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close to 300 °C, which are far above the temperatures used in Tc It is also of worth highlighting in this preliminary sVtuiewdyArttihclae tOnthlinee
measurements (ESI).
[N5C66][OTs] appeared to be capable of sDtaObI:il1iz0i.n10g39p/rDo0tCeCin0;4t4h17aBt
As we successfully developed cycloammonium sulfonates as is, after concentration, the resulting TIL-GFP solution stayed
TILs carrying LCST phase behavior, we turned our attention to bright green for over one week at room temperature.
undertake a preliminary study to see if these TILs are
In summary, we reported in this work the combinatorial
compatible with proteins with the hope that, upon discovery of new cycloammonium TILs that unambiguously
temperature-triggered phase separation, proteins may illustrated and highlighted the real value of fine structural
preferentially partition in ionic liquid layer, which would tunability of ionic liquids. All TILs discovered were identified to
ultimately make biomolecular interaction analysis possible. situate on the rim of totally hydrophilic and hydrophobic ionic
Isolation, separation and purification of proteins are usually liquids (Figure 2). Albeit no single TIL was found from the
performed using various forms of chromatographic, [N4CRR][OTMBS] sub-library, rational incorporation of
electrophoretic, ultrafiltration, precipitation and dialysis appropriate but asymmetric alkyl groups on pyrrolidine made
procedures, and used in almost all branches of biosciences and rational identification of new TILs possible (Figure 5c). All TILs
biotechnologies.10 New methods capable of treating dilute are eminently capable of performing LCST phase transitions
solutions or solutions containing only minute amounts of target with low Tc values in water. Combinatorial chemistry allows the
biomolecules should be of indispensable need in studies of synthesis of compounds simultaneously and has been an
precious proteins. In this work, TILs were considered and used important tool in many areas of research, including ionic liquid
as an immediate application for enriching dilute protein synthesis.11,12 To our knowledge, this work is the first
solutions. Here, [N5C66][OTs] was selected as a model TIL, combinatorial approach of small-molecule cycloammonium
primarily because it undergoes a LCST phase transition below ionic liquids that exhibit LCST property with water. The results
room temperature with water. We chose cytochrome c from presented in this work hold compelling possibilities of the use
equine heart and green fluorescent protein (GFP) of the jellyfish of TILs as functional materials for advancing biomolecule
Aequorea victoria for their ease of visualization by naked eyes separation and potential interaction analysis.
and with light box, respectively, to test to see if their dilute
This work was supported by a grant (MOST 106-2113-M-
concentration enrichments could be realized by TILs. Pleasingly, 194-006-MY3) from the Ministry of Science and Technology of
as shown in Figure 6, both proteins were readily concentrated Taiwan, Republic of China.
by adding [N5C66][OTs] into and mixing with dilute protein
solutions. In our hand, cytochrome c and GFP were enriched 39-
and 11-fold, respectively. From spectra shown in Figure 6, upper
Conflicts of interest
aqueous phases (blue spectra) contained essentially free of
proteins; that is, most, if not all, of proteins were preferentially
partitioned and concentrated in ionic liquid-rich bottom phases.
There are no conflicts to declare.
Notes and references
1
2
3
4
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6
7
8
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Figure 6. Photos and spectra of temperature-dependent phase
transition were exploited for demonstrating protein enrichment
from its aqueous mixtures (1:15, w/w) of [N5C66][OTs] with (a) a
solution of equine heart cytochrome c (4 mg/mL, H2O) and (b) a
solution of jellyfish Aequorea victoria green fluorescent protein
9
K. Fujita, R. Nakano, R. Nakaba, N. Nakamura and H. Ohno,
Chem. Commun., 2019, 55, 3578.
10 R. Hatti-Kaul and B. Mattiasson, Isolation and Purification of
Proteins, Marcel Dekker, New York, USA, 2005.
11 L. Zhang, S. Luo, X. Mi, S. Liu, Y. Qiao, H. Xu and J.-P. Cheng,
Org. Biomol. Chem., 2008, 6, 567.
(GFP, 16 g/75 L, 3 mM tris buffer). UV-vis spectrometry and
12 P. Wasserscheid, B. Drießen-Hölscher, R. van Hal, H. C.
fluorometry were used for quantitatively determining degree of
enrichment in cytochrome c (39-fold increase, Soret band at
406 nm) and GFP (11-fold increase, emission band at 511 nm),
respectively.
Steffensa and J. Zimmermann, Chem. Commun., 2003, 2038.
4 | J. Name., 2012, 00, 1-3
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