Direct syntheses of cucurbit[7]uril-anchored polyacrylic acid microspheres and adsorption of basic dyes by the derivative

Abstract

A one-pot strategy was employed to synthesize a series of water-soluble cucurbit[7]uril-anchored polyacrylic acids (Q[7]-anchored PAAs) with high yields in the presence of an ammonium persulfate salt as both an initiator and an oxidant. The effects of acrylic acid, Q[7] feed mass and APS concentration on the formation of polymer nanocapsules have been investigated. The Q[7]-anchored PAAs can be further esterified with 2-naphthol to yield water-insoluble derivatives, which are characteristic of the stationary phase for absorption of basic dyes, especially neutral red.

---

Introduction

Although cucurbit[n]urils (Q[n]s), like calixarenes and cyclodextrins, are potentially useful as well-known host molecules, their practical applications and use in separation science have been limited, mainly due to the difficulty of introducing functional groups on their surfaces.¹ In 2003, Kim and co-workers made an important breakthrough in Q[n] chemistry by directly oxidizing Q[n]s to yield perhydroxycucurbit[n]urils ((OH)_2nQ[n]s) with K₂S₂O₈ in water.² Since then, there have been great achievements in Q[n]-based host–guest chemistry, particularly with regard to Q[n]-based polymers.^3–15 For example, (HO)₁₂Q[6] can be grafted to silica gel for use as stationary phase in hydrophilic-interaction chromatography,^3,4 (HO)₁₂Q[6] can also be used to synthesize polymer nanocapsules with a noncovalently tailorable surface that exhibits a highly stable structure and relatively narrow size distribution without the need for a pre-organized structure, emulsifier, or template.⁵ Such Q[6]-based nanoparticles or polymers could serve as new efficient vehicles for the delivery of hydrophobic drugs.⁶ In preserving the imparted fluorescence,⁸ encapsulation and release of a fluorescent dye are achieved by such techniques as swelling and deswelling cycles,⁹ analysis of membrane proteins,^11,13 and selective metal ion extraction.¹² Generally, to synthesize Q[n]-based polymers, Q[n]s must first be oxidized to ((OH)_2nQ[n]s), after which they can be functionalized with various groups that can be polymerized to form Q[n]-based polymers. This method is principally applicable to any Q[n], and the polymer shell allows facile tailoring of its surface properties in a noncovalent and modular manner via accessible molecular cavities exposed on the surface.¹⁶ However, the direct oxidation of Q[n]s appears to be efficient for smaller Q[n]s, such as Q[5] and Q[6] (yields of 42% and 45%, respectively). For the larger Q[n]s, such as Q[7], Q[8] (ca. 5%),² and partial alkyl-substituted Q[n]s,^17–27 the cause of the low yields in these reactions is still unclear.

In 2013, Su and co-workers demonstrated a one-pot, direct method for the preparation of Q[6]-anchored acrylamide polymers without functionalizing Q[6], which is applicable for monomers used in radical polymerization in aqueous solution.²⁸ This method does not require separate (OH)_2nQ[n]s, because the oxidation and polymerization reactions occur in the same reaction system. Our group further confirmed that this strategy is efficient for various Q[n]s, including Q[5], Q[6], Q[7] and Q[8] with a high yield (50–70%).^29,30 As a functional polymer, the direct synthesis of cucurbit[7]uril-anchored polyacrylic acid (Q[7]-PAA) using the one-pot strategy will provide a high-capacity utilization method for Q[7] in polymer chemistry. Here, we not only introduce water-soluble Q[7]-PAAs, which have a typical vesicular structure, but also water-insoluble esterified Q[7]-PAAs with 2-naphthol, which have stationary phase characteristics for the absorption of basic dyes, especially neutral red (Scheme 1).

Scheme 1 Synthesis of Q[7]-PAA-(2-naphthol) radical polymerization mechanism.

Results and discussion

Characterization of Q[7]-anchored PAAs

Using the one-pot strategy, Q[7] was selected as the anchor, and acrylic acid (AA) was polymerized in the presence of ammonium persulfate salt (APS) as an initiator and oxidant to produce Q[7]-anchored PAA. Five via free radical polymerization in aqueous solution in the presence of APS were selected as typical samples to illustrate the features of the Q[7]-anchored PAA (Table 1). The representative samples were synthesized with increasing feed mass of acrylic acid gradually, as shown in Table 1. In general, the average molecular weight (MW) increased with an increasing amount of a APS used. In particular, when the ratio of feed mass of Q[7] to acrylic acid reached 1 : 80, the MW of the Q[7]-anchored PAA sharply increased by-up to 10⁶.

Table 1 Feed composition and typical features of Q[7]-PAA samples

---

NMR of Q[7]-anchored PAA

Fig. 1a shows the ¹H NMR spectrum of the obtained Q[7]-PAA-2 polymer in D₂O as a representative. Relatively broad proton signals at ∼1.2–2.4 ppm were attributed to the methylene protons (Hf) and methine protons (He) of the PAA segment, respectively (inset in Fig. 1a). The proton signals at ∼2.5 and ∼4.2 ppm represented the protons (Hd) and methine protons (Hc) of the PAA moiety that bridged the Q[7] moieties and the PAA segment, which was confirmed by the rosey spectrum shown in Fig. S1 in the ESI.† The proton signals Ha and Hb at ∼4.3 and 4.4 ppm, respectively, represented the methylene protons of the ungrafted and grafted units. The ¹³C NMR spectrum (Fig. 1b) shows a carbonyl carbon signal from PAA at ∼184 ppm (1), and the resonances of two different carbonyl carbons from grafted (8) and ungrafted (9) glycoluril moieties of Q[7] at ∼156 and ∼155 ppm, respectively. The HMQC spectrum of Q[7]-PAA-2 (Fig. S2 in the ESI†) can assist in assigning the ¹³C NMR spectrum: signals at 98.6, 79.7 and 70.5 ppm were attributed to three waist carbons 4, 5, 10 of Q[7], respectively. The cross peaks at 4.12/46.6 and 4.33/50.6 ppm were assigned to CH₂ close to the grafted (6) and ungrafted (7) units, respectively. The cross peaks at 4.11/62.2(11) and 2.41/45.7(2) ppm correspond to the bridged AA moiety. The cross peaks at higher fields can be assigned to the PAA segment of the polymer.

Fig. 1 (a) ¹H and (b) ¹³C NMR spectra of the Q[7]-PAA-2.

Using the host–guest interaction properties of Q[7] moieties in Q[7]-PAA polymers

To investigate the host–guest interaction properties of Q[7] moieties in Q[7]-PAA polymers, 2-[N-(1-adamantyl)]aminomethyl benzene (G) was selected as an interesting guest with dual probes (adamantyl and aminomethyl benzene moieties) to Q[7].^31,32 Fig. 2 shows the ¹H NMR spectra for the titration of Q[7]-PAA-2 with guest G, and one can see that the phenyl moiety of G experienced a downfield shift and the single resonance split into three peaks, suggesting that the phenyl moiety of G was outside the portal of the Q[7] moiety, whereas the adamantyl moiety of G was included in the cavity of the Q[7] moiety in the polymer when the Q[7]/G molecule ratio was 1: ∼0.6 (referring to Fig. 2(2)). It is interesting that the further additon of G resulted in the appearance of extra split phenyl resonances, which experienced an upfield shift, suggesting that the phenyl moiety of G was included in the cavity of the Q[7] moiety in the polymer when the Q[7]/G molecule ratio was 1: ∼0.3 (referring to Fig. 2(3)). Generally, adamantyl is a typical probe for Q[7] due to the formation of a very stable host–guest inclusion complex (K_a in the range 10¹² to 10¹⁵),³³ and the preference for inclusion of the phenyl moiety of G suggests that the portals of Q[7] moieties in the polymer are not fully opened which could be partially or even entirely covered. On the basis of the integral intensity of components in Q[7]-PAA-2 and the host–guest interaction of the Q[7] moiety and G (Fig. 2), it is easy to calculate the amount of Q[7] moiety with different interaction models in the polymer at different Q[7]/G molecule ratios. The Q[7] moiety reaches ∼30% in weight in the polymer (1), ∼18% of the Q[7] portals are fully opened (2), whereas ∼9% are partially covered. When the sample shown in Fig. 2(3) was heated at 80 °C for 5 hours, the extra split phenyl resonances disappeared, whereas the phenyl resonances experienced downfield shift increased by ∼9.5%, and reached ∼28.5%, it suggested that the portals of partially covered Q[7] can be recovered (Fig. 2(4)).

Fig. 2 ¹H NMR spectra of (1) Q[7]-PAA-2 with (2) G (10 g L⁻¹, 25 μL), (3) G1(10 g L⁻¹, 135 μL), (4 and 3) heated at 80 °C for 5 h and (5) neat G.

Size and morphology of Q[7]-anchored PAA

The size and morphology of the polymer aggregates are important factors for their applications. Here, the morphology of Q[7]-PAA-based aggregates were studied using TEM and SEM measurements.^34–36 Fig. 3 shows the micrographs of Q[7]-PAA-2. One can see that the aggregates of Q[7]-PAA-2 presented spherical vesicles with a membrane thickness in the range 10−20 nm, and a diameter of 150−200 nm based on the TEM image (Fig. 3a). The SEM image of Q[7]-PAA-2 in Fig. 3b confirmed the coexistence of a large amount of spherical vesicle aggregates. Based on the above mentioned observations and the general formation mechanism of vesicles, we proposed a model for the formation of the nanocapsule, which is similar to the formation model of lipid vesicles.³⁴ Combination of ¹H NMR titration results revealed that vesicular structures are multi-layered structures. Nanometer-sized hollow polymer spheres or polymer nano-capsules are important for a wide range of applications including drug delivery, encapsulation, and imaging.

Fig. 3 Aggregate micrographs of Q[7]-PAA-2 ((a) TEM; (b) SEM), Q[7]-PAA-2-(2-naphthol) ((c) SEM) and corresponding diagrams.

Characterization of Q[7]-PAA's derivative and its adsorption some of basic dyes

The above Q[7]-PAA cases have sufficiently shown the feasibility and simplicity of the one-pot synthesis strategy, and the Q[7]-anchored PAA with reactive carboxyl groups can be further derivated to yield water-insoluble materials. For example, esterification of Q[7]-PAA-2 with 2-naphthol yielded a Q[7]-PAA-2-(2-naphthol), which is insoluble in water, and can be used in the water treatment, and adsorption of organic molecules (Scheme 1). The detailed synthesis and characteristics of Q[7]-PAA-2, Q[7]-PAA-2-(2-naphthol) are described in the Experimental section. Fig. 3c shows an SEM image of Q[7]-PAA-2-(2-naphthol), which was significantly different from that of Q[7]-PAA-2 in Fig. 3b.

The Q[7]-PAA-2-(2-naphthol) has been characterized by IR and ¹³C CP-MAS NMR spectroscopy. The FT-IR spectrum (Fig. S5, ESI†) of blue line shows strong absorption bands at 1735 cm⁻¹ and 1190–1250 cm⁻¹ attributed to the portal carbonyl groups of Q[7] and polyacrylate, respectively. A broad band between 1450 to 1650 cm⁻¹ and the peak at 760 cm⁻¹ proves the existence of 2-naphthol, and esterification of the OH groups of polyacrylic acid leads to the decrease of the peak at 922 cm⁻¹. The Q[7]-PAA-2-(2-naphthol) can be further confirmed by ¹³C CP-MAS NMR spectroscopy (Fig. 4). The peaks at 159.7, 97.1, 88.5, 73.6 and 44.3 ppm correspond to the carbonyl, methine and methylene carbon atoms, respectively, of the Q[7] framework. The peaks at 181.7, 44.3, 32.5 and 17.8 ppm could correspond to the polyacrylic acid unit linking the Q[7] framework and 2-naphthol as assigned in Fig. 4. The peaks at 60.7 ppm correspond to the methylene carbon atoms (2 in Fig. 4) attached to the oxidized Q[7] framework, and the polyacrylic acid, respectively. The peaks at 100–140 nm correspond to 2-naphthol carbons.

Fig. 4 ¹³C CP-MAS NMR spectrum of the Q[7]-PAA-2-(2-naphthol).

Considering the novel host–guest interaction features of Q[7], in particular with various dyes,^37–41 the adsorption of Q[7]-PAA-(2-napthol) for basic dyes was monitored by UV-visible spectroscopy. The absorbance of the basic dyes generally decreased with the addition of solid Q[7]-PAA-(2-napthol), leading to a significant change in color fading (Fig. 5 and S6–S8 in the ESI†), suggesting the adsorption of Q[7]-PAA-(2-napthol) for the basic dyes. Based on these results, we were able to obtain the adsorption of a unit mass of Q[7]-PAA-(2-napthol) for some basic dyes (acridine orange: 15.42 mg g⁻¹, thionine: 13.06 mg g⁻¹, proflavine: 12.29 mg g⁻¹, neutral red: 48.13 mg g⁻¹, basic yellow: 11.39 mg g⁻¹, thiazole orange: 26.48 mg g⁻¹ (Fig. S9 in the ESI†)). The unit amount adsorption data show the preference of Q[7]-PAA-(2-napthol) for neutral red. Most impressively, the Q[7]-PAA-(2-napthol) exhibits high selectivity for capture of neutral red in a neutral medium and releases the neutral red by washing with ethanol. Moreover, the experiments also revealed that the higher degree of polymerization could result in an increase the number of pore space structure for more favourable the adsorption of the dye (referring to Fig. S9 and S10 in ESI†).

Fig. 5 Schematic diagram of the adsorption basic dyes and UV-visible absorption spectrum of thiazole orange.

Experimental section

Q[7] was prepared according to methods previously reported in the literature.^42,43 Ammonium persulfate (APS, AR) and acrylic acid (AA, AR) were obtained from the Aladdin Industrial Corporation (Naoqiao Town, Fengxian, Shanghai, China).

Synthesis of Q[7]-anchored PAA

As a typical example, Q[7]-PAA-2 is synthesized by dissolving, Q[7] (8 × 10⁻³ mol L⁻¹ in distilled water, 25 mL, 0.2 mmol) and ammonium persulfate (0.23 g, 1 mmol). The solution was stirred with a small magnetic stir bar under an inert nitrogen atmosphere and heated to 80 °C in a water bath. After thermal equilibrium had been reached and N₂ had been bubbled through the solution for 1 h, a 0.4 mol L⁻¹ aqueous solution of acrylic acid (5 mL, 2 mmol) was added by drop-wise. After stirring the reaction mixture for another 8 h, the solution was cooled to room temperature. The resulting precipitate (salts formed during reaction) was removed by filtration. The filtrate was purified by dialysis (MWCO 2000) against water for 24 h to yield a polymer solution, and then concentrated to a volume of 2 mL on a rotary evaporator. The product was precipitated from the concentrated solution by the addition of acetone, and purified by three dissolution–precipitation cycles. The white precipitate was washed three times with acetone. The final precipitate was collected, immersed in acetone for 4 h, and then dried under reduced pressure at 40 °C for 48 h. The yield was ∼60%. Other Q[7]-anchored polyacrylic acid samples were synthesized under similar conditions except with varying feed masses of acrylic acid (yields = 30–80%). The detailed conditions are listed in Table 1.

Synthesis of Q[7]-PAA-(2-naphthol)

The Q[7]-PAA-2 (1 g) and thionylchloride (5 mL) was add in 150 mL round bottom flask. The solution was stirred with a small magnetic stir bar and heated to 80 °C in a air bath for 12 h, then concentrated to a solid on a rotary evaporator. The obtained solid, methylbenzene (30 mL) and p-toluenesulfonic acid (0.01 g) was added in three-neck flask, and 2-naphthol (1 g) dissolved in methylbenzene (10 mL) was slowly added dropwise into the solution, then heated to 70 °C and refluxed for 12 h. The product by vacuum filtration, it was added to sodium hydroxide solution (pH = 9, 50 mL) and dispersion for 0.5 h, then vacuum filtration to obtain a solid. The above solid was washed 4 times with ether, placed in an oven dried, weighed, a yield of about 60%.

Conclusions

In summary, microspherical aggregates of Q[7]-anchored PAAs were synthesized using a one-pot free radical polymerization strategy in the presence of APS as an initiator and oxidant. As a representative Q[7]-PAA-2, Q[7] in the polymer reached 30% in weight, and 28% of them have opened or partially opened portals. The vesicle features of Q[7]-anchored PAAs could be used in drug delivery, preserving fluorescence, encapsulation and release of a fluorescent dye and so on. Moreover, the water soluble Q[7]-anchored PAAs can be esterified to form water insoluble materials, which could be used as a stationary phase for separation or purification.

Acknowledgements

We acknowledge the support of the National Natural Science Foundation of China (Nos 21272045, 51463004), the “Chun-Hui” Funds of the Chinese Ministry of Education (No. Z2014093), the Graduate Innovation Funds of Guizhou University (No. 2015036).

Notes and references

1. W. J. Cheong, J. H. Go and K. Kim, Bull. Korean Chem. Soc., 2008, 29, 1941.
2. S. Y. Jon, N. Selvapalam, D. H. Oh, J. K. Kang, S. Y. Kim, Y. J. Jeon, J. W. Lee and K. Kim, J. Am. Chem. Soc., 2003, 125, 10186.
3. S. M. Liu, L. Xu, C. T. Wu and Y. Q. Feng, Talanta, 2004, 64, 929.
4. E. R. Nagarajan, D. H. Oh, N. Selvapalam, Y. H. Ko, K. M. Park and K. Kim, Tetrahedron Lett., 2006, 47, 2073.
5. D. Kim, E. Kim, J. Kim, K. M. Park, K. Baek, M. Jung, Y. H. Ko, W. Sung, H. S. Kim, J. H. Suh, C. G. Park, O. S. Na, D. Lee, K. E. Lee, S. S. Han and K. Kim, Angew. Chem., Int. Ed., 2007, 46, 3471.
6. K. M. Park, K. Suh, H. Jung, D.-W. Lee, Y. Ahn, J. Kim, K. Baek and K. Kim, Chem. Commun., 2009, 71.
7. M. Munteanu, S. W. Choi and H. Ritter, Macromolecules, 2009, 42, 3887.
8. M. J. Li, M. B. Zaman, D. Bardelang, X. H. Wu, D. S. Wang, J. C. Margeson, D. M. Leek, J. A. Ripmeester, C. I. Ratcliffe, Q. Lin, B. Yang and K. Yu, Chem. Commun., 2009, 6807.
9. E. Kim, J. Lee, D. Kim, K. E. Lee, S. S. Han, N. Lim, J. Kang, C. G. Park and K. Kim, Chem. Commun., 2009, 1472.
10. D. Kim, E. Kim, J. Lee, S. Hong, W. Sung, N. Lim, C. G. Park and K. Kim, J. Am. Chem. Soc., 2010, 132, 9908.
11. D.-W. Lee, K. M. Park, M. Banerjee, S. H. Ha, T. Lee, K. Suh, S. Paul, H. Jung, J. Kim, N. Selvapalam, S. H. Ryu and K. Kim, Nature Chem., 2011, 154.
12. S. Kushwaha and P. P. Sudhakar, Analyst, 2012, 137, 3242.
13. K. M. Park, J.-A. Yang, H. Jung, J. Yeom, J. S. Park, K. H. Park, A. S. Hoffman, S. K. Hahn and K. Kim, NANO, 2012, 2960.
14. K. Baek, G. Yun, Y. Kim, D. Kim, R. Hota, I. Hwang, D. Xu, Y. H. Ko, G. H. Gu, J. H. Suh, C. G. Park, B. J. Sung and K. Kim, J. Am. Chem. Soc., 2013, 135, 6523.
15. C. J. Zou, T. Gu, P. F. Xiao, T. T. Ge, M. Wang and K. Wang, Ind. Eng. Chem. Res., 2014, 53, 7570.
16. K. Kim, N. Selvapalam, Y. H. Ko, K. M. Park, D. Kim and J. Kim, Chem. Soc. Rev., 2007, 36, 267.
17. A. I. Day, A. P. Arnold and R. J. Blanch, Molecules, 2003, 8, 74.
18. Y. J. Zhao, S. F. Xue, Q. J. Zhu, Z. Tao, J. X. Zhang, Z. B. Wei, L. S. Long, M. L. Hu, H. P. Xiao and A. I. Day, Chin. Sci. Bull., 2004, 49, 1111.
19. J. J. Zhou, X. Yu, Y. C. Zhao, X. Xiao, Y. Q. Zhang, Q. J. Zhu, S. F. Xue, Q. J. Zhang, J. X. Liu and Z. Tao, Tetrahedron, 2014, 70, 800.
20. J. J. Zhou, X. Yu, Y. C. Zhao, X. Xiao, Y. Q. Zhang, S. F. Xue, Z. Tao, J. X. Liu and Q. J. Zhu, Eur. J. Inorg. Chem., 2014, 5771.
21. L. M. Zheng, J. N. Zhu, Y. Q. Zhang, Q. J. Zhu, S. F. Xue, Z. Tao, J. X. Zhang, Z. Xin, Z. B. Wei, L. S. Long and A. I. Day, Supramol. Chem., 2008, 20, 709.
22. X. L. Ni, Y. Q. Zhang, Q. J. Zhu, S. F. Xue and Z. Tao, J. Mol. Struct., 2008, 876, 322.
23. V. Lewin, J. Rivollier, S. Coudert, D. A. Buisson, D. Baumann, B. Rousseau, F. X. Legrand, H. Kouřilová, P. Berthault, J. P. Dognon, M. P. Heck and G. Huber, Eur. J. Org. Chem., 2013, 3857.
24. H. Isobe, S. Sato and E. Nakamura, Org. Lett., 2002, 4, 1287.
25. C. Z. Wang, W. X. Zhao, Y. Q. Zhang, S. F. Xue, Q. J. Zhu and Z. Tao, RSC Adv., 2015, 5, 17354.
26. M. M. Ahmed, K. Koga, M. Fukudome, H. Sasaki and D. Q. Yuan, Tetrahedron Lett., 2011, 52, 4646.
27. L. H. Wu, X. L. Ni, F. Wu, Y. Q. Zhang, Q. J. Zhu, S. F. Xue and Z. Tao, J. Mol. Struct., 2009, 920, 183.
28. X. L. Huang, F. L. Hu and H. Q. Su, Macromolecules, 2013, 46, 1274.
29. B. Xiao, Y. Fan, N. N. Ji, X. J. Cheng, C. Z. Wang, J. J. Zhou, S. F. Xue, Z. Tao and Q. J. Zhu, Supramol. Chem., 2015, 27, 4.
30. B. Xiao, Y. Fan, R. H. Gao, P. Cheng, J. X. Zhang, Q. D. Zhou, S. F. Xue, Q. J. Zhu and Z. Tao, RSC Adv., 2015, 5, 33809.
31. P. H. Ma, J. Dong, S. C. Xiang, S. F. Xue, Q. J. Zhu and Z. Tao, Sci. China, Ser. B: Chem., 2004, 34, 133.
32. P. H. Ma, Z. Tao, S. F. Xue, Q. J. Zhu, S. K. Wang, S. W. Yuan, J. X. Zhang and X. Zhou, Chin. J. Org. Chem., 2007, 27, 414.
33. S. M. Liu, C. Ruspic, P. Mukhopadhyay, S. Chakrabarti, P. Y. Zavalij and L. Isaacs, J. Am. Chem. Soc., 2005, 127, 15959.
34. D. Kim, E. Kim, J. Kim, K. M. Park, K. Baek, M. Jung, Y. H. Ko, W. Sung, H. S. Kim, J. H. Suh, C. G. Park, O. S. Na, D. Lee, K. E. Lee, S. S. Han and K. Kim, Angew. Chem., Int. Ed., 2007, 46, 3471.
35. D. Kim, E. Kim, J. Lee, S. Hong, W. Sung, N. Lim, C. G. Park and K. Kim, J. Am. Chem. Soc., 2010, 132, 9908.
36. E. Kim, D. Kim, H. Jung, J. Lee, S. Paul, N. Selvapalam, Y. Yang, N. Lim, C. G. Park and K. Kim, Angew. Chem., Int. Ed., 2010, 49, 4405.
37. P. M. Navajas, A. Corma and H. Garcia, ChemPhysChem, 2008, 9, 713.
38. G. Ghale, V. Ramalingam, A. R. Urbach and W. M. Nau, J. Am. Chem. Soc., 2011, 133, 7528.
39. J. Mohanty, A. C. Bhasikuttan, W. M. Nau and H. Pal, J. Phys. Chem. B, 2006, 110, 5132.
40. M. Shaikh, J. Mohanty, A. C. Bhasikuttan, V. D. Uzunova, W. M. Nau and H. Pal, Chem. Commun., 2008, 3681.
41. M. Shaikh, S. D. Choudhury, J. Mohanty, A. C. Bhasikuttan and H. Pal, Phys. Chem. Chem. Phys., 2010, 12, 7050.
42. A. I. Day and A. P. Arnold, WO 2000068232 A1, 2000.
43. J. Kim, I. S. Jung, S. Y. Kim, E. Lee, J. K. Kang, S. Sakamoto, K. Yamaguchi and K. Kim, J. Am. Chem. Soc., 2000, 122, 540.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra09064d

---