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Crowned Ionic Liquids for Biomolecular Interaction Analysis Ming-Chung Tseng, Tsu-Chun Yuan, Zhuo Li, and Yen-Ho Chu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03323 • Publication Date (Web): 02 Nov 2016 Downloaded from http://pubs.acs.org on November 2, 2016

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Analytical Chemistry

Graphical Abstract

Crowned Ionic Liquids for Biomolecular Interaction Analysis Ming-Chung Tseng, Tsu-Chun Yuan, Zhuo Li and Yen-Ho Chu*

This work reports the synthesis of a new class of coronal ionic liquids and their application for selective recognition and affinity binding of peptides and proteins.

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Crowned Ionic Liquids for Biomolecular Interaction Analysis

Ming-Chung Tseng,‡ Tsu-Chun Yuan,‡ Zhuo Li and Yen-Ho Chu*

Department of Chemistry and Biochemistry, National Chung Cheng University, Minhsiung, Chiayi 62102, Taiwan, Republic of China

* Corresponding author. Tel: 886 52729139; fax: 886 52721040; e-mail: [email protected] ‡ Both authors contributed equally to this work.


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Abstract Based on affinity recognition with positively charged side chains in peptides and proteins, a series of crowned 1,2,3-triazolium ionic liquids CIL 1-6 were developed and found to be capable of quantitatively extracting peptides and proteins from aqueous layer into ionic liquid phase. All CIL 1-6 synthesized are liquid at room temperature. This is the first example of biomolecular recognition of both lysine- and arginine-containing peptides and proteins by CILs in pure ionic liquid phase.



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This paper describes the development of a system based on coronal ionic liquids—1,2,3-triazolium-based, crowned ionic liquids (CILs)—specifically tailored for selective recognition and affinity binding of peptides and proteins. This chemoselective binding interaction of peptides and proteins was achieved by affinity recognition of basic residues in peptides and proteins using crowned ionic liquids CIL 1-6 (Figure 1).

O

O N

NTf2

N N

O

N

Bu

NTf2

N N

CIL 1, [b-6C2-tr][NTf2]

O

O

N

O

Bu


O

Bu


O

O

NTf2

N N

O

O

O

O

O O

O N O

NTf2

N N

Bu

N

O

NTf2

N N

Bu

O



N

NTf2

N N

O

Bu

O CIL 6, [b-21C7-tr][NTf2]

Figure 1. Structures of crowned ionic liquids CIL 1-6.

Ionic liquids are a group of polar solvents entirely made of ions.1 Ionic liquids offer a platform of tunable structures on which the properties of both cation and anion can be independently engineered.1 We have a longstanding interest in developing newfangled ionic liquids with aims to discover advanced materials carrying novel functions for chemical and biochemical applications.2 Based specifically on the intramolecular Huisgen 1,3-dipolar [3+2] cycloaddition reaction,3 this work reports concise synthesis of a new class of ionic liquids CIL 1-6 (Figure 1) and demonstrates the usefulness of these affinity ionic liquids as crown ether mimetics for biomolecular recognition of peptides and proteins.


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Crown ethers are cyclic oligomers of ethylene oxide.4 Along with their synthetic coronands, crown ethers have found many applications in extraction systems that are selective for natural and synthetic molecules depending on the size of the crown rings; that is, a complex is formed when a cation size matches the size of the crown cavity.4 In literature, crown ethers such as 18-crown-6 (18C6) are also known to recognize ammonium ions of histamine and dopamine,5 and the positively charged side chain of lysine characteristically stabilized through a network of hydrogen bond interactions with three donor oxygen atoms on the macrocyclic ether rings.5 We envisaged that crown ether ring embedded in 1,2,3-triazolium ionic liquids6 such as CIL 1-6 could be readily assembled by an intramolecular Huisgen [3+2] cycloaddition and should therefore be valuable as novel crown ether mimetics for selective recognition and affinity extraction of targeted biomolecules.

Scheme 1 illustrates our synthesis of CIL 1-6 of which the crowned 1,2,3-triazoles 2a-f (Figure S1, Supporting Information) as the recognition elements could be assembled from O-(2-azidoethyl)-O’-propargyl-oligo(ethylene glycol) 1a-f via an intramolecular Huisgen [3+2] cycloaddition reaction in dilute toluene solution (12.5 mM) under reflux condition. The synthesis was straightforward and the overall isolated yields for this 6-step synthesis of CIL 1-6 were acceptable: 22-37% (Scheme 1). We were pleased that by-products from intermolecular triazole forming reactions were not observed under our experimental conditions. It is of note that small crowned ionic liquids CIL 1-3 synthesized were only in 1,5-isoform, and the larger crowned ionic liquids CIL 4-6 were composed of 1,4- and 1,5-isomers in which the latter is the major product.7 Detailed experimental procedures, spectra and data of CIL 1-6 are summarized in the Supporting Information.



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HO

N3

O

O

OH n-1

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propargyl bromide KOH, 0 oC to rt, 0.5 h

1. MsCl, Et3N, CH2Cl2 0 oC to rt, 2 h

61% (n=1), 95% (n=2), 69% (n=3), 70% (n=4), 47% (n=5), 60% (n=6)

2. NaN3, DMF (1 M) 40 oC, 8 h O

toluene (12.5 mM), reflux, 18 h

O n-1

O in 3 steps: 58% (n=1), 39% (n=2), 58% (n=3), 72% (n=4), 75% (n=5), 55% (n=6)

1a-f (n = 1-6)

1. Bu-OMs, 70 oC, 3 h 2. LiNTf2, H20, rt, 12 h

N N

n-1 2a-f (n = 1-6)

O O

in 2 steps: 74% (n=1), 72% (n=2), 81% (n=3), 74% (n=4), 63% (n=5), 74% (n=6)

N

N n-1

N

NTf2

N CIL 1-6 (n = 1-6)

Scheme 1. Synthesis of crowned ionic liquids CIL 1-6.

All CIL 1-6 obtained are pale yellow liquid at room temperature. This grafting of ethylene glycol units onto ionic liquids not only lowers their viscosities and melting temperatures, but also likely makes them potentially more biocompatible than alkyl substituted ionic liquids.1 In this work, we used CIL 1-6 and tested their effectiveness in binding interactions of selected biomolecules. Our system utilizes water-immiscible CILs to extract target biomolecules from aqueous solution through the complexation of targeted ions with CILs. To the best of our knowledge, these coronal ionic liquids for selective biomolecular interaction analysis have not been reported in the literature.

We first examined all six CILs for their effectiveness in affinity extraction of six N-dabsylated lysine/K-containing hexapeptide amides. In these experiments, equal volumes of ionic liquid and water were first mixed, the resulting mixtures were then vigorously shaken and lastly centrifuged at room temperature to afford phase


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separation and accomplish extraction. Results are summarized in Figure 2. We found that the efficiency of peptide extraction was highly dependent on the size of crown rings in CILs; that is, CIL 4-6 chemoselectively recognized side-chain ammonium groups of all six lysine peptides in water (upper phase) and, as a result, effectively extracted it into ionic liquid phase (bottom layer). Moreover, lysine peptides remained largely unaffected in water layer upon extracting by CIL 1-3 of smaller ring sizes (Figure 2), clearly indicating that the liquid-liquid partitioning of labeled peptides into ionic liquid phase was insignificant and, regardless of the number of lysine residues in peptides, the recognition of AIL 4-6 toward all six lysine peptides are chemospecific.8 Using Dab-AKKKKK-NH2 peptide as a representative example, Figure 3 unambiguously demonstrated that the transfer of this peptide from the aqueous phase into the ionic liquid layer was indeed entirely due to affinity recognition by CIL 6. This affinity peptide extraction by CIL 6 and total peptide retention in water when used by CIL 2 were apparent to the naked eyes (Figure 3). Furthermore, it is worth noting that the peptide in ionic liquid layer could be totally recovered into the upper aqueous phase by competitive extraction using 1 M KCl.9

Figure 2. Screening of water-immiscible crowned ionic liquids CIL 1-6 (10 µL) used



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to affinity extract N-dabsylated lysine/K-containing hexapeptide amides in aqueous solutions (1 mM in 10 mM Li2SO4 with 10% DMSO, 10 µL). The solution mixture was vigorously agitated and centrifuged to induce phase separation. The upper aqueous layer was then measured at 502 nm (NanoDrop 2000) to determine the amount of peptide remained in aqueous phase. The extraction efficiency was defined as [1-(Aafter/Abefore)]×100%, where Abefore and Aafter were absorbances at 502 nm before and after extraction by CIL 1-6. Estimated RSD, 10%.

Figure 3. Optical photographic images of affinity extraction of Dab-AKKKKK-NH2 peptide (1 mM in 10 mM Li2SO4 with 10% DMSO, 100 µL) by CIL 2 and CIL 6 (100 µL each), respectively. Competitive extraction of the peptide in CIL 6 ionic liquid layer into the aqueous phase could be readily achieved using KCl (1 M).

Albeit there is no ideal match between the ammonium cation size of lysine residue in peptides and cavity diameters of CIL 4-6, the reason for their successful affinity extractions likely reflects the flexibility of the larger crown ethers, which can adapt, wrap around and optimize its interaction with the ammonium ion, eventually giving rise to stable and stronger complexes. For CILs with smaller cavities, the complexation, if observed, could happen outside the crown cavity and the ammonium cation completes its coordination sphere with a second ionic liquid to form


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“sandwich-like” complexes. This effectiveness of affinity binding is driven by both the thermodynamically favored ammonium ion complexation by ionic CILs (ion-dipole interactions and hydrogen bonds) and the solvation of the peptide ions by the ionic solvent. These dual properties of ionic liquids make them efficient solvents for the extraction of ionic species from aqueous solutions.

As illustrated in Figure S2 (Supporting Information), result of lysine positional scanning experiments on N-dabsylated mono-K peptides showed that CIL 6 interacted equally well with hexapeptides having the lysine residue at any position. Using 1H NMR, we also investigated the binding stoichiometry of both the crowned 1,2,3-triazole 2f (n = 6) and CIL 6 with ammonium triflate in CDCl3, confirming their 1 : 1 binding interactions. These NMR titration results are summarized in Figure S3 (Supporting Information). Overall, we were pleased that both the triazole 2f and its triazolium ionic liquid CIL 6 effectively extracted lysine peptides and tightly associated with the ammonium ion to form a stable 1 : 1 complex.

As we were encouraged by the results of the use of CILs as affinity ionic liquids for efficient extractions of lysine-containing peptides, we went further to investigate CIL 6 to test its binding interactions with arginine/R-containing peptides. A N-dabsylated ARRRRR-NH2 hexapeptide amide was examined, and the result is summarized in Figure 4. We were pleased that this CIL 6 works well in recognizing and efficiently extracting the alkyl-guanidinium side chain of arginine residues in peptide.4b,10 As expected, the control CIL 2 was too small in crown ring size to accommodate the guanidinium group in the arginine peptide, clearly indicating specific binding interaction between the arginine peptide and CIL 6. In addition, this peptide in CIL 6 could be completely extracted back to the upper aqueous phase by competitive



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extraction using 1 M ammonium sulfate. Furthermore, because side chain functional groups of lysine and arginine residues in peptides carry different pKa’s (10.5 and 12.5, respectively), we envisaged that this significant difference in pKa should allow to provide convenient separation of lysine and arginine peptides. To our delight, Figure 5 showed that, at pH 11.8, the Dab-ARRRRR-NH2 peptide could be completely extracted into the ionic liquid layer by CIL 4 but the Dab-AKKKKK-NH2 peptide was totally unaffected and retained in aqueous layer (Figure S4 in the Supporting Information).

Figure 4. Optical photographic images of affinity extraction of Dab-ARRRRR-NH2 peptide (1 mM in 10 mM Li2SO4 with 10% DMSO, 20 µL) by CIL 2 and CIL 6 (20 µL each), respectively. Competitive extraction of the peptide in ionic liquid phase back to the upper aqueous layer was achieved using 1 M ammonium sulfate.

Figure

5.

Differential

affinity

extraction

of

Dab-ARRRRR-NH2


and

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Dab-AKKKKK-NH2 peptides (1 mM each in 100 mM Li2CO3 with 10 % DMSO, 20 µL) using CIL 4 (20 µL) under highly basic condition (pH 11.8). Extraction efficiencies of Dab-ARRRRR-NH2 and Dab-AKKKKK-NH2 by CIL 4 were 91% and 13%) in basic residues and their solutions are red brown that are apparent to naked eyes, they should be suited for CILs experiments. Results of the SDS-PAGE experiments illustrated in Figure 6A showed that myoglobin protein was readily recognized and quantitatively extracted by CIL 6 from the upper aqueous phase into the bottom ionic liquid layer, and subsequently could be competitively extracted using lysine (1 M) and fully recovered back into the aqueous solution. Our preliminary result indicated that, after centrifugal dialysis and protein refolding by ultrafiltration, myoglobin protein amount was fully retained but, on the basis of measurements of Soret absorption at 409 nm and circular dichroism spectra (Figure S5, Supporting Information), its correct conformation was only partially restored (Figure 6B). We noted that a sharp Soret absorption at 409 nm was observed for myoglobin after its refolding (Figure S5). This retention of the characteristic Soret band suggested that the heme environment of protein after competitive extraction and refolding was similar to that of the native conformations.11 Results on affinity extraction, protein recovery and refolding of cytochrome c were similar to that of myoglobin (Figures S6 and S7, Supporting Information).



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This protein dissolution in ionic liquids at the molecular level is highly complex and its understanding is far from complete, what could be certain is that in hydrophobic CIL 6 both proteins have shown their high solubility (not suspension). This experimental observation may be explained by the fact that CIL 6 has a strong affinity for K/R-rich proteins and therefore tightly associates with it to enable the complex formation, ultimately leading to the complete departure of the protein from the aqueous layer and quantitative transfer to the ionic liquid phase. Like peptides that are completely soluble in crowned ionic liquids CIL 4-6, our affinity extraction of proteins nevertheless shows that they remain soluble in ionic liquid CIL 6 and, albeit partially restored in conformation, can be completely extracted and recovered using the protocol developed in this work.

Figure 6. Extraction of myoglobin by CIL 6. (A) Protein measurements by SDS-PAGE of the upper aqueous solutions upon extracting myoglobin (0.2 mM, 10 µL) using CIL 6 (10 µL): 100% (before extraction), 0% (after extraction), 97% (competitive extraction by 1 M lysine), and 94% (desalting and refolding after Amicon ultra-3K filtration), respectively. (B) Soret absorption measurements at 409 nm (NanoDrop 2000): 100% (before extraction), 0% (after extraction), and 38% (desalting and refolding after Amicon ultra-3K filtration), respectively. Estimated RSD, 20%.


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In summary, we described in this work the development of new crowned ionic liquids CIL 1-6 and demonstrated that CIL 4-6 of medium to large ring sizes are eminently capable of performing affinity binding interactions with lysine/arginine-containing peptides and proteins totally in ionic liquid phase. This is the first example of biomolecular recognition of K/R-containing peptides and proteins by CILs in pure ionic liquid phase.12 The results presented in this work hold compelling possibilities for advancing biosensors as well as chemosensors targeting a new range of analytes and applications in biotechnology.13 Study of proteins with higher refolding yields upon interacting with CILs is being actively pursued and the result will be reported in due course.

We gratefully acknowledge support of this work by a grant from the Ministry of Science and Technology of Taiwan, ROC (MOST 103-2113-M-194-002-MY3). We thank Professor Cheng-I Lee (Department of Life Science) for the use of CD instrument, and our colleagues Chien-Yuan Chen and Tzu-Hsuan Hsu for assistance during the early development of this research. We also thank reviewers for their constructive comments.

Supporting Information Figures S1-S7; full experimental and spectral characterization details of crowned triazoles 2a-f and ionic liquids CIL 1-6; solid-phase synthesis of dabsylated lysineand arginine-containing peptides; stoichiometry measurements by 1H NMR; affinity extraction experiments of peptides and proteins.

REFERENCES



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1. (a) Cecchini, M. M.; Charnay, C.; Angelis, F. D.; Lamaty, F.; Martinez, J.; Colacino, E. ChemSusChem 2014, 7, 45‒65. (b) Ho, T. D.; Zhang, C.; Hantao, L. W.; Anderson, J. L. Anal. Chem. 2014, 86, 262‒285. (c) Cevasco, G.; Chiappe, C. Green Chem. 2014, 16, 2375‒2385. 2. (a) Chang, Y.-P.; Liu, W.-C.; Tseng, M.-C.; Chu, Y.-H. Rev. Anal. Chem. 2015, 34, 77‒86. (b) Sowmiah, S.; Cheng, C. I.; Chu, Y.-H. Curr. Org. Syn. 2012, 9, 74‒95. (c) Shi, Y.; Liu, Y.-L.; Lai, P.-Y.; Tseng, M.-C.; Tseng, M.-J.; Li, Y.; Chu, Y.-H. Chem. Commun. 2012, 48, 5325‒5327. 3. Thirumurugan, P.; Matosiuk, D.; Jozwiak, K. Chem. Rev. 2013, 113, 4905−4979. 4. (a) Christy, F. A.; Shrivastav, P. S. Crit. Rev. Anal. Chem. 2011, 41, 236‒269. (b) Gokel, G. W.; Leevy, W. M.; Weber, M. E. Chem. Rev. 2004, 104, 2723‒2750. 5. Spath, A.; Konig, B. Beilstein J. Org. Chem. 2010, 6, No. 32. doi: 10.3762/bjoc.6.32. 6. Tseng, M.-C.; Cheng, H.-T.; Shen, M.-J.; Chu, Y.-H. Org. Lett. 2011, 13, 4434‒4437. 7. Attempts to produce exclusively the 1,5-isomers using the protocol of the Cp*RuCl(PPh3)2-catalyzed cycloadditions developed by Jia and Fokin were however unsuccessful in our hand: Boren, B. C.; Narayan, S.; Rasmussen, L. K.; Zhang, L.; Zhao, H.; Lin, Z.; Jia, G.; Fokin, V. V. J. Am. Chem. Soc. 2008, 130, 8923‒8930. 8. In the case of CIL 3 with undersized ring cavity (Figure 2), high mole excess of CIL 3 vs. lysine residues in peptides would be required in order to afford much weak binding associations with mono-, di-, and tri-K peptides, but not tetra-to-hexa K peptides, to form untied sandwich-like complexes and, as a result, achieve poor extraction efficiency. 9. Potassium ion (1.33 Å) and ammonium ion (1.43 Å) have very similar ionic


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radius.4,5 10. It was reported that the 27-crown-9 ether is the optimum size for guanidinium cation: (a) Madan, K.; Cram, D. J. J. Chem. Soc., Chem. Comm. 1975, 427‒428. (b) Cram, D. J.; Cram, J. M. Acc. Chem. Res. 1978, 11, 8‒14. 11. Thiansilakul, Y.; Benjakul, S.; Richards, M. P. Food Chem. 2011, 124, 254‒261. 12. Using crown ethers as additives in ionic liquids likely encounters the issue of being leaked into aqueous phase, in principle at least, and the incomplete transfer of polypeptides during ion-specific extractions (Shimojo, K.; Kamiya, N.; Tani, F.; Naganawa, H.; Naruta, Y; Goto, M. Anal. Chem. 2006, 78, 7735‒7742). Our CILs with crown moiety embedded in [NTf2]-based ionic liquids should therefore give minimal leakage, if any. 13. Our preliminary result on the regeneration and repetitive use of CILs was promising. Its systematic investigation is ongoing and the result will be reported in due course.


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