- Email: [email protected]
Copper wire as a clean and efficient catalyst for click chemistry in supercritical CO2
Catalysis Today xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Catalysis Today journal homepage: www.elsevier.com/locate/cattod
Copper wire as a clean and efficient catalyst for click chemistry in supercritical CO2 E. Gracia, M.T. García, A. De Lucas, J.F. Rodríguez, I. Gracia
⁎
Institute of Chemical and Environmental Technology (ITQUIMA), Department of Chemical Engineering, University of Castilla-La Mancha, Avda. Camilo José Cela 12, 13071 Ciudad Real, Spain
A R T I C LE I N FO
A B S T R A C T
Keywords: Click chemistry Supercritical CO2 Copper wire Polymer functionalization
Use of copper wire bits as simple catalyst in a click chemistry reaction using supercritical CO2 has been achieved. The use of this catalyst in the reaction between polylactic acid (PLA) and coumarin allows to remove the whole amount of catalyst in the final product with a simple purification step using a green solvent where no toxic solvent is used in order to synthetize the product.
1. Introduction Total elimination of harmful or allergenic catalysts residues in functionalized products destined for biomedical applications is a critical issue. Depending on the mechanism of reaction selected, a different type of catalyst is required [1]. In the case of polymers functionalized using click chemistry a copper compound is the most frequently catalyst used. This reaction consists on the linkage of an azido group to an alkyne group (AAC) catalyzed by copper (CuAAC), being Huisgen 1,3-dipolar cycloaddition the most employed in polymer chemistry. In the last years, it has emerged as one of the most promising reactions because it is considered as a very specific, efficient and versatile reaction which allows obtaining high product yields [2–4]. The selection of the right solvent in this reaction is another important factor not only because it allows the dissolution of reactants and products but enhances their reactivity. Another important issue for the solvent selection is the easiness of solvent separation from the Active Pharmaceutical Ingredients (API) [1]. For the Huisgen 1,3-dipolar cycloaddition the most common solvents are THF or DMF [5]. The presence of residual solvents even in traces is not an option when a biomedical use is the final application of the click product. For this reason, cleaner and more sustainable alternatives have to be developed in order to obtain products totally free of harmful residues [6,7]. Among the different green solvents described in bibliography, specific supercritical fluids, like carbon dioxide, are finding application in the production of pharmaceutical related products due to their ability to solve particle formation processes and formulation problems. The use of supercritical carbon dioxide (scCO2) has the potential to be an excellent solvent when it is used with polymers with release purpose ⁎
[8–10]. Its properties such as lack of reactivity, dry conditions, low viscosity and high diffusivity make it an excellent solution for environmental and patient friendly processes [11–14]. In this work it is presented as a clean technology which makes possible to avoid the use of organic solvents to carry out successfully polymers functionalization with the only use of scCO2 as the solvent and a catalyst which can be easily and completely removed from the API. In a previous work, click chemistry in scCO2 was described for first time for the functionalization of a biodegradable polymer eliminating the need of an organic solvent apart from CO2 in the reaction, obtaining yields higher than 95% without any catalyst purification step [15]. In the conventional click chemistry cooper is employed as homogeneous catalyst. Apart from the limitations of its complete elimination from the final clicked product, the use of copper as a catalyst presents some additional difficulties depending on its state of oxidation. The first limitation is fresh Cu+1 is required for each new click reaction. Second limitation is Cu+1 is oxidized in the presence of air to Cu+2. Finally the third limitation is the removal of Cu+1 and Cu+2 from the reactional medium, fact which is very tedious and difficult for air-sensitive systems [16]. Both problems are solved in this work by the use of copper wire bits as catalyst (Cu (0) wire) in scCO2. The selection of copper wire in this reaction allows the elimination of the whole amount of catalyst charged in the reaction with a sieving step or simply with the use of tweezers. This catalyst adds also the possibility of reusing the same catalyst in several reactions, what could mean a decrease of the operational cost of this process [17,18]. The polymer chosen in this work is a biodegradable polymer approved by the US Food and Drug Administration, Polylactic acid (PLA)
Corresponding author. E-mail address: [email protected] (I. Gracia).
https://doi.org/10.1016/j.cattod.2018.12.021 Received 27 August 2018; Received in revised form 6 December 2018; Accepted 11 December 2018 0920-5861/ © 2018 Published by Elsevier B.V.
Please cite this article as: Gracia, E., Catalysis Today, https://doi.org/10.1016/j.cattod.2018.12.021
Catalysis Today xxx (xxxx) xxx–xxx
E. Gracia et al.
Fig. 1. Scheme of click reaction using copper wire as a catalyst.
any solvent.
[19]. Its properties make this polymer an excellent candidate to be used in biomedical field as a manufacture tissue engineering scaffolds, delivery system materials, and it was also used widely in human medicine [20–23]. This polymer was chosen in this work in order to compare the yield obtained with Copper (II) acetate monohydrate and copper wire as catalysts. On the same way, the organic compound chosen in this work is the coumarin. This substance is a plant-derived natural product which consists of an aromatic ring fused to a condensed lactone ring [24]. Chemistry of coumarins and their derivates have arisen considerable interest due to their wide range of biological and pharmacological properties [25,26]. It is a well-known their anti-inflammatory activity, where it is able to remove protein and oedema fluid from injured tissues, anticoagulant activity due to coumarin is vitamin K antagonist producing anticoagulant effect or antiviral activity because this compound is considered as anti-HIV agent [27]. In this work, the functionalization of polylactic acid (PLA) acetylene via click chemistry in supercritical carbon dioxide using copper wire bits as catalyst has been studied for first time.
2.4. Purification of click product Once click product was obtained from click chemistry reaction it was separated from copper wire through a sieve step where the whole catalyst is removed from the click product. 2.5. Characterization 2.5.1. Nuclear magnetic resonance (NMR) 1 H NMR was measured with Varian Gemini FT-400 spectrometer using CDCl3 as solvent. 2.5.2. Maldi-Toff Matrix-assisted laser desorption/ionization time-of-flight (MALDITOF) mass spectrometry (MS) was carried out using a Bruker Autoflex II TOF/TOF spectrometer (Bremen, Germany) using CDCl3 as solvent and dithranol (1,8,9-trihydroxyanthracene) as matrix material.
2. Experimental 2.5.3. Atomic absorption spectrophometry Cu metal loading was determined by atomic absorption spectrophotometry, using a SPECTRA 220FS analyzer. The sample (ca. 0.5 g) was treated in 2 mL HCl, 3 mL HF and 2 mL H2O2 followed by microwave digestion (T = 250 ◦C).
2.1. Materials Sodium azide (> 99,5%, Sigma Aldrich), 4-Bromomethyl-7methoxy-coumarin (97%, Sigma Aldrich), Polylactic Acid acetylene (98.3%, Specific polymers), Copper wire and carbon dioxide (Carburos metálicos, S.A., Spain) with a purity of 99.5%. All other reagents and solvents used in the study were of analytical grade and used as delivered.
3. Results In order to check if copper wire is a suitable catalyst for the click functionalization with coumarin of polylactide several reactions were carried out using carbon dioxide in supercritical conditions. This reaction can be observed in the scheme shown in Fig. 1. The main novelty in this research is the substitution of the catalyst previously used in supercritical CO2, copper (II) acetate monohydrate, by copper wire bits. This fact supposes a great advance from purification point of view because a chromatographic column will not be necessarily set up to perform the purification. In this case a simple sieve step or the use of tweezers enough for click product separation. Initially, a first click chemistry reaction was carried out in order to check if copper wire works as catalyst using a low loading of catalyst when it is used with a biodegradable polymer. The diameter of the used metallic copper wire was 0.18 mm and the operational conditions were chosen according to optimized conditions studied in a previous work [15]. 1 H NMR spectrum of click product using copper wire as a catalyst is shown in Fig. 2. In the spectrum can be identified the characteristic signals corresponding to the proton of the 1,2,3,-trizole ring at 7.70 ppm and the correspondent to CH2 neighbouring the 1,2,3,-trizole group at 5.30 ppm. These observations confirms that click ligation of coumarin to PLA has been achieved using only cooper wires as catalysts in absence of solvents other than scCO2. To complete the characterization of the product and get stronger confirmation about the formation of the target product and no side
2.2. Synthesis of 4-azidomethyl-7-methoxycoumarin The synthesis of this compound was carried out following a bibliography route [28]. A mixture of NaN3 (1.2 g) and 4-bromomethyl-7methoxycoumarin (1 g) in acetone/acetonitrile (1:1, 120 ml) solution was added to a 250 ml flask. The mixture was stirred at 50 °C for 48 h. Then, solvents were removed under vacuum. The organic extracts were washed with water to precipitate the 4-bromomethyl-7-methoxycoumarin which did not react. Then, product was filtered and washed with heptane and dried under vacuum. 2.3. Synthesis of click product in supercritical CO2 Click product in scCO2 was synthetized using copper wire as catalyst. The procedure of synthesis is the following: An equimolar quantity of PLA acetylene and 4-azidomethyl-7-methoxycoumarin were added into a stirring tank reactor using a catalyst loading among 40–72% mol, being 40% mol the reaction where a lowest yield was obtained in this study, and 71.32% the yield in the optimized reaction. Once sample is introduced into the reactor, it is heated and loaded with CO2 until temperature (50 °C) and pressure (130 bar) conditions are reached. When reaction time is completed (24 h), the reactor is depressurized to remove CO2 from the reactor in order to obtain click product without 2
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Fig. 2. 1H NMR spectra of click product synthetized in supercritical CO2 using copper wire as catalyst (40% mol).
Fig. 3. MALDI-TOF mass spectrum of coumarin functionalized PLA in scCO2, cat: 40% mol, t: 24 h.
byproducts Maldi-Tof, mass spectrum of the click product was carried out. In Fig. 3 it can be confirmed that the peaks of the coumarin-PLA appear spaced at the proper MW distance, corresponding to the MW of the coumarin, Also the yield of the coumarin click ligation could be calculated. Yield was determined according to a procedure explained in literature In this reaction the yield reached was only of 26.36%. This yield is somewhat low compared with the yield higher than 95% that was obtained in a previous work where copper acetate monohydrate was employed as catalyst in scCO2 [15]. Copper concentration in the final click product was determined by ICP analysis., The presence of copper in the coumarin-PLA was undetectable. This result confirms that copper wire can be an excellent and non-harmful catalyst for the preparation of functionalized biopolymers for drug delivery by a click procedure. The amount of catalyst employed was modified in order to check if this increase improves the yield of the reaction. In Table 1 are shown the yields of the click reaction employing growing amounts of catalyst using copper wire chips of the same diameter.
Table 1 Study of catalyst loading using copper wire. Entry
Diameter (mm)
Catalyst (%)
Final Cu (ppm)
Yield (%)
1 2 3
0.18 0.18 0.18
61.82 67.55 71.32
0.00 0.00 0.00
71.28 85.64 93.02
As can be observed in Table 1, an increase of catalyst loading produces the increase of the yield achieving a 93.02% in the case of using the highest weight of copper wire. The amounts of residual copper in the final product were also measured by ICP and as can be observed in Table 1, copper is totally absent in the final product, independently of amount of catalyst used in the reaction. This fact supposes an important improvement with respect to the previous click reaction where copper catalyst in powder form was used [15]. The high purity of the product obtained in the reaction3 can be appreciated in Fig. 4, where Maldi-Tof spectra shows that the peaks correspondent to click product have a much higher intensity with 3
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E. Gracia et al.
Fig. 4. MALDI-TOF mass spectrum of coumarin functionalized PLA in scCO2, cat: 71.32% mol, t: 24 h.
respect to the peaks correspondent to PLA acetylene. As the specific surface of copper wire is substantially smaller than specific surface of the catalyst employed as powder, the yield obtained for the same reaction time is lower. This fact could be an inconvenient since a higher amount of catalyst would be required to obtain the same yield. However, as copper wire is easily and totally removed in the purification step, independently of catalyst quantity, copper wire can be considered as an excellent alternative catalyst for this reaction. An example is given when an increase of molar percentage from 26% to 71% is chosen. The yield value increases up to a value of 93% with 0.00 ppm of copper in the final product.
[6]
[7] [8] [9]
[10] [11]
4. Conclusions [12]
The functionalization via click chemistry of polylactic acid (PLA) with coumarin in supercritical CO2 has been achieved using copper wire as catalyst, obtaining similar yield to the reaction carried out with copper catalyst in powder form [15]. The highest yield was achieved at 130 bar and 50 °C obtaining a clickation efficiency of 93% in 24 h of reaction. After an easy purification step, the final amount of copper in the product was analysed obtaining a result of 0.00 ppm. This fact stablishes copper wire as an excellent candidate for further reactions in click chemistry.
[13] [14]
[15] [16] [17]
Acknowledgments
[18]
We gratefully acknowledge funding from the Ministerio de Economía y Competitividad through the projects Ref. CTQ2013-46380P and CTQ2016-79811-P. The authors also acknowledge the support of the Ministerio de Economía y Competitividad for the fellowship of Mr. Gracia Cortes Ref. BES-2014-069313. The authors state that they have not conflict of interest with any public or private body.
[19] [20] [21] [22] [23]
References [24] [1] M.D. Argentine, P.K. Owens, B.A. Olsen, Strategies for the investigation and control of process-related impurities in drug substances, Adv. Drug Delivery Rev. 59 (1) (2007) 12–28. [2] C.D. Hein, X.-M. Liu, D. Wang, Click chemistry, a powerful tool for pharmaceutical sciences, Pharm. Res. 25 (10) (2008) 2216–2230. [3] G. Franc, A.K. Kakkar, "Click" methodologies: efficient, simple and greener routes to design dendrimers, Chem. Soc. Rev. 39 (5) (2010) 1536–1544. [4] J.F. Lutz, Z. Zarafshani, Efficient construction of therapeutics, bioconjugates, biomaterials and bioactive surfaces using azide-alkyne "click" chemistry, Adv. Drug Deliv. Rev. 60 (9) (2008) 958–970. [5] V., R.V, et al., A stepwise Huisgen cycloaddition process: copper(I)‐Catalyzed
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4
regioselective “Ligation” of azides and terminal alkynes, Angew. Chem. Int. Ed. 41 (14) (2002) 2596–2599. N. Noshiranzadeh, et al., Green click synthesis of β-hydroxy-1,2,3-triazoles in water in the presence of a Cu(ii)-azide catalyst: a new function for Cu(ii)-azide complexes, New J. Chem. 41 (7) (2017) 2658–2667. W. Zhang, et al., Cu(OAc)2·H2O—an efficient catalyst for Huisgen-click reaction in supercritical carbon dioxide, Tetrahedron Lett. 56 (19) (2015) 2472–2475. L.I. Cabezas, et al., Production of biodegradable porous scaffolds impregnated with indomethacin in supercritical CO2, J. Supercrit. Fluids 63 (2012) 155–160. L.I. Cabezas, et al., Novel model for the description of the controlled release of 5fluorouracil from PLGA and PLA foamed Scaffolds impregnated in supercritical CO2, Ind. Eng. Chem. Res. 53 (40) (2014) 15374–15382. L.I. Cabezas, et al., Production of biodegradable porous scaffolds impregnated with 5-fluorouracil in supercritical CO2, J. Supercrit. Fluids 80 (2013) 1–8. O.R. Davies, et al., Applications of supercritical CO2 in the fabrication of polymer systems for drug delivery and tissue engineering, Adv. Drug Deliv. Rev. 60 (3) (2008) 373–387. K. Grodowska, A. Parczewski, Organic solvents in the pharmaceutical industry, Acta Poloniae Pharm. Drug Res. 67 (1) (2010) 3–12. Q. Lang, C.M. Wai, Supercritical fluid extraction in herbal and natural product studies — a practical review, Talanta 53 (4) (2001) 771–782. Y. Medina-Gonzalez, S. Camy, J.-S. Condoret, ScCO2/Green solvents: biphasic promising systems for cleaner chemicals manufacturing, ACS Sustain. Chem. Eng. 2 (12) (2014) 2623–2636. E. Gracia, et al., Functionalization and optimization of PLA with coumarin via click chemistry in supercritical CO2, J. CO2 Util. 20 (2017) 20–26. A., B.C, et al., Modulating catalytic activity of polymer‐based cuAAC “click” reactions, J. Polym. Sci. Part A: Polym. Chem. 49 (21) (2011) 4539–4548. C.N. Urbani, et al., Convergent synthesis of second generation AB-type miktoarm dendrimers using “Click” chemistry catalyzed by copper wire, Macromolecules 41 (4) (2008) 1057–1060. Y. Jiang, et al., Metallic copper wire: a simple, clear and reusable catalyst for the CuAAC reaction in supercritical carbon dioxide, RSC Adv. 5 (90) (2015) 73340–73345. R.A. Jain, The manufacturing techniques of various drug loaded biodegradable poly (lactide-co-glycolide) (PLGA) devices, Biomaterials 21 (23) (2000) 2475–2490. K. Hamad, et al., Properties and medical applications of polylactic acid: a review, eXPRESS Polym. Lett. 9 (5) (2015) 435–455. S. Kumar, et al., Controlled drug release through regulated biodegradation of poly (lactic acid) using inorganic salts, Int. J. Biol. Macromol. 104 (2017) 487–497. M.A. Ghalia, Y. Dahman, Biodegradable poly(lactic acid)-based scaffolds: synthesis and biomedical applications, J. Polym. Res. 24 (5) (2017). D. Zhu, et al., Docetaxel (DTX)-loaded polydopamine-modified TPGS-PLA nanoparticles as a targeted drug delivery system for the treatment of liver cancer, Acta Biomater. 31 (2016) 144–154. P.K. Jain, H. Joshi, Coumarin: chemical and pharmacological profile, J. Appl. Pharm. Sci. 2 (6) (2012) 236–240. J. Yang, et al., Synthesis and biological evaluation of hydroxylated 3-phenylcoumarins as antioxidants and antiproliferative agents, Bioorg. Med. Chem. Lett. 21 (21) (2011) 6420–6425. Y. Zhang, et al., Synthesis and antioxidant activities of novel 4-Schiff base-7-benzyloxy-coumarin derivatives, Bioorg. Med. Chem. Lett. 21 (22) (2011) 6811–6815. K.N. Venugopala, V. Rashmi, B. Odhav, Review on natural coumarin lead compounds for their pharmacological activity, Biomed Res. Int. (2013). M.M. Velencoso, et al., Click-ligation of coumarin to polyether polyols for polyurethane foams, Polym. Int. 62 (5) (2013) 783–790.
Contents lists available at ScienceDirect
Catalysis Today journal homepage: www.elsevier.com/locate/cattod
Copper wire as a clean and efficient catalyst for click chemistry in supercritical CO2 E. Gracia, M.T. García, A. De Lucas, J.F. Rodríguez, I. Gracia
⁎
Institute of Chemical and Environmental Technology (ITQUIMA), Department of Chemical Engineering, University of Castilla-La Mancha, Avda. Camilo José Cela 12, 13071 Ciudad Real, Spain
A R T I C LE I N FO
A B S T R A C T
Keywords: Click chemistry Supercritical CO2 Copper wire Polymer functionalization
Use of copper wire bits as simple catalyst in a click chemistry reaction using supercritical CO2 has been achieved. The use of this catalyst in the reaction between polylactic acid (PLA) and coumarin allows to remove the whole amount of catalyst in the final product with a simple purification step using a green solvent where no toxic solvent is used in order to synthetize the product.
1. Introduction Total elimination of harmful or allergenic catalysts residues in functionalized products destined for biomedical applications is a critical issue. Depending on the mechanism of reaction selected, a different type of catalyst is required [1]. In the case of polymers functionalized using click chemistry a copper compound is the most frequently catalyst used. This reaction consists on the linkage of an azido group to an alkyne group (AAC) catalyzed by copper (CuAAC), being Huisgen 1,3-dipolar cycloaddition the most employed in polymer chemistry. In the last years, it has emerged as one of the most promising reactions because it is considered as a very specific, efficient and versatile reaction which allows obtaining high product yields [2–4]. The selection of the right solvent in this reaction is another important factor not only because it allows the dissolution of reactants and products but enhances their reactivity. Another important issue for the solvent selection is the easiness of solvent separation from the Active Pharmaceutical Ingredients (API) [1]. For the Huisgen 1,3-dipolar cycloaddition the most common solvents are THF or DMF [5]. The presence of residual solvents even in traces is not an option when a biomedical use is the final application of the click product. For this reason, cleaner and more sustainable alternatives have to be developed in order to obtain products totally free of harmful residues [6,7]. Among the different green solvents described in bibliography, specific supercritical fluids, like carbon dioxide, are finding application in the production of pharmaceutical related products due to their ability to solve particle formation processes and formulation problems. The use of supercritical carbon dioxide (scCO2) has the potential to be an excellent solvent when it is used with polymers with release purpose ⁎
[8–10]. Its properties such as lack of reactivity, dry conditions, low viscosity and high diffusivity make it an excellent solution for environmental and patient friendly processes [11–14]. In this work it is presented as a clean technology which makes possible to avoid the use of organic solvents to carry out successfully polymers functionalization with the only use of scCO2 as the solvent and a catalyst which can be easily and completely removed from the API. In a previous work, click chemistry in scCO2 was described for first time for the functionalization of a biodegradable polymer eliminating the need of an organic solvent apart from CO2 in the reaction, obtaining yields higher than 95% without any catalyst purification step [15]. In the conventional click chemistry cooper is employed as homogeneous catalyst. Apart from the limitations of its complete elimination from the final clicked product, the use of copper as a catalyst presents some additional difficulties depending on its state of oxidation. The first limitation is fresh Cu+1 is required for each new click reaction. Second limitation is Cu+1 is oxidized in the presence of air to Cu+2. Finally the third limitation is the removal of Cu+1 and Cu+2 from the reactional medium, fact which is very tedious and difficult for air-sensitive systems [16]. Both problems are solved in this work by the use of copper wire bits as catalyst (Cu (0) wire) in scCO2. The selection of copper wire in this reaction allows the elimination of the whole amount of catalyst charged in the reaction with a sieving step or simply with the use of tweezers. This catalyst adds also the possibility of reusing the same catalyst in several reactions, what could mean a decrease of the operational cost of this process [17,18]. The polymer chosen in this work is a biodegradable polymer approved by the US Food and Drug Administration, Polylactic acid (PLA)
Corresponding author. E-mail address: [email protected] (I. Gracia).
https://doi.org/10.1016/j.cattod.2018.12.021 Received 27 August 2018; Received in revised form 6 December 2018; Accepted 11 December 2018 0920-5861/ © 2018 Published by Elsevier B.V.
Please cite this article as: Gracia, E., Catalysis Today, https://doi.org/10.1016/j.cattod.2018.12.021
Catalysis Today xxx (xxxx) xxx–xxx
E. Gracia et al.
Fig. 1. Scheme of click reaction using copper wire as a catalyst.
any solvent.
[19]. Its properties make this polymer an excellent candidate to be used in biomedical field as a manufacture tissue engineering scaffolds, delivery system materials, and it was also used widely in human medicine [20–23]. This polymer was chosen in this work in order to compare the yield obtained with Copper (II) acetate monohydrate and copper wire as catalysts. On the same way, the organic compound chosen in this work is the coumarin. This substance is a plant-derived natural product which consists of an aromatic ring fused to a condensed lactone ring [24]. Chemistry of coumarins and their derivates have arisen considerable interest due to their wide range of biological and pharmacological properties [25,26]. It is a well-known their anti-inflammatory activity, where it is able to remove protein and oedema fluid from injured tissues, anticoagulant activity due to coumarin is vitamin K antagonist producing anticoagulant effect or antiviral activity because this compound is considered as anti-HIV agent [27]. In this work, the functionalization of polylactic acid (PLA) acetylene via click chemistry in supercritical carbon dioxide using copper wire bits as catalyst has been studied for first time.
2.4. Purification of click product Once click product was obtained from click chemistry reaction it was separated from copper wire through a sieve step where the whole catalyst is removed from the click product. 2.5. Characterization 2.5.1. Nuclear magnetic resonance (NMR) 1 H NMR was measured with Varian Gemini FT-400 spectrometer using CDCl3 as solvent. 2.5.2. Maldi-Toff Matrix-assisted laser desorption/ionization time-of-flight (MALDITOF) mass spectrometry (MS) was carried out using a Bruker Autoflex II TOF/TOF spectrometer (Bremen, Germany) using CDCl3 as solvent and dithranol (1,8,9-trihydroxyanthracene) as matrix material.
2. Experimental 2.5.3. Atomic absorption spectrophometry Cu metal loading was determined by atomic absorption spectrophotometry, using a SPECTRA 220FS analyzer. The sample (ca. 0.5 g) was treated in 2 mL HCl, 3 mL HF and 2 mL H2O2 followed by microwave digestion (T = 250 ◦C).
2.1. Materials Sodium azide (> 99,5%, Sigma Aldrich), 4-Bromomethyl-7methoxy-coumarin (97%, Sigma Aldrich), Polylactic Acid acetylene (98.3%, Specific polymers), Copper wire and carbon dioxide (Carburos metálicos, S.A., Spain) with a purity of 99.5%. All other reagents and solvents used in the study were of analytical grade and used as delivered.
3. Results In order to check if copper wire is a suitable catalyst for the click functionalization with coumarin of polylactide several reactions were carried out using carbon dioxide in supercritical conditions. This reaction can be observed in the scheme shown in Fig. 1. The main novelty in this research is the substitution of the catalyst previously used in supercritical CO2, copper (II) acetate monohydrate, by copper wire bits. This fact supposes a great advance from purification point of view because a chromatographic column will not be necessarily set up to perform the purification. In this case a simple sieve step or the use of tweezers enough for click product separation. Initially, a first click chemistry reaction was carried out in order to check if copper wire works as catalyst using a low loading of catalyst when it is used with a biodegradable polymer. The diameter of the used metallic copper wire was 0.18 mm and the operational conditions were chosen according to optimized conditions studied in a previous work [15]. 1 H NMR spectrum of click product using copper wire as a catalyst is shown in Fig. 2. In the spectrum can be identified the characteristic signals corresponding to the proton of the 1,2,3,-trizole ring at 7.70 ppm and the correspondent to CH2 neighbouring the 1,2,3,-trizole group at 5.30 ppm. These observations confirms that click ligation of coumarin to PLA has been achieved using only cooper wires as catalysts in absence of solvents other than scCO2. To complete the characterization of the product and get stronger confirmation about the formation of the target product and no side
2.2. Synthesis of 4-azidomethyl-7-methoxycoumarin The synthesis of this compound was carried out following a bibliography route [28]. A mixture of NaN3 (1.2 g) and 4-bromomethyl-7methoxycoumarin (1 g) in acetone/acetonitrile (1:1, 120 ml) solution was added to a 250 ml flask. The mixture was stirred at 50 °C for 48 h. Then, solvents were removed under vacuum. The organic extracts were washed with water to precipitate the 4-bromomethyl-7-methoxycoumarin which did not react. Then, product was filtered and washed with heptane and dried under vacuum. 2.3. Synthesis of click product in supercritical CO2 Click product in scCO2 was synthetized using copper wire as catalyst. The procedure of synthesis is the following: An equimolar quantity of PLA acetylene and 4-azidomethyl-7-methoxycoumarin were added into a stirring tank reactor using a catalyst loading among 40–72% mol, being 40% mol the reaction where a lowest yield was obtained in this study, and 71.32% the yield in the optimized reaction. Once sample is introduced into the reactor, it is heated and loaded with CO2 until temperature (50 °C) and pressure (130 bar) conditions are reached. When reaction time is completed (24 h), the reactor is depressurized to remove CO2 from the reactor in order to obtain click product without 2
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Fig. 2. 1H NMR spectra of click product synthetized in supercritical CO2 using copper wire as catalyst (40% mol).
Fig. 3. MALDI-TOF mass spectrum of coumarin functionalized PLA in scCO2, cat: 40% mol, t: 24 h.
byproducts Maldi-Tof, mass spectrum of the click product was carried out. In Fig. 3 it can be confirmed that the peaks of the coumarin-PLA appear spaced at the proper MW distance, corresponding to the MW of the coumarin, Also the yield of the coumarin click ligation could be calculated. Yield was determined according to a procedure explained in literature In this reaction the yield reached was only of 26.36%. This yield is somewhat low compared with the yield higher than 95% that was obtained in a previous work where copper acetate monohydrate was employed as catalyst in scCO2 [15]. Copper concentration in the final click product was determined by ICP analysis., The presence of copper in the coumarin-PLA was undetectable. This result confirms that copper wire can be an excellent and non-harmful catalyst for the preparation of functionalized biopolymers for drug delivery by a click procedure. The amount of catalyst employed was modified in order to check if this increase improves the yield of the reaction. In Table 1 are shown the yields of the click reaction employing growing amounts of catalyst using copper wire chips of the same diameter.
Table 1 Study of catalyst loading using copper wire. Entry
Diameter (mm)
Catalyst (%)
Final Cu (ppm)
Yield (%)
1 2 3
0.18 0.18 0.18
61.82 67.55 71.32
0.00 0.00 0.00
71.28 85.64 93.02
As can be observed in Table 1, an increase of catalyst loading produces the increase of the yield achieving a 93.02% in the case of using the highest weight of copper wire. The amounts of residual copper in the final product were also measured by ICP and as can be observed in Table 1, copper is totally absent in the final product, independently of amount of catalyst used in the reaction. This fact supposes an important improvement with respect to the previous click reaction where copper catalyst in powder form was used [15]. The high purity of the product obtained in the reaction3 can be appreciated in Fig. 4, where Maldi-Tof spectra shows that the peaks correspondent to click product have a much higher intensity with 3
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Fig. 4. MALDI-TOF mass spectrum of coumarin functionalized PLA in scCO2, cat: 71.32% mol, t: 24 h.
respect to the peaks correspondent to PLA acetylene. As the specific surface of copper wire is substantially smaller than specific surface of the catalyst employed as powder, the yield obtained for the same reaction time is lower. This fact could be an inconvenient since a higher amount of catalyst would be required to obtain the same yield. However, as copper wire is easily and totally removed in the purification step, independently of catalyst quantity, copper wire can be considered as an excellent alternative catalyst for this reaction. An example is given when an increase of molar percentage from 26% to 71% is chosen. The yield value increases up to a value of 93% with 0.00 ppm of copper in the final product.
[6]
[7] [8] [9]
[10] [11]
4. Conclusions [12]
The functionalization via click chemistry of polylactic acid (PLA) with coumarin in supercritical CO2 has been achieved using copper wire as catalyst, obtaining similar yield to the reaction carried out with copper catalyst in powder form [15]. The highest yield was achieved at 130 bar and 50 °C obtaining a clickation efficiency of 93% in 24 h of reaction. After an easy purification step, the final amount of copper in the product was analysed obtaining a result of 0.00 ppm. This fact stablishes copper wire as an excellent candidate for further reactions in click chemistry.
[13] [14]
[15] [16] [17]
Acknowledgments
[18]
We gratefully acknowledge funding from the Ministerio de Economía y Competitividad through the projects Ref. CTQ2013-46380P and CTQ2016-79811-P. The authors also acknowledge the support of the Ministerio de Economía y Competitividad for the fellowship of Mr. Gracia Cortes Ref. BES-2014-069313. The authors state that they have not conflict of interest with any public or private body.
[19] [20] [21] [22] [23]
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