Authors: Hironobu Murata | Stefanie L. Baker | Bibifatima Kaupbayeva | Dylan J. Lewis | Libin Zhang | Susanne Boye | Albena Lederer | Alan J. Russell
Source: Journal of Polymer Science (article, 2020-01-01)
Field: Chemistry — Organic Chemistry

Ligands and characterization for effective bio‐atom‐transfer radical polymerization Three new morpholine-based ligands were synthesized and used in the ATRP reactions of free polymers and polymers grown from the surface of a protein in aqueous media. The new ligands were designed in a way that decreased their Lewis basicity with each additional incorporation of a morpholine group (1, 2, and 3). The morpholine-based ligands significantly slowed the polymerization rates and led to lower dispersities than Et6TREN. Increasing the control of aqueous ATRP correlated well with decreasing ligand basicity. ATRP is a reversible deactivation radical polymerization method commonly used to synthesize polymers with defined architectures and narrow molar mass distributions. 1 Of particular interest is the growth of polymers from the surface of a protein serving as a macroinitiator to create uniform protein–polymer conjugates. Since the first studies of protein-ATRP, 2, 3 protein–polymer conjugates with different polymer architectures and functionalities have been synthesized to manipulate the activity, stability, and substrate binding affinity of the conjugate. 4-12 These previous studies indicated that to provide consistent and reliable performance, conjugates must be homogeneous in the number of chains attached and have low molar mass dispersities. A challenge in creating well-defined protein–polymer conjugates by grafting from a protein's surface is the requirement to use conditions where the protein's secondary and tertiary structures are not altered. These conditions include reduced protein (initiator) concentrations and the use of water as a solvent.13 The challenges in performing ATRP in aqueous media have been reviewed recently.14-17 Briefly, the concentration of radicals is higher in water than in typical organic solvents, leading to faster polymerizations and broader molar mass distributions. Disproportionation of the water-insoluble activating CuI ion and dissociation of halide from the CuII-ligand–halide complex additionally lead to a reaction with less control. Phosphate buffers commonly used with protein solutions add another challenge, wherein phosphate ions may combine with CuI ions to form insoluble Cu3PO4 compounds, reducing the concentration of active species and slowing the polymerization reaction.13 Finally, and perhaps most critically, no effective characterization tool for protein–polymer conjugates has been developed, so even if the chemistry has been optimized, the characterization of the products does not accurately describe what has been made. This is especially true of light-scattering-based techniques that use unproven assumptions to calculate an apparent molar mass of the conjugate and of GPC that does not function effectively because of the unknown changes in conjugate–column interactions as a function of modification. New analytic techniques need to be tested first with ATRP-synthesized polymers. Compared with tris(2-pyridylmethyl)amine (TPMA) and 2,2′-bipyridine (bpy), common pyridyl ligands for aqueous ATRP that typically result in a slow reaction with moderate control, alkyl amines, such as 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA), result in a rapid polymerization18 with slightly less control (Supporting Information Fig. S1). The activity of neutral nitrogen ligands can be altered by changing the number of nitrogen coordination sites and the number of carbon atoms that separate nitrogen atoms.19 Knowledge of heteroatom substitution within the nitrogen ligand on the polymerization rates and dispersities is not as fully developed. Herein, we describe the synthesis of three new morpholine-based ATRP ligands and their application in polymerizations of oligo(ethylene glycol) methyl ether methacrylate (OEGMA) and zwitterionic carboxybetaine methacrylate (CBMA) in aqueous, buffered conditions at room temperature. The influence of electron withdrawing groups (Lewis basicity) on the rate of polymerization and the degree of control (evaluated by the polymer dispersity) was examined. The newly prepared ligands showed improved control of the molar mass distribution and decreased polymerization rates compared with tris(2-(diethylamino)ethyl)amine (Et6TREN). In addition, pCBMA and pOEGMA polymers of two different molar masses synthesized utilizing the new 4-(bis(N,N-diethylaminoethyl)aminoethyl)morpholine (MMA) ligand were characterized by asymmetric flow field-flow fractionation coupled with multiangle laser light scattering (AF4-MALLS). Subsequently, uniform pCBMA and pOEGMA polymers were synthesized by grafting from the surface of avidin using MMA ligand to create well-defined protein–polymer conjugates. Copper-mediated ATRP kinetics in aqueous media depend on the equilibrium (KATRP = kact/kdeact) between the production of radicals (kact) and the generation of alkyl halides (kdeact) (Fig. 1). Controlling this equilibrium process enables the fine-tuning of polymerization rates, chain-end termination, and dispersities (Đ).1 This is extremely important for decreasing the heterogeneity of a sample of either free polymer or grafted-from protein–polymer conjugates. There are potential side reactions when performing aqueous ATRP, however, including the disproportionation of the active species, CuILm, to Cu0 and CuIILm and loss of the halide (X═Br or Cl) from the deactivating species, X─CuIILm. Therefore, in order to slow the ATRP kinetics (decrease KATRP) and gain better control (lower Đ), the CuILm complex should be stabilized to prevent disproportionation and the concentration of deactivating species, X-CuIILm, should be increased. The ligand (Lm) is responsible for increasing the solubility and stability of CuI,20 so we focused on tuning the ligand structure to control bio-ATRP kinetics. Ligands with pyridine groups such as bpy are known to have slower polymerization kinetics than alkyl amine derivative ligands such as HMTETA.20 Interestingly, pyridines are weak Lewis bases and alkyl amines are strong Lewis bases21 (Fig. 1) that complex with CuI, which is a weak Lewis acid. We were therefore interested in synthesizing ligands that varied in Lewis basicity(Fig. 1) and hypothesized that ligands that were weaker Lewis bases would increase the stability of the CuI species to decrease KATRP and Đ. The Lewis basicities of the ligands were varied by incorporation of 1, 2, or 3 morpholine groups from a reference structure of an alkyl amine ligand, Et6TREN (Fig. 1). Incorporation of additional morpholine groups weakens the Lewis basicity of the ligand, since the oxygen atoms are electron-withdrawing, which decreases the charge density on the ring nitrogens and increases stability.21 All ligands were soluble in aqueous media and would be amenable to protein-ATRP. pOEGMA [Fig. 2(a)] and pCBMA [Fig. 2(b)] were synthesized using the various ligands and the ATRP kinetics of the reactions were monitored by taking aliquots of the reactions at increasing time points and measuring the number-average molar mass (Mn) and dispersities (Mw/Mn) using GPC (GPC with three columns is thought to be an effective tool for polymer analysis and is used ubiquitously). The targeted degree of polymerizations (DPs) for pOEGMA and pCBMA were 100, which would correspond to the Mn values of 50,000 and 22,900 Da for pOEGMA and pCBMA, respectively. Et6TREN had the highest polymerization rate reaching 72% conversion within the first 5 min and 92% conversion after 60 min for pOEGMA [Fig. 2(a) and Supporting Information Tables S1–S4). The polymerization was not well controlled as indicated by the relatively high values of Đ around 1.60 and nonlinearity of Mn versus time. Incorporation of just one morpholine group (MMA) into the ligand decreased the polymerization rate and the rates were steadily decreased with each additional morpholine group. The reactions with the morpholino ligands (MMA, N,N-diethylaminoethyl-bis(2-morpholinoethyl)amine [DMA], and tris(2-morpholinoethyl)amine [TMA]) were also more controlled as indicated by the linear increases in Mn over time and the corresponding lower values of Đ. The percent conversion with MMA was 54% after 60 min with an average Đ of 1.25. DMA had a percent conversion of 30% after 60 min with an average Đ of 1.12 and TMA had a percent conversion of 11% after 60 min with an average Đ of 1.12. The promising results with pOEGMA were mimicked with pCBMA [Fig. 2(b) and Supporting Information Tables S5–S8]. Percent conversions after 60 min and average Đs of pCBMA were 136% (1.51), 105% (1.31), 41% (1.14), and 24% (1.13) for Et6TREN, MMA, DMA, and TMA, respectively. Slower polymerization rates and lower values of Đ with the morpholino ligands indicated better control of the ATRP reaction and the reaction rates correlated with the Lewis basicity of the ligand (Et6TREN > MMA > DMA > TMA). Moreover, since the percent conversions of pOEGMA and pCBMA with the TMA ligand were low after 60 min, the ATRP reaction times were extended to 6 h in order to determine the point at which the polymerization terminated (Supporting Information Tables S9 and S10). The values of Mn plateaued after 4 h with 14% conversion and 33% conversion for pOEGMA and pCBMA, respectively, while maintaining low values of Đ. In general, the data show that polymerization rates can be tuned by tuning the Lewis basicity of the ligand and that weaker Lewis bases, with increased numbers of morpholine groups, leads to more controlled ATRP. Additionally, these morpholine-based ligands were able to polymerize different monomer types, namely OEGMA and CBMA, and are expected to work well with other monomer types as well. In addition to GPC analysis, the molar masses of pCBMA and pOEGMA were also determined by AF4-MALLS. The main advantages of using the channel-based separation technique of AF4-MALLS are avoiding interactions with separation columns, very broad separation ranges, as well as gentler separation due to significantly reduced shear forces.22, 23 Another advantage is that, using MALLS, one can obtain the absolute molar mass of the sample based on the Rayleigh ratio Rθ, without the need to use calibration standards.24-26 (as is needed for GPC). Since the MMA ligand showed the higher conversion of polymerization while maintaining lower values of Đ, it was selected to prepare the polymers for AF4-MALLS analysis. Two different molar masses of pCBMA and pOEGMA with the target DPs of 50 and 200 were synthesized using the MMA ligand. To estimate the molar masses of polymers, GPC calibration with Pullulan standards was applied. From GPC, it was determined that the relative Mn values of pCBMA polymers were 20.6 and 49.3 kDa, while pOEGMA were 24.6 and 49.5 Da for target DPs of 50 and 200, respectively. The values of Đ of these polymers varied from 1.26 to 1.55. (Supporting Information Table S11 ). Interestingly, AF4-MALLS results were different from the results obtained with GPC. After optimizing the AF4 conditions for separation of pCBMA and pOEGMA polymers by adjusting the flow rates, all sample fractograms showed a baseline separation from the void peak (at 8 min) (Supporting Information Fig. S2). For both polymer types, pCBMA (Fig. 3a) and pOEGMA (Fig. 3b), a shift to longer elution times and higher molar masses was observed depending on the polymer chain length. Furthermore, the intensity of the light scattering signal increased with increase in polymer molar mass due to its sensitivity to the molecule's size. Molar masses of pCBMA and pOEGMA were higher than the molar masses obtained from GPC (Supporting Information Table S11). pCBMA molar masses increased from 49.5 to 134 Da, while pOEGMA molar masses were 71.8 and 189 kDa for a DP of 50 and 200, respectively. GPC molar masses can be underestimated for several reasons. First, it can be a result of comparing the pCBMA and pOEGMA samples to Pullulan, which has different chemical structures and solution properties. In addition, it is impossible to verify the adsorptive interactions of the sample with separation columns using only a concentration sensitive detector. These interactions can result in a delay in elution time or partial adsorption of the sample inside the SEC column. The peak shape and the dispersities obtained from AF4-MALLS indicated a narrow distribution (Đ 1.10–1.25) of the samples and absence of any aggregation in the sample (Fig. 3). Hydrodynamic radii of the samples (Rh) determined using DLS also showed an increase from 6.8 nm (pCBMA short) to 9.4 nm (pCBMA long). However, the result for the short pOEGMA had a higher error because of a high data scattering during the measurement. Using the combination of AF4 separation and absolute molar mass determination by MALLS helped us gain a deeper insight into the properties of the synthesized polymers that cannot be determined using GPC alone. We note that AF4 molar mass detection is a complex endeavor, but finally delivers a tool that accurately provides the intrinsic molar mass of bio-ATRP products without the need for standards. Next, we were interested if using morpholine-based ligands could prepare protein–polymer conjugates with narrow dispersities as well. Even after 15 years since the development of grafting polymers from protein surfaces,2, 3 room for improvement in the synthesis remains. We synthesized four avidin–polymer conjugates using CBMA and OEGMA monomers with target DPs of 50 and 200. Avidin is a tetrameric protein with 10 primary amine groups per monomer. Avidin was first modified using the N-2-bromo-2-methylpropanoyl-β-alanine N′-oxysuccinimide bromide ATRP initiator. MALDI-ToF-MS showed that an average of eight initiators per avidin monomer were covalently attached (Supporting Information Fig. S3). After modification with ATRP initiators, two types of monomers with two different target lengths were grown directly from the surface of avidin using the MMA ligand. Synthesized conjugates were characterized using bicinchoninic acid (BCA) protein assay to determine the protein content in the conjugates and estimate the total molar mass of the protein–polymer conjugates (Table 1). 10 From the BCA assay, it was determined that the total conjugates molar masses increased with increasing chain lengths of attached polymers (Supporting Information). Then, DLS was used to determine the hydrodynamic diameters (Dh) of avidin-pCBMA and avidin-pOEGMA conjugates. The values of Dh of the conjugates increased from 18.1 nm for avidin-pCBMA50 to 29.7 nm for avidin-pCBMA200 (Table 1 and Supporting Information Fig. S4). The sizes of the avidin-pOEGMA conjugate increased in a similar manner from 19.8 to 30.9 nm with an increased chain length of pOEGMA (Table 1). After characterizing the sizes and estimating the total conjugates molar masses, we next cleaved the grafted polymers from avidin surface using 6 N HCl and separated the polymers using dialysis. Polymer molar masses and dispersities were then characterized using GPC. From GPC results, we also observed that the molar masses of the cleaved polymers increased from 9.4 to 39.4 kDa for pCBMA and from 9.3 to 51.8 kDa for pOEGMA polymers with the target DPs of 50 and 200, respectively. The dispersities of these polymers were at the lower end of ATRP approaches compared with polymers grown from avidin surfaces using other ligands (PDI = 1.7 by using bpy 3 and 1.6–1.9 by HMTETA 7) (Table 1). 3, 7 A colorimetric assay based on the binding of 2-(4-hydroxyphenylazo)benzoic acid (HABA) was used to determine the binding activity of avidin–polymer conjugates.27 Avidin–polymer conjugates retained 50–70% of activity (Table 1). Intrinsic tryptophan fluorescence studies of avidin in the presence of Et6TREN, MMA, DMA, and TMA showed that these ligands did not affect the tertiary structure of the protein (Supporting Information Fig. S5). As described above, AF4-MALLS enables the determination of absolute molar masses, dispersities, sizes, and conformations of the conjugates without many of the assumptions made during GPC-based analysis and we are now characterizing a large family of protein polymer conjugates with AF4. Herein, we have presented the development of three novel ligands for controlled aqueous ATRP based on ligand structure–function relationships. ATRP rates are faster in aqueous media than organic solvents; however using morpholine-based ligands presented in this work, one can slow down the polymerization rates in water. Developments of these ligands open new avenues to the synthesis of well-defined next-generation protein–polymer conjugates with even more controlled dispersities. We are currently investigating morpholine-based ligands effects on kinetics of bio-ATRP under various conditions. To obtain a deeper understanding of the synthesized polymer properties with new ligands, we have also characterized the polymers using the AF4-MALLS system. AF4-MALLS allowed us to gather information on polymer hydrodynamic sizes, their absolute molar masses, and dispersities. While advancing the synthetic techniques for preperation of well-defined protein–polymer conjugates, we also need to be able to reliably characterize these conjugates. Currently, we are working on characterizing protein–polymer conjugates with AF4-MALLS. Combining the DLS with MALLS data, we will be able to determine the conformation of the surface attached polymers. The ability to properly characterize protein–polymer conjugates will improve the understanding of their in vivo performances. Four ATRP ligands were obtained by alkylation of amine with alkyl halide (N,N,N′,N′-tetraethyldiethylenetriamine and 2-bromo-N,N-diethylethylamine for Et6TREN; 4-(2-aminoethyl)morpholine and 2-bromo-N,N-diethylethylamine for MMA; N,N-diethylethylenediamine and 4-(2-chloroethyl)morpholine for DMA; and 4-(2-aminoethyl)morpholine and 4-(2-chloroethyl)morpholine for TMA, respectively) in the presence of DBU. Synthetic details are described in the Supporting Information. Kinetics of ATRP using the various morpholine-based ligands with CBMA and OEGMA were monitored by taking aliquots of the polymerization solution at given time points. The polymerization condition was fixed as [M] 0:[I]0:[Cu(II)Cl2]0:[NaAsc]0:[ligand]0 = 100:1:10:1:12. Aliquots were immediately exposed to the air, and the obtained polymer was isolated by dialysis. The number-average molar masses (Mn) and dispersities (Mw/Mn) were measured by GPC. AF4 measurements were performed to determine the intrinsic molar masses and hydrodynamic radii of polymers with an Eclipse DualTec system (Wyatt Technology Europe, Germany). Avidin-polymer conjugates were synthesized by surface-initiated ATRP of CBMA and OEGMA with target DPs of 50 and 200 using MMA as a ligand, after preparing Avidin-ATRP macroinitiators using N-2-bromo-2-methylpropanoyl-β-alanine N′-oxysuccinimide bromide. The conjugates were isolated by dialysis with a 25-kDa molar mass cutoff dialysis then lyophilized. The grafted polymers from avidin's surface were cleaved by acidic hydrolysis, and the molar masses and dispersities were measured by GPC. Total molar mass of conjugate was estimated from a BCA protein assay and molar masses of cleaved polymer. This work was supported by DTRA grant: HDTRA1−18−1−0028, Carnegie Mellon FRBAA14-BR-TA7-G19−2−0124. D. L. would like to acknowledge funding from the H. William and Ruth Hamilton Prengle and Thomas and Adrienne Klopack Graduate Fellowships. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Atom-transfer radical-polymerization ; Morpholine ; Chemistry ; Polymer ; Molar mass ; Polymerization ; Polymer chemistry ; Aqueous solution ; Reversible addition−fragmentation chain-transfer polymerization ; Conjugate ; Radical polymerization ; Combinatorial chemistry ; Organic chemistry Advanced Polymer Synthesis and Characterization ; biodegradable polymer synthesis and properties ; Polymer Surface Interaction Studies