Cu Elimination from Cu-Coordinating Macromolecules - ACS Macro

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Cu Elimination from Cu-Coordinating Macromolecules Morten F. Ebbesen,* Dana Itskalov, Mischa Baier, and Laura Hartmann Heinrich-Heine-Universität Düsseldorf, Institut für Organische Chemie und Makromolekulare Chemie, Universitätsstr. 1, 40225 Düsseldorf, Germany S Supporting Information *

ABSTRACT: We demonstrate a simple, fast, and efficient process for the elimination of Cu impurities from water-soluble Cu-coordinating macromolecules that are difficult to purify via standard polymer purification techniques. The process is based on the complexation and precipitation of Cu by sodium diethyldithiocarbamate and was investigated for two different compound classes known to coordinate to Cu in aqueous solution. More than 99.9% of the Cu impurity was eliminated, with a remaining level below the detection limit (0.0005 wt %). Further analysis by 1H NMR, MALDI, ATR-IR, and SEC showed no degradation or side reactions of the polymers induced by the treatment. This process thus compliments the growing toolbox of Cucatalyzed conjugation techniques as a mild, effective, and scalable tool for the removal of Cu from water-soluble and Cu-coordinating polymers.

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cyclooctynes that are not easily adapted for all applications.8 Development of proper techniques for Cu-removal from Cubinding macromolecules thus remains of great importance for broadening the utility of the CuAAC reaction but potentially also for other Cu-catalyzed reactions such as atom transfer radical polymerization (ATRP). Methods for efficient Cu removal from organic reaction products exist with one prominent approach being based on aqueous electrolysis of a Cu-coordinating polymer that in 24 h removes Cu impurities down to a concentration of 0.042 wt %.11 Another technique utilizes adsorption of Cu ions onto the surface of iron oxide nanoparticles under oxygen free conditions that reduces the Cu level in noncoordinating and low molecular weight compounds down to 0.00046 wt % after two consecutive treatments.14 Being elegant approaches for Cu removal, other methods not relying on specialized equipment and materials as well as not requiring prolonged purification times should further broaden the applicability of these methods. Here, we demonstrate a fast and user-friendly, yet, highly efficient and scalable method for the elimination of Cu from water-soluble polymers based on the complexation and precipitation of Cu by sodium diethyldithiocarbamate (DDC) in water that enables removal of more than 99.9% of Cu from Cu-binding polymers to a level below 0.0005 wt %. The purification method arose from the need for proper Cu removal from polytriazoles synthesized via CuAAC-mediated addition polymerization of oligo(amidoamine) macromonomers (Scheme 1).15 Catalyzed by in situ-generated Cu(I) in a DMF/water mixture, bis-alkyne-terminated macromonomers of

he discovery of selective and versatile synthetic conjugation methods, the so-called click reactions, has been essential for the design and synthesis of increasingly complex macromolecules within a broad scientific area.1 One of the central tools in this development is the Cu-catalyzed azide/ alkyne cycloaddition (CuAAC) that has attracted immense attention due to its high efficiency and tolerance to functional groups and reaction solvents. It has thus become the method of choice for many difficult macromolecule conjugation or functionalization reactions.2−6 One limitation of CuAAC, however, is the Cu-based catalyst that, if not adequately removed from the macromolecule, poses a risk, for example, through Cu-induced cellular oxidative damage in biomedical applications.7,8 Further issues include remaining color, the ability to induce macromolecular physicochemical changes by Cu complexation, and side reactions such as Glaser coupling induced by Cu(II)-species generated for CuAAC reactions exposed to (trace) amounts of oxygen, for example, during aqueous dialysis.9 Remaining Cu, therefore, has to be efficiently eliminated from the product after catalysis. For many cases this can be accomplished via standard polymer purification techniques such as dialysis, extraction, or filtration over gels or aluminum oxide, however, for polar macromolecules containing N-donor motifs such as in poly(amidoamines), chitosan or for 2-ureido-4Hpyrimidone units, Cu coordination, and complexation significantly hampers effective purification.10,11 Moreover, high amounts (∼100 equiv) of Cu might be necessary for successful catalysis in certain cases, such as for very dilute reactions or to saturate Cu-binding sites and release freely available catalyst when Cu-coordinating macromolecules are involved.12,13 To circumvent these issues, Cu-free azide/alkyne cycloadditions have emerged as alternatives to the Cu-catalyzed variant but typically involve laborious synthesis of bulky and less stable © XXXX American Chemical Society

Received: February 19, 2017 Accepted: March 21, 2017

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DOI: 10.1021/acsmacrolett.7b00124 ACS Macro Lett. 2017, 6, 399−403

Letter

ACS Macro Letters

to the reaction mixture or later during the workup. After ∼10 min stirring, the DDC-Cu precipitate is filtered off through a maximally 0.2 μm pore-size filter. These two steps comprise the actual Cu elimination. Removal of any excess DDC is, however, still necessary, but easily carried out as part of standard purification protocols for removal of other low molecular weight impurities such as remaining DMF and sodium ascorbate used in this work. Due to the amount of water used for the initial Cu elimination, ultrafiltration is here a convenient technique and can be carried out in a few hours. For polymers generated by CuAAC-mediated conjugation or polymerization reactions, DDC-mediated Cu removal is, therefore, a fast, mild, and efficient way for eliminating Cu from the polymer product. Moreover, the retained DDC-Cu complex can be dissolved and extracted to recover the DDC and Cu.21,22 Visually comparing polytriazole samples purified via DDC precipitation (2 #1−3) to those purified by dialysis via ultrafiltration only (2 #4) illustrates the effect of DDC with the green color remaining in 2 #4 after thorough dialysis but being absent from 2 #1−3 (Figure 1). This is also reflected by the remaining Cu levels measured by AAS (Table 1), showing

Scheme 1. CuAAC-Mediated Polymerization of the Macromonomer (1) and Linker 1,11-Diazido-PEG3 (L)

1.5 kDa were copolymerized with 1.11-diazido-PEG3 to obtain polytriazoles with molecular weights, Mn, up to ∼9 kDa (Mw/ Mn = 1.5). Subsequent purification was, however, difficult as standard purification techniques such as dialysis via ultrafiltration, gel-filtration or preparative HPLC, proved insufficient for catalyst removal. A light green color was always present in the polymer product (Figure 1), and furthermore, slight

Table 1. DDC-Mediated Cu-Removal from the Polytriazole (2) and the mPEG-Sialic Acid Conjugate (4)a entry 2 2 2 2 2 4 4 4 4 4

Figure 1. Appearance of polymer products purified using DDC and dialysis via ultrafiltration (2 #1−3) and dialysis via ultrafiltration only (2 #4); see Table 1.

DDC/Cu [eq]

dialysis

#1 #2 #3 #4

2 5 10

√ √ √ √

#1 #2 #3 #4

2 5 10

√ √ √ √

Cubremaining [wt %] 3.83 99.9 42.5

a The Cu catalyst was removed from 2 and 4 by 10 min stirring with an aqueous solution of the specified amount of DDC, followed by filtration and dialysis via ultrafiltration. bRemaining Cu in the purified/ nonpurified polymers was measured by AAS from 4 mL ultrapure water solutions with 8 mg sample each. For nonpurified samples 2 and 4, Cu wt % values were calculated from the amount added. c% Cu removed was calculated based on the Cu wt % for the nonpurified polymer (2 and 4). dBelow AAS detection limit; the lowest detectable Cu-standard concentration was 0.01 ppm corresponding to a Cu detection limit of 0.0005 wt % (dry) and 99.9% Cu removed for a 8 mg polymer sample dissolved in 4 mL of ultrapure water.

variations in SEC elution behavior were observed for samples subjected to short and extended dialysis sessions. As the polytriazoles are both highly polar and also contain a significant number of N-donor motifs (Scheme 1), the formation of polymer−Cu complexes was possible, and varying amounts of Cu still being present in the polymer was suspected to be the origin for the observed effects.10,11 DDC in organic solution, for example, in DMF is a routine approach for washing out residual Cu after CuAAC coupling on solid support.16 In aqueous solutions, however, DDC and Cu form a complex, which efficiently precipitates out of solution. This behavior of DDC with Cu and other heavy metals such as Pd, Ni, Zn, Hg, Cd, and Pb17−19 in their free form is known and has, for instance, been used for preconcentration of Cu for improved quantification and for purifying Cu-polluted liquid waste streams, for example, from the paint industry.18,20,21 In this work, we apply the DDC-mediated Cu precipitation to a challenging polymer purification and demonstrate that the complexation effect is sufficiently strong for completely sequestering Cu ions from two classes of otherwise Cu-binding macromolecules in water, importantly, without generating any side reactions or polymer degradation events. This simple procedure starts with Cu precipitation induced by the addition of an aqueous solution of DDC either directly

incomplete Cu removal (30.9%) for 2 #4 and essentially complete Cu removal to below the AAS sensitivity limit (0.0005 wt %) already for 2 equiv DDC. This demonstrates that the DDC-Cu complexation and precipitation is sufficiently strong for quantitatively sequestering Cu from the strongly Cucoordinating polymers. The presence of residual Cu was suspected to influence the physicochemical properties of the polymers by inducing a more restricted solution conformation and smaller hydrodynamic radius governed by coordinate bonding between the polymer and the Cu ions. Following this hypothesis, the effect should be reversed upon removing the bound Cu, which is supported by the SEC elugram (Figure 2) showing an increase in hydrodynamic radius for purified polymers 2 #1−3 compared 400

DOI: 10.1021/acsmacrolett.7b00124 ACS Macro Lett. 2017, 6, 399−403

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ACS Macro Letters

Figure 2. SEC traces of 2 with DDC-mediated Cu removal (2, 5, and 10 equiv DDC, 2 #1−3) or dialysis via ultrafiltration only (2 #4). Changes in elution profile upon removal of Cu from the polymer indicates that remaining Cu in the polymer influences the physicochemical properties such as hydrodynamic size and/or charge. dRI solvent peak is indicated (*). See Figure S1 for the reverse effect of readding Cu to the polymer.

Figure 3. 1H NMR (600 MHz, D2O) spectrum of the polymer product (2) purified by DDC-mediated Cu removal (2 #3) and by dialysis via ultrafiltration only (2 #4). The Cu impurity in 2 #4 leads to broadening of the resonance signals in particular for the triazole proton (*, δ 7.9−8.2 ppm).

to 2 #4. In addition, no effect is shown by increasing the DDC level above 2 equiv, indicating that the DDC treatment is not further affecting the polymer besides removing the bound Cu. Further evidence for the reversibility of this effect was obtained by readdition of Cu to the purified polymer 2 #3, which recreated the restricted conformation resulting in a lower hydrodynamic radius (Figure S1). Further investigations of the process were carried out to exclude any DDC-induced degradation events or side reactions. Polymers purified with a high amount of DDC (10 equiv, 2 #3) and without DDC (dialysis via ultrafiltration only, 2 #4) were compared. MALDI-MS spectra of 2 #3 and 2 #4 show the same pattern of peaks, indicating that the DDC treatment does not change the polymer (Figure S2). IR spectra also appear similar, however, with slightly affected amide absorptions at 3500 cm−1 for 2 #4, likely due to interactions with complexed Cu (Figure S3). Generally, the 1H NMR spectra (Figure 3) of 2 #3 and 2 #4 are identical, indicating no DDC-induced degradations or side reactions. A significant peak broadening is, however, observed for 2 #4, in particular, for the triazole proton from δ 7.9 to 8.2 ppm, which is ascribed to the paramagnetic environment provided by the complexed Cu(II) ions and is additional evidence for the Cu complexation of the polymers.23 To widen the scope, the Cu-removal process was additionally investigated for a different Cu-coordinating system composed of α-methoxy-ω-azido-polyethylene glycol (mPEG-N3) conjugated to propargylated sialic acid (3; Schemes 2 and Scheme S1). Glyco-PEG macromolecules (4) are used, for example, for studies of carbohydrate-biomolecular interactions and their synthesis typically require high amounts of Cu catalyst (>1 equiv).24−28 DDC-mediated Cu removal proved effective for this compound class as well with essentially complete (>99.9%) Cu removal to levels below the AAS sensitivity limit (0.0005 wt %) (Table 1). 4 was also investigated for any evidence of sidereactions from the DDC treatment. ATR-IR spectra of compounds 4 #3−4 (Figure S4) are essentially alike with minor differences due to N-donation in the Cu-polymer complex for 4 #4. 1H NMR spectra of 4 #3−4 (Figure S5) also exclude any side reactions from the cleaning procedure as

Scheme 2. CuAAC-Mediated Conjugation of Propargyl Sialic Acid (3) to mPEG-N3

well as indicate similar paramagnetic effects as discussed for 2 #4. SEC and MALDI-MS illustrate the increase in molecular weight and hydrodynamic radius after conjugation of sialic acid (3) showing only the expected peaks and masses with no indications of side reactions after treatment with DDC (4 #3− 4, Figures S6 and S7). SEC additionally displays minor tailing toward lower hydrodynamic radii (Figure S6, 4 #4), indicating possible Cu-complex formation as also discussed for the polytriazole compound 2 #4. Overall, we demonstrate here a mild, fast, and highly efficient process for the elimination of Cu ions from strongly Cucoordinating polymers that cannot be adequately purified by standardized means such as dialysis, gel-filtration, or preparative HPLC. Via the addition of DDC in water, the Cu impurities are efficiently sequestered from the polymers, precipitated, and simply filtered off, while any excess DDC is removed via standard dialysis via ultrafiltration together with other low molecular weight impurities. DDC is known to induce precipitation of a range of heavy metals that provides the opportunity for possibly extending the Cu removal method to other metal catalysts. The process is demonstrated here for two different compound classes, proving a simple and efficient route for the removal of more than 99.9% of Cu from Cu-binding polymers to a level below 0.0005 wt %. No additional and undesired effects were induced by the treatment, as determined by 1H NMR, MALDI, ATR-IR, and SEC. The process thus constitutes an important tool that compliments aqueous Cucatalyzed chemistries as a mild, efficient, and scalable technique for Cu removal from Cu-binding polymers. 401

DOI: 10.1021/acsmacrolett.7b00124 ACS Macro Lett. 2017, 6, 399−403

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EXPERIMENTAL SECTION

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ASSOCIATED CONTENT

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The polytriazole (2) was prepared from a bis-alkyne oligo(amidoamine) (1) and a 1,11-bis-azido-PEG3-linker (L) via CuAAC-mediated addition polymerization using an equimolar amount of CuSO4 together with 10 equiv sodium ascorbate in a DMF/water 9/1 mixture (see Supporting Information and Scheme 1). Compound 1 was synthesized as described previously.15 After Cu removal and freeze-drying, the green (Cu-containing) or off-white solids (Figure 1) were obtained in yields between 85−95 wt % and analyzed by 1H NMR, MALDI, and ATR-IR spectroscopy and SEC. The remaining Cu levels were measured by AAS. The PEG-sialic acid conjugate (4) was prepared via CuAAC conjugation of mPEG-N3 and 3 using CuSO4 (2 equiv) and sodium ascorbate (20 equiv) in a tBuOH/water 1/1 mixture (see Supporting Information and Scheme 2). The synthesis and characterization of 3 is described in the Supporting Information. After Cu removal and freezedrying, the resulting polymer conjugates were obtained as green or white solids in yields between 93 and 98 wt % and analyzed as described for 2. The Cu catalysts were removed via DDC-mediated Cu precipitation and filtration. Different DDC to Cu molar ratios were used to evaluate the efficiency of the method (Table 1) and whether excess amounts of DDC would lead to any undesired effects. DDC coordinates to Cu in a 2:1 stoichiometry17 and was therefore added as an aqueous solution (1.3 mL) in the range of 2, 5, and 10 equiv (2 #1−3 and 4 #1−3) directly to the reaction mixture (∼150 μL). Organic reaction solutions such as DMF or tBuOH need to be sufficiently diluted with water for the precipitation of the apolar DDC-Cu complex to take place and it is thus convenient to add the DDC in an aqueous volume ∼10-fold larger than the reaction volume. After 10 min stirring, the formed precipitate was carefully filtered off using 0.2 μm syringe filters, the filtrate extensively dialyzed via ultrafiltration against ultrapure water (2 kDa MWCO Vivaspin 2 spin columns, 7 repetitions of concentration, and refilling with water) and freeze-dried.

REFERENCES

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* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.7b00124. Materials, synthesis protocols, characterization methods, and supporting analytical data (PDF).

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Letter

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Morten F. Ebbesen: 0000-0001-5416-7429 Laura Hartmann: 0000-0003-0115-6405 Author Contributions

All authors have given approval to the final version of the manuscript. Funding

M.F.E. thanks the Danish Council for Independent Research for support through the grant DFF-4005-00023. M.B. and L.H. thank the DFG for funding through the FOR2327. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Assoc. Prof. Nicole Snyder (Davidson College, North Carolina) for fruitful discussions. 402

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DOI: 10.1021/acsmacrolett.7b00124 ACS Macro Lett. 2017, 6, 399−403