfer (RAFT) polymerization has led to the synthesis of a plethora of well-defined, size-controlled polymers. The RAFT process and the ultimate properties of the resultant polymer rely significantly on the nature and integrity of the RAFT agent that is used. The role of the R and Z groups of the RAFT agent are well understood in relation to their effect on polymerization of particular monomers, their reaction kinetics, and polymerization conditions required.1 Polymer end-group fidelity imparted by the RAFT agent is also of great importance.2 Not only does the end-group result from the Rand Z-groups of the RAFT agent but also these will determine to a greater or lesser extent the properties of the polymer with regard to solubility,3 polymer−polymer interactions, branched polymers,4 and postpolymerization reactivity.5 End-group reactivity and defined polymer properties are highly important aspects in many areas of polymeric materials especially nanomedicines and theranostics.6 Generally, the RAFT process is a popular choice for controlled living radical polymerizations. This is largely due to the tolerance of the RAFT agent to oxidative stresses, hydrophobic or hydrophilic environments, and other harsh processes commonly encountered during polymer design and synthesis. It is known, however, that under the right conditions RAFT agents undergo aminolysis,7 can be light sensitive,8,9 and are prone to hydrolysis10,11 and various cycloaddition side reactions.12,13 Many commonly used and commercially available RAFT agents containing tertiary R groups are derived from the © XXXX American Chemical Society
Received: February 11, 2017 Accepted: March 2, 2017
287
DOI: 10.1021/acsmacrolett.7b00100 ACS Macro Lett. 2017, 6, 287−291
Letter
ACS Macro Letters
Scheme 1. Proposed Acid-Catalyzed Cyano-Hydrolysis Mechanism with ESI Mass Spectra of the Starting and Isolated Impurity of Compound 1a
a
Arrows point to the sodium adduct of each respective compound.
through incorrect handling and storage. We also highlight the hydrolysis products that arise upon degradation and the effect on polymer structure when they are mistakenly used in RAFTcontrolled polymerizations. We aim to highlight the importance of RAFT agent purity so that the resultant polymers contain the desired chemical and physical properties. A series of common cyano-containing trithiocarbonates investigated in this study are shown in Scheme 1 (inset), where the dodecyl variant (1) is also commercially available. 1− 4 were synthesized as published previously by substituting the 1-dodecanethiol with either 1-butanethiol, ethanethiol, or 2phenylethanethiol.16,18 An example procedure for the synthesis of 4 may be found in the Supporting Information. It was initially noticed that all of the above RAFT agents, after a period of storage in recommended conditions, i.e., −20 °C, handling, or even nonuse, gave a series of identical, yet slightly shifted signals in the 1H NMR spectrum (Figure S1). These signals originated from a second species isolatable by either column chromatography (1 and 4) or filtration from diethyl ether (2 and 3). The 1H and 13C NMR of the pure and isolated unknown degradation product of compound 1 can be seen in Figure 1 and compounds 2−4 in Figures S1−S6. The 1H spectra show a significant shift of the methyl protons (c) and the methylene protons (a and b) between the pure cyano RAFT agent and the isolated impurity. Furthermore, it can be seen in the 13C NMR spectrum (Figure 1b) that there is an appearance of a second carbonyl peak at 172 ppm (h′), along with the disappearance of the cyano carbon at 119.1 ppm (e), and a shift of the quaternary carbon from 46.7 to 61.2 ppm (d′). The same trends were witnessed with all other compounds (2−4) (Figures S1−S6). The observed behavior seen in the NMR analysis suggests that there is a loss of the cyano functionality causing a significant shift to the surrounding proton and carbon nuclei. To further investigate this observation, electrospray ionization mass spectroscopy (ESI-MS) of compound 1 and its isolated degradation product (1a) was performed and is shown in Scheme 1.
Figure 1. 1H (a) and 13C (b) NMR spectra of Compound 1 (top) and its isolated impurity (bottom). Spectra recorded in DMSO-d6 at 500 MHz.
Through the analysis of the NMR and ESI-MS data, it can be concluded that the degradation product of 1 is the amide formation occurring via a proposed acid-catalyzed hydrolysis of the cyano functionality (Scheme 1). The appearance of the second carbonyl peak in the 13C NMR spectrum at 172 ppm is 288
DOI: 10.1021/acsmacrolett.7b00100 ACS Macro Lett. 2017, 6, 287−291
Letter
ACS Macro Letters
[RAFT]:[initiator] ratios were maintained at 153:1:0.2 in THF, 75 °C for 4.5 h. The hydrolysis product of each respective RAFT agent (1a in 1 or 4a in 4) was doped at mole fractions of 0 (pure cyano-RAFT agent), 0.1, 0.5, and 1.0 (pure amideRAFT agent). The molar mass distribution curves, obtained by GPC (Figure 3), show a gradual increase in Mn and dispersity
consistent with the presence of an amide, as too is the upfield shift of protons a, b, and c in Figure 1. Analysis of all of the cyano-containing trithiocarbonates and their amide-containing degradation products with ESI-MS (Table S1) showed that the obtained experimental masses match well with the theoretical masses of both the cyano-containing RAFT agents and the proposed amide hydrolysis products for a variety of cation adducts. Scheme 1 suggests that the presence of water acts as the nucleophile on the electrophilic carbon of the protonated cyano group. This mechanism is a well-known method of amide formation and may be found in many organic chemistry textbooks.19 We propose that since the RAFT agent contains an acidic proton, and through the presence of water acquired during handling or storage in aqueous environments, e.g., refrigerator/freezer, that the amide will eventually form under standard storage conditions. Indeed, the recommended storage conditions of compound 1 (available through Sigma-Aldrich, Technical Bulletin AL-26420) are an airtight container, kept away from light at −20 °C. Through experience, we recommend storage under vacuum and protected from light. Baussard et al.10 have studied the hydrolysis of 4-(thiobenzoylthio)-4-cyanopentanoic acid and witnessed various degradation products forming at elevated temperatures in D2O at pH 6. They putatively assign the degradation to have occurred to the CS to form the CO analogue. They also mention that the electron-withdrawing CN group in the α-position to the dithioester may be responsible for the comparatively faster hydrolysis seen compared to other RAFT agents studied. The rate of hydrolysis was measured by the addition of D2O (5 uL) to a solution of RAFT agent 4 (10 mg) in 550 uL of DMSO-d6 (Figure 2). The in situ 1H NMR measurements
Figure 3. GPC traces of PMMA polymers synthesized with increasing mole percentage of RAFT agent 1 (a) or 4 (b). Inset shows dispersity variations across the polymer series. Mole fractions: (−) 0, (- - -) 0.1, (---) 0.5, (− - − ) 1.0
(Đ) until, at 100% hydrolysis product RAFT agent (1a or 4a, PMMA_1_1.0 and PMMA_4_1.0), there is a loss of polymerization control at approximately equal monomer conversion in each case (dispersity of 2.1 and 1.8 for 1a and 4a, respectively). While conversion percentages remain somewhat consistent at approximately 60% for 1 and 40% for 4, molar masses increase drastically as the mole fraction of the amide variant is increased (Table 1). It is interesting to note that at 0.1 mole fraction dispersities remain similar in both cases to the pure CTA, and the Mw and Mn both slightly increase. The importance of this becomes apparent when further modification to the polymer is intended, for example when utilizing the end group in further ligation processes. Thus, polymer end-group integrity imparted by the RAFT agent is key. The polymerizations that contain 100% of the hydrolyzed RAFT agent both displayed large increases in dispersity. It is believed that while these polymerizations may still exhibit “living” characteristics the chain transfer coefficients are likely low. To investigate this, we semiquantitatively approximated the apparent transfer constants (Ctrapp) of RAFT agents 1 and 4
Figure 2. Rate of hydrolysis of RAFT agent 4 in the presence of D2O. Inset is the 1H NMR measured over time in DMSO-d6 at 500 MHz.
clearly show the gradual increase in the α-methyl protons (Figure S1, protons d′) over the course of 50 days. When normalized to proton d, the integral of proton d′ shows that 11 mol % of the hydrolysis product (4a) occurs over the course of 50 days. Of importance is how (unknowingly) having a CTA that contains these hydrolysis products affects the outcome of a RAFT-mediated polymerization. To study this, the polymerization of methyl methacrylate (MMA) was performed whereby different mole fractions of the amide were doped into the pure RAFT agent analogue. Standard RAFT polymerization conditions were employed (see SI) whereby the [monomer]: 289
DOI: 10.1021/acsmacrolett.7b00100 ACS Macro Lett. 2017, 6, 287−291
Letter
ACS Macro Letters
Table 1. Tabular Summary of PMMA Polymers Synthesized Containing Various Mole Fractions of the Hydrolyzed RAFT Agent
a
dodecyl-CVA TTC
Mw
Mn
ĐM
conv (%)a
ethyl-phenyl-CVA TTC
Mw
Mn
ĐM
conv (%)a
PMMA_1 PMMA_1_ replicate PMMA_1_0.1 PMMA_1_0.5 PMMA_1_1.0
9370 8920 10270 15560 43780
8010 7700 8670 11340 20830
1.17 1.16 1.18 1.37 2.10
55 60 62 52 54
PMMA_4 PMMA_4_0.1 PMMA_4_0.5 PMMA_4_1.0
8550 10060 12400 35490
6940 8120 9230 19670
1.23 1.24 1.34 1.80
36 43 35 45
Conversion determined gravimetrically.
polymers (Figure 4a), and the underlying signals appearing between 7.15 and 7.30 ppm for PMMA_4 polymers (Figure 4b). While these peaks are difficult to assign, on comparison with the pure 1H NMR of 1a, 2a, 3a, and 4a (Figures 1a, S3, S5, and S1, respectively), they can be tentatively assigned to the two amide protons (−NH2) and its tautomer.24 It is clear, however, that as the mole fraction of the amide-RAFT agent increases, so does the prevalence of the amide end group, thus confirming that the hydrolyzed RAFT agent doped into the polymerization is present and takes part in the RAFT polymerization. The consequence of this leads to poor confidence in polymer chain size uniformity and the reactivity of chain end groups. These factors are crucial in applications of polymer science, particularly in the fields of polymeric nanomedicines and complex polymer architechtures. In summary, we have identified an important degradation product of cyano-containing trithiocarbonate RAFT agents. The degradation appears to occur through an acid-catalyzed hydrolysis of the cyano moiety and was confirmed through NMR and MS analysis. The hydrolysis product, when doped into a conventional RAFT polymerization of MMA, caused a gradual increase in dispersity inherited from its reduced chain transfer coefficient. Furthermore, polymer end-group analysis revealed that the amide-containing hydrolysis product was present in the polymers synthesized. This highlights the importance of identifying and removing any of the hydrolysis product and that storage and handling of these cyanocontaining acid RAFT agents must be done under environments where water is avoided. The commercial availability of cyano-containing, acid-bearing RAFT agents demonstrates their popularity and widespread use. The identification and removal of the hydrolysis product combined with correct storage is essential for the successful synthesis of well-defined RAFTmediated polymerizations.
along with their amide equivalent (1a and 4a). The following equation was used21 2−χ 1 Đ≈1+ + χn χC tr (1) The dispersity (Đ), polymer number-average chain length (χn), and conversion (χ) were used as given in Table 1 (note: χn was calculated by dividing Mn by the Mr of MMA). Consequently, RAFT agents 1 and 4 gave a Ctr of 16.7 and 21.1 respectively. Comparatively lower Ctr values were calculated (2.5 and 4.3 for 1a and 4a, respectively) for the amide variants. This suggests that under these polymerization conditions the rate constant for chain transfer (ktr) in the initialization equilibria is slower and therefore leads to higher molar masses and dispersities.17,22 A reduction in C tr has been reported previously for dithiobenzoate derivatives where RAFT agents were compared that contained either an α-amide at the Z position or the cyano equivalent.23 To confirm that the R-group of the amide-RAFT agent is present and contributes to the end-group functionality of the polymer, 1H NMR analysis was performed on the polymers synthesized above (Figure 4). The 1H NMR spectra were
■
ASSOCIATED CONTENT
S Supporting Information *
Figure 4. Normalized 1H NMR diffusion spectra of polymers (a) PMMA_1 and (b) PMMA_4.
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.7b00100.
acquired as diffusion-weighted spectra to remove any erroneous signals originating from nonpolymeric and solvent species. The gradual appearance of several sets of signals between 6.7 and 7.5 ppm can be clearly seen as the mole fraction increases (top of Figure 4 to the bottom). In the case of PMMA polymers synthesized with RAFT agent 4 (Figure 4b), the phenyl moiety is present (7.35−7.15 ppm) and overlaps the underlying complex set of signals that become more pronounced as the amide-RAFT agent (4a) mole fraction is increased. Among these are three sharp singlets at 6.94, 7.04, and 7.14 ppm, broad signals at 6.85, 7.09, 7.22, and 7.42 ppm for PMMA_1
Synthesis, experimental details, and supplementary results (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Adrian V. Fuchs: 0000-0002-8112-0527 Kristofer J. Thurecht: 0000-0002-4100-3131 290
DOI: 10.1021/acsmacrolett.7b00100 ACS Macro Lett. 2017, 6, 287−291
Letter
ACS Macro Letters Author Contributions
(22) Moad, G.; Rizzardo, E.; Thang, S. H. Polymer 2008, 49, 1079− 1131. (23) Chong, Y. K.; Krstina, J.; Le, T. P. T.; Moad, G.; Postma, A.; Rizzardo, E.; Thang, S. H. Macromolecules 2003, 36, 2256−2272. (24) Laurella, L. S.; Sierra, M. G.; Furlong, J. J. P.; Allegretti, P. E. Open J. Phys. Chem. 2013, 3, 138−149.
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We acknowledge funding from the National Health and Medical Research Council (APP1099321, KJT) and the Australian Research Council (FT110100284 (KJT), DP140100951 (KJT)). This research was conducted and funded by the ARC Centre of Excellence in Convergent BioNano Science and Technology (CE140100036). AVF would like to thank the University of Queensland for the UQECR grant (UQECR1607665). This work was performed in part at the Queensland node of the Australian National Fabrication Facility, a company established under the National Collaborative Research Infrastructure Strategy to provide nano and microfabrication facilities for Australia’s researchers.
■
REFERENCES
(1) Keddie, D. J. Chem. Soc. Rev. 2014, 43, 496−505. (2) Willcock, H.; O’Reilly, R. K. Polym. Chem. 2010, 1, 149−157. (3) Barner-Kowollik, C.; Blinco, J. P.; Perrier, S. In Macromolecular engineering via RAFT chemistry: from sequential to modular design; Wiley-VCH Verlag GmbH & Co. KGaA: 2012; pp 601−626. (4) Alfurhood, J. A.; Bachler, P. R.; Sumerlin, B. S. Polym. Chem. 2016, 7, 3361−3369. (5) Boyer, C.; Stenzel, M. H.; Davis, T. P. J. Polym. Sci., Part A: Polym. Chem. 2011, 49, 551−595. (6) Fairbanks, B. D.; Gunatillake, P. A.; Meagher, L. Adv. Drug Delivery Rev. 2015, 91, 141−152. (7) Deletre, M.; Levesque, G. Macromolecules 1990, 23, 4733−4741. (8) Quinn, J. F.; Barner, L.; Barner-Kowollik, C.; Rizzardo, E.; Davis, T. P. Macromolecules 2002, 35, 7620−7627. (9) McKenzie, T. G.; Costa, L. P. d. M.; Fu, Q.; Dunstan, D. E.; Qiao, G. G. Polym. Chem. 2016, 7, 4246−4253. (10) Baussard, J.-F.; Habib-Jiwan, J.-L.; Laschewsky, A.; Mertoglu, M.; Storsberg, J. Polymer 2004, 45, 3615−3626. (11) Thomas, D. B.; Convertine, A. J.; Hester, R. D.; Lowe, A. B.; McCormick, C. L. Macromolecules 2004, 37, 1735−1741. (12) Nebhani, L.; Sinnwell, S.; Lin, C. Y.; Coote, M. L.; Stenzel, M. H.; Barner-Kowollik, C. J. Polym. Sci., Part A: Polym. Chem. 2009, 47, 6053−6071. (13) Chen, M.; Moad, G.; Rizzardo, E. Aust. J. Chem. 2011, 64, 433− 437. (14) Thang, S. H.; Chong, Y. K.; Mayadunne, R. T. A.; Moad, G.; Rizzardo, E. Tetrahedron Lett. 1999, 40, 2435−2438. (15) Bouhadir, G.; Legrand, N.; Quiclet-Sire, B.; Zard, S. Z. Tetrahedron Lett. 1999, 40, 277−280. (16) Moad, G.; Chong, Y. K.; Postma, A.; Rizzardo, E.; Thang, S. H. Polymer 2005, 46, 8458−8468. (17) Keddie, D. J.; Moad, G.; Rizzardo, E.; Thang, S. H. Macromolecules 2012, 45, 5321−5342. (18) Fuchs, A. V.; Tse, B. W. C.; Pearce, A. K.; Yeh, M.-C.; Fletcher, N. L.; Huang, S. S.; Heston, W. D.; Whittaker, A. K.; Russell, P. J.; Thurecht, K. J. Biomacromolecules 2015, 16, 3235−3247. (19) Clayden, J.; Greeves, N.; Warren, S.; Wothers, P. Organic Chemistry; Oxford University Press: New York, 2008. (20) http://www.sigmaaldrich.com/catalog/product/aldrich/723274 (accessed 28/2/2017). (21) Derboven, P.; Van Steenberge, P. H. M.; Reyniers, M.-F.; Barner-Kowollik, C.; D’Hooge, D. R.; Marin, G. B. Macromol. Theory Simul. 2016, 25, 104−115. 291
DOI: 10.1021/acsmacrolett.7b00100 ACS Macro Lett. 2017, 6, 287−291
