4-Hydroxy- l -Proline as a General Platform for Stereoregular Aliphatic Polyesters: Controlled Ring-Opening Polymerization, Facile Functionalization, and Site-Specific Bioconjugation

Open AccessCCS ChemistryRESEARCH ARTICLE1 Oct 20204-Hydroxy-l-Proline as a General Platform for Stereoregular Aliphatic Polyesters: Controlled Ring-Opening Polymerization, Facile Functionalization, and Site-Specific Bioconjugation Jingsong Yuan, Dong Shi, Yi Zhang, Jianhua Lu, Letian Wang, Er-Qiang Chen and Hua Lu Jingsong Yuan Beijing National Laboratory for Molecular Sciences, Center for Soft Matter Science and Engineering, Key Laboratory of Polymer Chemistry and Physics of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China). , Dong Shi Beijing National Laboratory for Molecular Sciences, Center for Soft Matter Science and Engineering, Key Laboratory of Polymer Chemistry and Physics of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China). , Yi Zhang Beijing National Laboratory for Molecular Sciences, Center for Soft Matter Science and Engineering, Key Laboratory of Polymer Chemistry and Physics of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China). , Jianhua Lu Beijing National Laboratory for Molecular Sciences, Center for Soft Matter Science and Engineering, Key Laboratory of Polymer Chemistry and Physics of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China). , Letian Wang Beijing National Laboratory for Molecular Sciences, Center for Soft Matter Science and Engineering, Key Laboratory of Polymer Chemistry and Physics of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China). , Er-Qiang Chen *Corresponding authors: E-mail Address: email protected, E-mail Address: email protected Beijing National Laboratory for Molecular Sciences, Center for Soft Matter Science and Engineering, Key Laboratory of Polymer Chemistry and Physics of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China). and Hua Lu *Corresponding authors: E-mail Address: email protected, E-mail Address: email protected Beijing National Laboratory for Molecular Sciences, Center for Soft Matter Science and Engineering, Key Laboratory of Polymer Chemistry and Physics of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China). https://doi.org/10.31635/ccschem.020.201900119 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail Degradable polyesters have long been regarded as eco-friendly materials, useful for various applications while meeting the growing needs of sustainability. However, it is still challenging to synthesize functional aliphatic polyesters from abundant and cheap renewable sources. Our present study reports a readily available and versatile platform for producing functional and stereoregular aliphatic polyesters from 4-hydroxy-l-proline(4-HYP). We synthesized a bicyclic bridged lactone monomer, namely, NR-PL, by a simple and scalable two-step process allowing facile side-chain functionalization and derivatization. The ring-opening homopolymerization and copolymerization for the generation of NR-PL were controlled fully by using organobases such as 1,8-diazabicyclo5.4.0-undec-7-ene (DBU) without any detectable epimerization. This process afforded stereoregular polyesters PNRPE with molar mass (Mn) up to 90 kg/mol and a narrow dispersity (Ð) generally below 1.10. The uniqueness of the backbone, which contains two chiral centers on a rigid propyl ring, together with the versatility of the side chain, offer tunable properties complementary to existing aliphatic polyesters. The utility of the polymers was showcased by the facile site-specific bioconjugation of PNEG3PE, a water-soluble polyester, to a protein. This work might open numerous opportunities in creating functional and sustainable polyesters for a wide range of applications, including degradable plastics, drug delivery, and protein therapeutics. Download figure Download PowerPoint Introduction The past half-century has witnessed the booming of petroleum-based polymeric materials. However, the everlasting life span and end-of-use issues of such plastics have caused vast environmental pollutions, making sustainable polymeric products derived from renewable feedstock increasingly desirable.1–10 Degradable aliphatic polyesters have long been regarded as eco-friendly materials useful for various applications, including food packing, containers, agricultural mulching film, and biomaterials.5,11 For instance, polylactide (PLA), produced by the ring-opening polymerization (ROP) of lactide, is an excellent material for surgical suturing and disposable packing owing to its biocompatibility, biodegradability, and mechanical properties.12–14 Additionally, polyhydroxyalkanoates (PHA) such as poly(3-hydroxybutyrate) (P3HB) either biosynthesized using fermentation or chemically synthesized via the ROP of lactones, have long been considered promising candidates for substituting petroleum-based plastics.15,16 Despite the immense promises, the current choices of bio-renewable aliphatic polyesters are still rare; therefore, there is a pressing need for an expanded repertoire of functionalizable polyesters.17–23 Accordingly, the recent development of the ROP of amino acid-derived O-carboxyanhydride (OCA) has gained considerable success in preparing functional poly(α-hydroxy acid)s.24–27 However, the handling of OCA requires sophisticated synthetic skills, in the sense that, α-amino acids need to be converted into their corresponding α-hydroxyl acids before the transformation into the unstable OCA monomers under anhydrous conditions. Moreover, the ROP of OCAs often leads to racemization due to the enhanced acidity of α-hydrogen, which was addressed only recently by using carefully designed organometallic or organic catalysts.26,28–30 As such, a novel monomer platform that could provide easier access to libraries of functional and stereoregular polyesters from bio-derived resources is still an urgent need. 4-Hydroxyl-l-proline (4-HYP) is abundant in collagen and many other proteins; it is a naturally occurring amino acid conveniently accessible at relatively low prices. Previously, 4-HYP-based polyesters with only low molar mass (Mn), typically 1.5, were reported by Langer et al.31,32 and Park et al.33 by step-growth condensation polymerization, whereas a well-controlled ROP process has never been reported. We envisioned that 4-HYP is an ideal building block to construct functional polyesters via ROP approach for the following reasons: (1) 4-HYP could be convert readily to a bicyclic bridged lactone, namely NR-PL (Scheme 1), with the amine group as a convenient chemical handle for functionalization by using a well-established protocol.34 (2) In general, the α-hydrogen of α-amino acids is less acidic, compared with α-hydroxyl acids due to a weaker induction effect, thus making epimerization less likely. (3) Recently, we showed that 4-HYP-derived bridged bicyclic thiolactones (NR-PTL) could undergo well-controlled ROP and afford high Mn and narrow Ð polythioesters.35 The control over ROP was achieved through the judicious molecular design, which involved highly strained monomers for rapid chain propagation and inert proline–proline thioester junctions in the polymer backbone for minimized chain transfer.36 Owing to the structural similarity, we speculated that NR-PL might also generate an outstandingly controlled ROP, thereby, representing a modulable platform for fabricating numerous functional, sustainable polyesters with tunable properties. Scheme 1 | Synthesis and ring-opening polymerization of a bicyclic bridged lactone monomer, NR-PL, for the subsequent fabrication of functional, stereoregular aliphatic polyesters. Download figure Download PowerPoint Results and Discussion Synthesis and controlled ROP of NR-PL As depicted in Scheme 1, various alcohols were coupled to the amino group of 4-HYP through a urethane group. Next, the precursors NR-HYP were converted to corresponding monomers bearing different side chains via Mitsunobu reaction with typical yields ∼53–80%.34 NBoc-PL, NC6-PL, and NC12-PL were selected as monomers bearing alkyl side chains of varying lengths and branching degrees; NCbz-PL, NEG3-PL, and NEGene-PL were synthesized to represent monomers carrying aryl, hydrophilic, and modifiable moieties, respectively. The characterization data of the precursors and monomers were obtained by 1H, 13C nuclear magnetic resonance (NMR), 1H–13C heteronuclear single quantum coherence (HSQC) NMR, and single-crystal X-ray diffraction (XRD), compiled in Supporting Information Figures S1–S21. We started our ROP investigation by screening proper organobases for the monomer NC12-PL using benzyl alcohol as a model initiator. We found that weak bases such as triethylamine (TEA), did not lead to any monomer conversion (entry 1, Supporting Information Table S1). Some phosphazene bases, such as 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine (BEMP; BEMP−H+ pKaMeCN = 27.6) and the cyclic trimeric phosphazene base (CTPB; CTPB−H+, pKaMeCN = 33.3), gave fast ROP and over 95% monomer conversion in tetrahydrofuran (THF).37 However, both phosphazene superbases mediated ill-controlled ROP, as revealed by multimodal peaks, low Mn, and broad Ð in size-exclusion chromatography (SEC; entries 2 and 3, Supporting Information Table S1). In contrast, when the base 1,8-diazabicyclo5.4.0-undec-7-ene (DBU, DBU−H+ pKaMeCN = 24.3) was used for the ROP at an initial monomer/initiator/base ratio (M0/I0/base0) of 50/1/1 in deuterated chloroform (CDCl3), a > 95% monomer conversion was realized within 24 h, affording poly(N-carboxydodecane 4-hydroxyl-l-prolyl ester) (PNC12PE) with a sharp and unimodal peak in SEC. Also, the ROP was confirmed by 1H NMR spectroscopy ( Supporting Information Figure S22), and the shift of the ester carbonyl stretch peak from 1800 cm−1 of the lactone to the corresponding 1748 cm−1 of the open-chain ester was demonstrated by Fourier-transform infrared (FT-IR) spectroscopy ( Supporting Information Figure S23). The Mn and Ð measurements obtained were 17.9 kg·mol−1 (expected Mn = 16.3) and 1.09, respectively (entry 1, Table 1). Notably, prolonging the reaction time to 72 h did not cause broadening of the peak in the SEC, suggesting minimal transesterification-induced chain transfer, even after monomer consumption. Besides, increasing the equivalent of DBU to M0/I0/base0 = 50/1/5 led to a significantly enhanced ROP rate (> 95% monomer conversion within 5 h) without jeopardizing the degree of control of the reaction (entry 2, Table 1). Further, DBU-mediated ROP in other common organic solvents, such as dichloromethane (DCM) and THF, also gave similar well-controlled results (entries 3 and 4, Table 1; Supporting Information Figure S24). Kinetic studies of the DBU-catalyzed ROPs depicted first-order kinetics over monomer concentration in all three solvents, with those in DCM and CDCl3 being considerably faster than that in THF (Figure 1a). Moreover, the well-controlled chain-growth feature of DBU-mediated ROP of NC12-PL was confirmed by the linear relationship of Mn with the monomer conversion (Figure 1b) or the feeding M0/I0 ratio (Figure 1c). SEC analyses exhibited sharp and narrow unimodal peaks for all the polymers (inset of Figure 1b and c). The Mn of PNC12PE obtained were in 95% monomer conversion. bCalculated molar mass based on feeding M0/I0 ratio. cObtained relative molar mass determined by SEC in THF using polystyrene as the standard; PNC6PE and PNC12PE were insoluble in DMF. dObtained absolute molar mass determined by SEC in DMF containing 0.1 M LiBr equipped with a multiangle laser light scattering (MALLS) detector. eDispersity, determined by SEC. To expand the monomer scope, we explored the ROP of NBoc-PL, NCbz-PL, NC6-PL, NEG3-PL, and NEGene-PL, respectively, under the reaction conditions mentioned above. Four out of the five monomers, namely NBoc-PL, NC6-PL, NEG3-PL, and NEGene-PL gave over 95% conversion and controlled the homopolymerization process satisfactorily (entries 11–17, Table 1; Supporting Information Figures S25–S29). For example, NEG3-PL was polymerized in a fully controlled manner and gave water-soluble PNEG3PE with linear growth of Mn as a function of feeding M0/I0 ratio up to 75/1 as unimodal SEC peaks ( Supporting Information Figure S30). Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) of benzyl alcohol-initiated ROP of NEG3-PL at a feeding M0/I0 ratio of 10/1 displayed a group of Poisson-distributed peaks, which were assigned to oligomeric PNEG3PE bearing the C6H5CH2–/–OH end groups (Figure 1d). Homopolymerization of NCbz-PL in most common organic solvents, including DCM, CDCl3, THF, and DMF, led to evident precipitation due to the poor solubility of resultant PNCbzPE. Nevertheless, a random copolymerization of NEG3-PL and NCbz-PL gave copolymers with good solubility and excellent control, shown by both Mn and Ð (entry 18, Table 1). Sequential ROP of NEG3-PL and NC12-PL in one-pot synthesis gave a well-defined block copolymer P(NEG3PE-b-NC12PE), once again highlighting the controllability of the ROP (entry 19, Table 1; Supporting Information Figure S31). Moreover, the structural diversity of the polymer was tailorable by postpolymerization modification of PNEGenePE via highly efficient UV-triggered thiol–ene reaction, which enabled the facile introduction of cationic, anionic, as well as zwitterionic moieties in an almost quantitative fashion ( Supporting Information Figures S32–S35). Overall, the ROP of NR-PL offered outstanding control and tunability over Mn, Ð, end groups, and side-chain functionalities. Stereoregularity of PNRPE The ROP of OCA could undergo epimerization easily due to the deprotonation of the acidic α-hydrogen. Thus, we conducted comprehensive NMR and hydrolysis studies to investigate the stereoregularity of NR-PL ROP (Figure 2). Unfavorably, 13C NMR spectrum of PNBocPE in CDCl3 at room temperature (Figure 2a) exhibited complicated splitting and multimodal peaks. Switching the solvent to DMF-d7, the same polymer gave a considerably cleaner 13C NMR spectrum pattern than that in CDCl3 (Figure 2a), but the peaks for carbon (c, d, and b) were still broad and difficult to assign. We reasoned that the broadening and splitting could be a combined effect of the trans–cis isomerization of the urethane carbonyl and the endo–exo conformation isomerization of the prolyl ring.38–40 This notion was later confirmed by the 13C NMR spectroscopy measurements of PNBocPE in DMF-d7 at 90 °C, which showed sharp and single peaks for major prolyl carbons (Figure 2a). We next hydrolyzed the monomer NEG3-PL and corresponding polymer PNEG3PE in alkaline D2O solution at 4 °C for 2 h to obtain more conclusive evidence regarding the tacticity of the polymers. Interestingly, completion of both hydrolysis reactions were attained, giving almost identical 1H NMR spectra, assignable to the pure enantiomer (2S,4S)-N-(2,5,8,11-tetraoxadodecanoyl)-4-hydroxypyrrolidine-2-carboxylic acid (top and middle spectra in Figure 2b), which was distinct from the 1H NMR of (2S,4R)-N-(2,5,8,11-tetraoxadodecanoyl)-4-hydroxypyrrolidine-2-carboxylic acid (trans-NEG3-HYP; bottom spectrum in Figure 2b). Together, we inferred from the solvent and temperature-dependent 13C NMR spectroscopy and the 1H NMR analysis of the hydrolysis product that there was indeed no epimerization during the polymerization process. Figure 2 | (a) Overlay of 13C NMR spectra of PNBocPE in CDCl3 (25 °C) and DMF-d7 (25 and 90°C). (b) Overlay of 1H NMR spectra of trans-NEG3-HYP and the hydrolyzed product of NEG3-PL and PNEG3PE in alkaline D2O (pD = 10 for NEG3-PL pD = 13 for PNEG3PE, respectively, 4 °C, 2 h). Download figure Download PowerPoint Thermal and mechanical properties of PNRPE We conducted thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) experiments to evaluate the thermal property and phase transition of PNRPE. The decomposition temperature (Td) to a 5% weight loss of PNEG3PE, PNC6PE, and PNC12PE were ∼300 °C, close to the Td of most aliphatic polyesters, while the Td of PNBocPE and PNEGenePE were 210 °C and 252 °C, respectively, likely due to the thermal instability of the Boc and allyl carbamate groups ( Supporting Information Figure S36 and Supporting Information Table S2). The DSC traces acquired during the second heating at a rate of 10 °C/min of PNRPE are depicted in Figure 3a. The glass transition temperatures (Tg) of PNEG3PE and PNEGenePE were detected at 40 °C and 64 °C, respectively. PNC12PE and PNC6PE showed complex transition behaviors; in the low-temperature range, both samples likely underwent the glass transition immediately, followed by a cold-crystallization process, resulting in an overall apparent endothermic process. Moreover, upon heating PNC12PE to high temperature (190 °C), sequential small endotherm, an exotherm, and a larger endotherm were observed, suggesting a minor melting/recrystallization at 196 °C and 198 °C, respectively occurred, proceeded by a major melting process with a melting temperature (Tm) at For PNC6PE, the larger endothermic peak was apparent at °C, after the small at The DSC results that PNC12PE and PNC6PE might phase at low and be into a by at °C ( Supporting Information Figure Besides, the of PNC12PE revealed a at and of at ( Supporting Information Figure Figure 3 | (a) DSC traces of PNRPE obtained during the second heating at a rate of 10 Inset: expanded DSC traces of PNEG3PE and PNEGenePE the glass (b) of PNRPE at room differential scanning bicyclic bridged lactone Download figure Download PowerPoint Figure depicted the of PNRPE in which the samples were at °C/min from the to room The results that both PNEG3PE and PNEGenePE were in with the DSC scattering were in the and of which the was to with the of the chain bearing rigid backbone and the relatively In contrast, both PNC12PE and PNC6PE showed sharp diffraction peaks. For three peaks following a ratio of scattering of were with the peak at This was an of a with a of diffraction was with PNC6PE, a side chain of and a of In the PNC12PE and PNC6PE showed similar diffraction the of The were different PNEG3PE, PNEGenePE and PNC6PE, which might from the in both their and side-chain the results that the thermal and mechanical properties of PNRPE could be readily in a broad range by the side thereby, for materials in the protein Previously, we as an for the ROP of amino acid to in a thioester group at the end of synthetic the polymers were to enhanced protein bearing an via a chemical we that a similar could be for a thioester could be at the end of PNEG3PE (Figure For this and DBU were selected to the ROP of NEG3-PL which afforded PNEG3PE bearing thioester with a Mn of 6.0 kg·mol−1 and a Ð of 1.07 in SEC ( Supporting Information Figure MALDI-TOF analysis of revealed the corresponding bearing the thioester group on the chain end (Figure Next, we the of to a temperature of the two at a molar ratio of in = for 12 h, afforded a conversion ( Supporting Information Figure and a of of the corresponding (Figure is that alkyl prolyl thioester was in by using an in prolyl we demonstrated that could at a good rate and without or Figure 4 | (a) Synthesis of and subsequent reaction with (b) MALDI-TOF mass spectrum of PNEG3PE initiated by M0/I0/base0 = (c) of chemical protein matrix-assisted laser desorption/ionization-time of flight. Download figure Download PowerPoint General for the synthesis of NR-PL We present NC12-PL as a model for the synthesis reaction, as solution of in THF was to a solution of and in THF over at the reaction was at room temperature for 4 h, following solvent by We the product by chromatography = to afford a The conditions for the characterization and the results are as 1H NMR = = = 10.6 = = 10.6 = 13 NMR mass spectrometry = found = General for the ROP of NR-PL We NC12-PL as an for the ROP, as In a the solution of NC12-PL 0.1 in CDCl3 was with benzyl alcohol M, in CDCl3, followed by the of M, in CDCl3, and the reaction at room temperature for 5 an of the solution was quenched by acid in CDCl3 before analysis by 1H NMR for the of the monomer conversion. The polymer was by precipitation in and for 5 the obtained was The polymer was as Mn and Ð were by size-exclusion chromatography 1H NMR We have demonstrated the facile synthesis of a of 4-HYP-derived functional aliphatic polyesters, bearing tunable side The polymerization process of the NR-PL was carefully controlled by using DBU as the and as affording stereoregular PNRPE polyesters with controlled Mn up to kg·mol−1 and well-defined end The solubility and properties of the resulting PNRPE were easily by the introduction of side-chain functionalization either from the monomer or via postpolymerization PNEG3PE, demonstrated excellent solubility and was to site-specific protein via highly efficient the controlled ROP and tunability of NR-PL, this approach has the to open opportunities in creating functional polyesters from renewable for a wide range of applications, including plastics, drug delivery, and protein therapeutics. 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