Synthesis of an Alternating Polycation with the Dense 1,2,3-Triazole Backbone

Abstract

Polycations are an important class of water-soluble polymers because they form polyion complexes with DNA. Thus, the synthesis of polycations with controlled monomer sequences will be of increasing importance for the formation of well-defined polyion complexes. In this study, cationic homopolymer and alternating copolymer with the dense triazole backbone were synthesized by copper(I)-catalyzed azide–alkyne cycloaddition polymerization. The polycations obtained were characterized by potentiometric and turbidimetric titrations, and by complex formation with poly(acrylic acid).

Polycations are an important class of water-soluble polymers. This is because polycations form polyion complexes with DNA, i.e., biological polyanions carrying genetic information, and can be applicable to gene therapy using the polyion complexes.[1] Synthesis of polycations with controlled monomer sequences will be of increasing importance for the formation of well-defined polyion complexes. However, there are fewer examples of sequence-controlled polycations compared with sequence-controlled polyanions presumably because of the low tolerance of controlled polymerization systems to cationic species.[2] We have been working on the synthesis of sequence-controlled polymers by stepwise copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) of 4-azido-5-hexynoic acid (AH) derivatives,[3] [4] because CuAAC is a powerful tool for polymer synthesis.[5] Notably, polymers of AH derivatives possess the dense 1,2,3-triazole backbone consisting of many 1,2,3-triazole units linked through a carbon atom. Since 1,2,3-triazole units exhibit structural features similar to those of amide groups,[6] the dense 1,2,3-triazole polymers are promising as functional polymers like polypeptides. Given that the simplest sequence-controlled polymers are alternating copolymers, this study deals with the synthesis of an alternating polycation carrying amino groups as side chains by CuAAC polymerization of AH derivatives.

The synthetic schemes for the monomer precursors (TBDMS-HE^Ac and TBDMS-AE^Boc) and monomer (AE^Boc) used in this study are shown in Scheme [1], where Ac and Boc denote the acetyl and tert-butoxycarbonyl protecting groups, respectively. TBDMS-HE^Ac and TBDMS-AE^Boc were synthesized by amide coupling of 6-tert-butyldimethylsilyl-4-azido-5-hexynoic acid (TBDMS-AH)[7] with the corresponding amines, which were prepared according to a reported procedure.[8] The tert-butyldimethylsilyl (TBDMS) protecting group of TBDMS-AE^Boc was removed using tetrabutylammonium fluoride (TBAF) to yield AE^Boc. Scheme [2] displays the synthetic scheme for the heterodimer (HE^Ac·AE^Boc); i.e., the monomer of alternating copolymer. TBDMS-HE^Ac and compound 1 were coupled through CuAAC using copper(I) bromide (CuBr) as a catalyst in the presence of sodium l-(+)-ascorbate (NaAsc) to form compound 2. Compound 2 was azidated on the hydroxy group using diphenylphosphoryl azide (DPPA) in the presence of triphenylphosphine (PPh₃) and diisopropyl azodicarboxylate (DIAD) to obtain compound 3. The tert-butyl (tBu) ester in compound 3 was hydrolyzed with trifluoroacetic acid (TFA) to give compound 4. Compound 4 was coupled with Boc-protected ethylenediamine (N-Boc-ethylenediamine) through amide bond formation to yield the TBDMS-protected heterodimer (TBDMS-HE^Ac·AE^Boc). HE^Ac·AE^Boc was finally synthesized by deprotection of the TBDMS protecting group using TBAF. All the intermediates, monomers, and heterodimer in Scheme [1] and Scheme [2] were characterized by ¹H NMR spectroscopy and high-resolution mass spectrometry (HRMS). Figure [1] shows the ¹H NMR spectra for AE^Boc and HE^Ac·AE^Boc. The spectra show signals ascribable to the ethynyl protons at ca. 3.8 ppm. There are signals due to the methine protons connected to the azide group at ca. 4.4 and 4.8 ppm in the spectra for AE^Boc and HE^Ac·AE^Boc, respectively. The signals in the region 8.1–7.8 ppm are assignable to the amide protons. Signals due to the protons in Ac and Boc protecting groups were observed at 2.0 and 1.4 ppm, respectively. The signals in the region of 4.0–2.0 ppm are ascribed to the methylene protons in the side chains. As described in the Experimental Section (see the Supporting Information), the HRMS data indicated the signals could be attributed to the sodium adducts of intermediates, monomers, and heterodimer. These characterization data confirmed the formation of the monomer and heterodimer.

The synthetic scheme of the polymers is shown in Scheme [3]. CuAAC polymerizations of AE^Boc and HE^Ac·AE^Boc were carried out in N,N-dimethylformamide (DMF) using CuBr as a copper(I) catalyst, according to our previous studies.[3] [7] The conditions and results are listed in Table [1]. The polymers obtained were purified by reprecipitation using DMF and diethyl ether as good and poor solvents, respectively. The polymer yields were higher (82 and 94%). The values of M _n and M _w/M _n were determined to be 3.3 × 10³ and 2.1 × 10³, and 1.7 and 1.5 for poly(AE^Boc) and poly(HE^Ac·AE^Boc), respectively, by size-exclusion chromatography (SEC) calibrated with poly(ethylene glycol) (PEG) and poly(ethylene oxide) (PEO) standards using dimethyl sulfoxide (DMSO) that contained LiBr (1.05 g L^–1) as the eluent. The polymerization conditions should be further optimized to obtain high-molecular-weight polymer samples in the future (CuAAC polymerization of AH derivatives may also form cyclic oligomers). Figure [1b] and Figure [1e] show the ¹H NMR spectra for poly(AE^Boc) and poly(HE^Ac·AE^Boc), respectively. In both the spectra, there are signals ascribable to the triazole protons at ca. 8.5 ppm, but no signals due to the ethynyl protons at ca. 3.8 ppm. The signals due to the methine protons were observed at low magnetic fields because of the effect of neighboring 1,2,3-triazole units formed through CuAAC. These spectra are indicative of successful CuAAC polymerization. The Boc and Ac protecting groups in the resulting polymers were removed using TFA and lithium hydroxide monohydrate (LiOH·H₂O), respectively. Figure [1c] and Figure [1f] show the ¹H NMR spectra for poly(AE) and poly(HE^Ac·AE). These spectra do not contain signals assignable to the tBu protons in the Boc protecting group, indicative of quantitative removal of the Boc groups. Figure [1g] shows the ¹H NMR spectrum for poly(HE·AE). Since this spectrum exhibits broad signals, the signal due to the Ac protons may overlap with those due to the methylene protons; however, it is likely that most of the Ac protecting groups were removed based on the integral values of signals around 6 and 2 ppm. Figure S1 in the Supporting Information shows the ¹³C NMR spectra for poly(AE) and poly(HE·AE). These spectra are also indicative of the successful formation of poly(AE) and poly(HE·AE). The dense 1,2,3-triazole polymers of AH derivatives strongly adsorb copper ions derived from the catalyst.[3] [7] In this study, the residual copper ions may perturb the results of the following experiments. The perturbation should likely be minor because the amount of residual copper ions is low (< ca. 10 mol% of the monomer units).

The basic characteristics of poly(AE) and poly(HE·AE) were established by potentiometric and turbidimetric titrations in aqueous media. The degrees of dissociation (α) were calculated from the molar ratios of the AE units in the polymer and the amount of NaOH added. The values of α and turbidity (100 – %T) were plotted in Figure [2] against pH; in both cases, α decreased with pH. Using the data, the apparent pK _a values of the conjugate acids, at which α = 0.5, were evaluated to be pH 8.9 and 8.0 for poly(AE) and poly(HE·AE), respectively. On the other hand, as the pH increased from pH 3 to 9, the turbidity increased gradually from ca. 5% to ca. 20%. The turbidity for poly(AE) was almost constant (ca. 40%) in the pH region 10–11. On the other hand, the turbidity for poly(HE·AE) reached a maximum at pH 9 and decreased with increasing pH from 9 to 11, indicating that the hydroxy groups in HE units also dissociated at higher pH.

The formation of polyion complexes of polycations (poly(AE) and poly(HE·AE)) with poly(acrylic acid) (poly(AA)), a typical polyanion, was investigated by turbidimetric titration. While an aqueous solution of poly(AA) was titrated with an aqueous solution of polycation (poly(AE) or poly(HE·AE)), or while an aqueous solution of polycation was titrated with an aqueous solution of poly(AA), the turbidities were recorded. In this study, two poly(AA) samples of different average molecular weights (2.5 × 10⁵ and 5.0 × 10³) were utilized. Figure [3a] shows a typical example of the turbidimetric titration data. When an aqueous solution of poly(AE) (10 mM AE units) was added to an aqueous solution of the higher molecular weight poly(AA) (5 mM AA units), the turbidity increased gradually with the volume of titrant (V _t) and then commenced to increase more rapidly at V _t ~ 0.85 mL. On the other hand, when an aqueous solution of the poly(AA) (5 mM AA units) was added to an aqueous solution of poly(AE) (5 mM AE units), the turbidity commenced to increase rapidly at a smaller V _t (ca. 0.12 mL). As can be seen in Figure [3a], the regions of gradual and steep changes were fitted with straight lines, respectively. From the intersection of the two straight lines, the critical points (i.e., combinations of the concentrations of AA and AE units) for the phase separation were determined. The critical points obtained are plotted in Figure [3b] and Figure [3c]. Contrary to our expectation, these figures indicate that the phase separation properties are practically the same for poly(AE) and poly(HE·AE). As can be seen in the figures, the phase-separation region is wider for the lower molecular weight poly(AA) than for the higher molecular weight poly(AA). In the case of excess poly(AA), a single chain of the lower molecular weight poly(AA) requires fewer polycation chains for the phase separation, in which the anionic charges are neutralized, than does the higher molecular weight poly(AA). In the case of excess polycation, on the other hand, phase separation occurs even upon the addition of a small amount of lower or higher molecular weight poly(AA), because the polycation chains attach to a poly(AA) chain until the complex is neutralized.

In this study, cationic homopolymer and alternating copolymer with the dense triazole backbone, i.e., poly(AE) and poly(HE·AE), respectively, were synthesized by CuAAC polymerization. Using the monomer precursors synthesized in our previous and present studies, stepwise CuAAC will allow the synthesis of sequence-controlled polymers consisting of nonionic, cationic, anionic, and hydrophobic side chains, with the aim of synthesizing highly functional polymers that are comparable to polypeptides.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgment

The authors thank Dr. Yasuto Todokoro, Analytical Instrument Facility, Graduate School of Science, Osaka University, for his helpful suggestions on the ¹³C NMR measurements.

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Corresponding Author

Publication History

Received: 26 October 2023

Accepted after revision: 20 November 2023

Accepted Manuscript online:
20 November 2023

Article published online:
08 January 2024

© 2023. Thieme. All rights reserved

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References and Notes

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• 1c Zhang Y, Wang Z, Gemeinhart RA. J. Controlled Release 2013; 172: 962
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• 1d Osada K. Polymers 2020; 12: 1603
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• 1e Vetter VC, Wagner E. J. Controlled Release 2022; 346: 110
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• 1f Huang P, Jiang L, Pan H, Ding L, Zhou B, Zhao M, Zou J, Li B, Qi M, Deng H, Zhou Y, Chen X. Adv. Mater. 2023; 35: 2207471
  CrossrefPubMedSearch in Google Scholar
• 2a Majumdar RN, Yang S.-L, Harwood HJ. J. Polym. Sci., Polym. Chem. Ed. 1983; 21: 1717
  CrossrefSearch in Google Scholar
• 2b Couture G, Ladmiral V, Améduri B. RSC Adv. 2015; 5: 10243
  CrossrefSearch in Google Scholar
• 3a Yamasaki S, Kamon Y, Xu L, Hashidzume A. Polymers 2021; 13: 1627
  CrossrefPubMedSearch in Google Scholar
• 3b Okuno K, Miura J, Yamasaki S, Nakahata M, Kamon Y, Hashidzume A. Polym. Chem. 2023; 14: 1488
  CrossrefSearch in Google Scholar
• 3c Xu L, Nakahata M, Kamon Y, Hashidzume A. J. Polym. Sci. 2024; in press
  Crossref
• 4 Kamon Y, Miura J, Okuno K, Yamasaki S, Nakahata M, Hashidzume A. Macromolecules 2023; 56: 292
  CrossrefSearch in Google Scholar
• 5a Click Polymerization . Qin A, Tang BZ. Royal Society of Chemistry; London: 2018
  Search in Google Scholar
• 5b Angelo NG, Arora PS. J. Am. Chem. Soc. 2005; 127: 17134
  CrossrefPubMedSearch in Google Scholar
• 5c Angelo NG, Arora PS. J. Org. Chem. 2007; 72: 7963
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• 5d Binauld S, Hawker CJ, Fleury E, Drockenmuller E. Angew. Chem. Int. Ed. 2009; 48: 6654
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• 5e Binauld S, Damiron D, Connal LA, Hawker CJ, Drockenmuller E. Macromol. Rapid Commun. 2011; 32: 147
  CrossrefPubMedSearch in Google Scholar
• 5f Nguyen HV. T, Jiang Y, Mohapatra S, Wang W, Barnes JC, Oldenhuis NJ, Chen KK, Axelrod S, Huang Z, Chen Q, Golder MR, Young K, Suvlu D, Shen Y, Willard AP, Hore MJ. A, Gómez-Bombarelli R, Johnson JA. Nat. Chem. 2022; 14: 85
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  CrossrefPubMedSearch in Google Scholar
• 6a Angell YL, Burgess K. Chem. Soc. Rev. 2007; 36: 1674
  CrossrefPubMedSearch in Google Scholar
• 6b Holub JM, Kirshenbaum K. Chem. Soc. Rev. 2010; 39: 1325
  CrossrefPubMedSearch in Google Scholar
• 6c Mohammed I, Kummetha IR, Singh G, Sharova N, Lichinchi G, Dang J, Stevenson M, Rana TM. J. Med. Chem. 2016; 59: 7677
  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 8a Wang J, Uttamchandani M, Li J, Hu M, Yao SQ. Chem. Commun. 2006; 3783
  CrossrefPubMed
• 8b Liu J, Kolar C, Lawson TA, Gmeiner WH. J. Org. Chem. 2001; 66: 5655
  CrossrefPubMedSearch in Google Scholar

• Supplementary Material