Thioacetate-Based Initiators for the Synthesis of Thiol-End-Functionalized Poly(2-oxazoline)s

Dr. G. Gil Alvaradejo, Dr. Y. Wang, Prof. G. Delaittre Institute of Biological and Chemical Systems (IBCS) Karlsruhe Institute of Technology (KIT) Hermann-von-Helmholtz-Platz 1, Eggenstein-Leopoldshafen 76344, Germany Dr. M. Glassner, Dr. V. R. de la Rosa, Prof. R. Hoogenboom Supramolecular Chemistry Group Centre of Macromolecular Chemistry (CMaC) Department of Organic and Macromolecular Chemistry Ghent University Krijgslaan 281-S4, Ghent 9000, Belgium Dr. R. Kumar, Dr. M. Hirtz Institute of Nanotechnology (INT) Karlsruhe Institute of Technology (KIT) Hermann-von-Helmholtz-Platz 1, Eggenstein-Leopoldshafen 76344, Germany


DOI: 10.1002/marc.202000320
Polymer grafting is a powerful method to control surface properties of materials in the biomedical and biotechnological realms, notably with respect to biofouling. Examples include medical implants, biosensors, drug delivery nanovehicles, where interfaces are required to both enhance specific binding and reduce non-specific binding. [1][2][3][4][5][6][7][8] In this context, poly(ethylene glycol) (PEG) has been for a long time considered the gold standard. [9,10] However, due to its degradation mode [11,12] and potential immunogenicity, [13,14] alternative polymers are sought after. Poly(2alkyl-2-oxazoline)s (PAOx), specifically poly-2-methyl-2-oxazoline (PMeOx) and poly-2-ethyl-2-oxazoline (PEtOx), share some characteristics with PEG, such as stealth behavior, biocompatibility, and low toxicity. [15][16][17] At the same time, PMeOx and New functional initiators for the cationic ring-opening polymerization of 2-alkyl-2-oxazolines are described to introduce a thiol moiety at the α terminus. Both tosylate and nosylate initiators carrying a thioacetate group are obtained in multigram scale, from commercial reagents in two steps, including a phototriggered thiol-ene radical addition. The nosylate derivative gives access to a satisfying control over the cationic ring-opening polymerization of 2-ethyl-2-oxazoline, with dispersity values lower than 1.1 during the entire course of the polymerization, until full conversion. Cleavage of the thioacetate end group is rapidly achieved using triazabicyclodecene, thereby leading to a mercapto terminus. The latter gives access to a new subgeneration of α-functional poly(2-oxazoline)s (butyl ester, N-hydroxysuccinimidyl ester, furan) by Michael addition with commercial (meth)acrylates. The amenability of the mercapto-poly(2-ethyl-2-oxazoline) for covalent surface patterning onto acrylated surfaces is demonstrated in a microchannel cantilever spotting (µCS) experiment, characterized by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary-ion mass spectrometry (ToF-SIMS).
PEtOx offer the advantage of higher stability, lower viscosity, less demanding synthesis, and large number of functionalization possibilities, notably thanks to functional side chains. [18][19][20] These features make PAOx promising alternatives for the functionalization of biomaterials. [17,[21][22][23] The synthesis of PAOx proceeds through cationic ringopening polymerization (cROP), which allows full control of the polymer properties via tuning of the molar mass distributions and functionality, depending on the choice of the 2-oxazoline monomer(s) and the specific end groups arising from initiation and termination. [19,[24][25][26][27] Several strategies to graft PAOx on surfaces have been reported. [28,29] A simple and popular method uses αor ω-functional PAOx prepared with either a functional initiator or a terminating agent, to introduce (directly or after additional modification) a coupling moiety at one chain end. [27] Functionalities introduced by direct end-capping of the living polymer species include trialkoxysilane, [30,31] alkyne, [32] di thiobenzoate, [33] xanthate, [34] methacrylate, [35] methacrylamide, [36] and norbornene, [37] among others, as anchors for the modification of different substrates via the ω end group of PAOx. For this same purpose, functional initiators carrying moieties such as disulfide, [38] allyl, [30] alkyne, [39] or coumarin, [40] to name but a few, were used to produce α-functionalized PAOx.
Here, we present a strategy for the synthesis of well-defined functional α-thiolated PAOx. Thiols are popular reactive groups because of their ability to engage in a wide range of reactions with compounds such as halides, [41] pentafluorophenylalkanes, [42] alkenes, [43] alkynes, [44] disulfides. [45] In the context of end-functional PAOx, thiol moieties have exclusively been introduced at the ω chain end, where it was introduced by direct termination with sodium hydrosulfide [46] or by postmodification of ω end groups such as xanthate [44,46] or diallylamino. [47] To the best of our knowledge, a thiol group was never introduced at the α position of PAOx chains. In the present contribution, we report the synthesis and characterization of two initiators for the cROP of 2-oxazolines and introduction of a masked thiol functionality at the α chain end. After deprotection, we demonstrate the reactivity of the thiol end group using various alkenes, enabling the introduction of alternative functionality to the α terminus. Finally, we show that the as-synthesized α-mercapto PEtOx is amenable to highly localized grafting onto acrylated substrates using a fine surface patterning method, i.e., microchannel cantilever spotting (µCS). [48][49][50][51] Since the thiol moiety is an excellent nucleophile, it may interfere with the cROP process. A classic method to circumvent such issues in organic and polymer chemistry is the recourse to protecting groups. This method has been rather parsimoniously used in the synthesis of α-functional PAOx. Example of protecting groups incorporated in cROP initiators include N-alkylphthalimide or tert-butyl N-monoalkylcarbamate (for amines), [52][53][54][55][56] tert-butyldiphenylsilyl ethers (for hydroxyl), [57] methyl ester (for carboxylic acid or others through amidation), [58][59][60][61][62][63] and furanyl Diels-Alder cycloadduct (for maleimide). [64] Here, the thioacetate group has been chosen as a stable and economic protection group for thiols. [65] In the case of the tosylate initiator, ThioAcTos (Scheme 1), the synthetic route started with the nucleophilic substitution of allyl alcohol with tosyl chloride to yield allyl tosylate 1 (Scheme S1, Supporting Information). The corresponding initiator ThioAcTos was obtained by phototriggered thiol-ene radical addition of 1 with thioacetic acid. An alternative route for the synthesis of the nosylate initiator (ThioAcNos) was designed, because purification of the products obtained following the previous strategy proved to be a more challenging task. ThioAcNos was obtained after nosylation of the product of the thiol-ene addition between thioacetic acid and allyl alcohol (2). This led to a more straightforward purification avoiding chromatographic steps and resulting in higher yields. Analysis of both initiators by 1 H and 13 C NMR spectroscopy, as well as electrospray ionization mass spectrometry (ESI-MS), confirmed that the targeted structures were obtained with high purity (Figures S1-S6, Supporting Information).
The polymerization was performed using optimized microwave conditions, [66] at 140 °C and with acetonitrile as reaction solvent (Scheme 1). To assess the ability of both initiators to start the polymerization, a first set of experiments was carried out for 7.5 minutes with [EtOx] = 4 m and [EtOx]/[initiator] = 50. Full conversion was achieved in both cases; however, the corresponding size-exclusion chromatography (SEC) traces revealed distinct dispersity values of 1.20 and 1.10 for ThioAcTos and ThioAcNos, respectively ( Figure S7, Supporting Information). The broader molar mass distribution obtained when initiating the polymerization with ThioAcTos may be the result of a lower electrophilicity of this initiator, leading to a lower initiation rate. [67] It is nevertheless conceivable that ThioAcTos is a suitable initiator for the polymerization of more nucleophilic monomers such as MeOx. However, we did not investigate this further and concentrated on ThioAcNos because it will offer more versatility in terms of accessible 2-alkyl-2-oxazoline monomers. The living character of the poly merization was successfully shown via kinetic studies using three different monomer-to-initiator ratios (20, 40, and 60) (Figure 1). As expected, the polymerization proceeds faster when a lower [EtOx]/[ThioAcNos] ratio is used ( Figure 1A). [68] All kinetics showed a linear behavior, indicating a constant concentration of the propagating species. The apparent k p values are rather similar (Table S1, Supporting Information) and therefore appear to be independent of the initial monomer-to-initiator concentration, as reported in previous studies. [68] Additionally, SEC analysis of the samples taken at various time intervals evidenced a linear increase of the number-average molar masses (M n ) and dispersity values consistently below 1.1 ( Figure 1B). A clear uniform shift towards higher retention times can be Scheme 1. Synthetic route for the synthesis of α-thiolated PEtOx by polymerization of EtOx using functional initiators ThioAcTos and ThioAcNos and base-triggered deprotection. observed ( Figure 1C; Figure S8, Supporting Information). The discrepancy between theoretical and experimental M n values comes from the fact that the latter are calculated relative to poly(methyl methacrylate) standards, which results in an overestimation of the molar mass. [69] A purified polymer, ThioAc-PEtOx, obtained with [EtOx]/ [ThioAcNos] = 40, was used to confirm the nature of the end groups, and for further deprotection and modification. In NMR spectra, although the acetate methyl group signal overlaps with those corresponding to the PEtOx side-chain methylene groups, signals of all methylene protons located between the sulfur atom and the first nitrogen atom of the polymer chain appear independently and confirm the presence of the end group ( Figure S9, Supporting Information). These methylene proton signals could also be used to calculate the degree of polymerization of the ThioAc-PEtOx and provided values closer to the expected ones than SEC (see Table S2 in the Supporting Information). ESI-MS also confirmed the structure of the polymer (Figure 2, black spectrum). Note that double-charged species are represented here. The main population was assigned to the thioacetate-functionalized PEtOx chains with an ω hydroxyl end group arising from termination with sodium carbonate. Besides, a population was observed arising from proton-initiation, either due to residual protic sources (e.g., water) or β-termination. [24] Two further minor populations were also detected, yet not assigned. It is however noteworthy that these vanish during subsequent steps, which suggests they correspond to chains carrying the thioacetate moiety.
Deprotection was initially attempted using sodium hydroxide and tetramethylammonium hydroxide, yet without any success (broad distributions). Eventually, triazabicyclodecene (TBD), a strong yet selective guanidine base, led to full deprotection and yielded Thio-PEtOx, as evidenced via ESI-MS measurement by the negative shift of 21 amu (i.e., 42/2), corresponding to the loss of the acetate group ( Figure 2, red spectrum). The analysis by SEC revealed more challenging. Indeed, the as-deprotected product led to a multimodal distribution, clearly indicating the presence of a population with molar masses similar to those of the parent ThioAcPEtOx, as well as a main distribution with approximately twice the molar mass (Figure 3, dotted red line). This can clearly be attributed to disulfide coupling. Incubating the deprotected polymer solution with a reducing agent such as dithiothreitol (DTT) did reduce the occurrence of coupled chains but did not fully suppress it (Figure 3, full red line).
As previously mentioned, the versatility of the thiol group allows a wide range of reactions. In this study, we first wanted to briefly showcase that Thio-PEtOx could be used as an access point to further α-functional PEtOx, particularly making use of the large variety of commercial functional Michael acceptors, such as acrylates and methacrylates. Conditions chosen for the base-catalyzed thiol-ene addition are depicted in Figure 2 (top): with dimethylphenylphosphine (DMPP) playing the dual role of reducing agent and base, in acetone at ambient temperature. The first reaction was performed with n-butyl acrylate (BA), leading to a slightly amphiphilic structure. ESI-MS measurement evidenced a shift of the main population corresponding to the addition of the acrylate (Figure 2, blue spectrum). Importantly, the SEC trace of BA-PEtOx displayed a near-monomodal distribution (Figure 3, blue line), which closely reproduced the distribution of the protected ThioAc-PEtOx, demonstrating a clean deprotection-functionalization sequence and confirming the origin of the second population observed for Thio-PEtOx.
In order to showcase the versatility of the new Thio-PEtOx as an entry point for further end-functional poly(2-oxazoline)s, we then performed a similar end-group modification with two (meth)acrylates that carry a reactive group in the ester side chain, namely acrylic acid N-hydroxysuccinimide ester (NHSA) and furfuryl methacrylate (FurMA). The respective products, NHSA-PEtOx and FurMA-PEtOx, were detected as main species in ESI-MS (Figure 2, green and dark blue spectra, respectively). SEC characterization indicated a similar outcome as for the functionalization with BA ( Figure S10, Supporting Information). For NHSA, however, a secondary distribution with approximately a doubled molar mass is visible. It could arise from undesired reaction of the NHS ester with thiol end groups, leading to covalently coupled chains, as some succinimidyl ester have been found to react with cysteine side chains for instance. [70] Yet, no vinyl signals were observed in 1 H NMR spectrum (not shown). It is therefore assumed that the present reaction did not go to completion.
We further aimed at demonstrating the possibility to pattern thiolated PEtOx onto flat surfaces by Michael addition. Indeed, PEtOx has been involved in several studies related to biointerfaces, particularly to control bioadhesion. [21,22,28,29] In the present case, we harnessed microchannel cantilever spotting (µCS) [48][49][50][51] to position microdroplets of a reactive PEtOx ink onto a functionalized surface, here an acrylated Si wafer (Figure 4A). The droplets then act as microreactors in which covalent grafting to the surface occurs. The ink consisted of Thio-PEtOx, DMPP and glycerol-to tune viscosity and evaporation-in DMSO. A control ink in which Thio-PEtOx was replaced with a nonfunctional PEtOx was also spotted. All samples were thoroughly washed after reaction. Figure 4B depicts the droplet pattern imaged by optical microscopy, directly after the µCS step: Dots with a diameter of ≈15 µm were arranged with a 30 µm spacing. X-ray photoelectron spectroscopy (XPS) analysis of the activated wafer showed a total carbon content of 4.3 at% at the surface, attributed to contaminants ( Figure 4C bottom and Table S2 in the Supporting Information). After functionalization with 3-(trimethoxysilyl)propyl acrylate, the total carbon content increased significantly to 19.7 at% and the appearance of a peak at 289.2 eV was observed ( Figure 4C middle), proving the presence of the OCO species of the acrylic polysiloxane layer with a contribution of 3.5 at%. After Thio-PEtOx patterning, an intensity decrease of this latter signal (1.1 at%) indicated successful surface modification (Figure 4C bottom). Concomitantly, the appearance of an additional peak at 288.4 eV, attributed to amide groups, [71] was observed with a concentration of 0.5 at% (Figure 4C top), proving functionalization with PEtOx. Also, the corresponding signal at 400.1 eV (0.5 at%) indicates the presence of nitrogen atoms on the surface with a 1:1 ratio to the amide carbon (Table S2, Supporting Information). Finally, time-of-flight secondary-ion mass spectrometry (ToF-SIMS), which offers a greater spatial resolution and can therefore independently probe the spotted areas and their surroundings, confirmed the presence of chemical patterns: Dots rich in CN,  CNO, S, and SH species on a background poor in these species are clearly identified ( Figure 4D top; Figures S11 and S12, Supporting Information). The pattern fidelity, with respect to the initial droplet array, is very high ( Figure 4D bottom; Figure S13, Supporting Information). Importantly, the control ink did not show any pattern, thereby demonstrating that ThioPEtOx covalently reacted with the surface, and did not just adsorb.
In the present contribution, we reported two new thioacetate-functionalized initiators for the cationic polymerization of 2-alkyl-2-oxazolines. The nosylate version revealed to be most suitable for the polymerization of 2-ethyl-2-oxazoline in acetonitrile, achieving near-quantitative conversion, low dispersity, and full α end-group retention, within minutes at 140 °C under microwave irradiation. Full deprotection could be performed using an organic guanidine base, namely triazabicyclodecene. Precise characterization of the polymer with a free thiol α end group proved challenging due to the occurrence of disulfide coupling. Nevertheless, further functionalization by Michael addition with functional (meth)acrylates unambiguously confirmed a clean transformation. It is incidentally demonstrated that the α-mercapto poly(2-ethyl-2-oxazoline) is an access point to generations of α-functional polymers by virtue of the large range of available and accessible (meth)acrylic derivatives, themselves being further potential access points, in the present case by further amidation or Diels-Alder cycloaddition.
Finally, the α-mercapto poly(2-ethyl-2-oxazoline) was successfully patterned in a covalent way onto acrylated surfaces using a soft micro/nanolithography method, namely microchannel cantilever spotting. This appears promising for the design of micro/nanostructured biointerfaces, in which poly(2-ethyl-2-oxazoline) could act as antibioadhesive barrier. Further methods of patterned immobilization, such as photoinitiated thiol-ene addition could be envisaged as well.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.