Exploiting 1,2,3-Triazolium Ionic Liquids for Synthesis of Tryptanthrin and Chemoselective Extraction of Copper(II) Ions and Histidine-Containing Peptides

Article abstract of DOI:10.3390/molecules21101355

Based on a common structural core of 4,5,6,7-Tetrahydro[1,2,3]triazolo[1,5-A]pyridine, a number of bicyclic triazolium ionic liquids 1-3 were designed and successfully prepared. In our hands, this optimized synthesis of ionic liquids 1 and 2 requires no chromatographic separation. Also in this work, ionic liquids 1, 2 were shown to be efficient ionic solvents for fast synthesis of tryptanthrin natural product. Furthermore, a new affinity ionic liquid 3 was tailor-synthesized and displayed its effectiveness in chemoselective extraction of both Cu(II) ions and, for the first time, histidine-containing peptides.

Full text of DOI:10.3390/molecules21101355

molecules
Communication
Exploiting 1,2,3-Triazolium Ionic Liquids for Synthesis
of Tryptanthrin and Chemoselective Extraction of
Copper(II) Ions and Histidine-Containing Peptides
Hsin-Yi Li †, Chien-Yuan Chen †, Hui-Ting Cheng and Yen-Ho Chu *
Department of Chemistry and Biochemistry, National Chung Cheng University, Chiayi 62102, Taiwan;
lanbarla0708@gmail.com (H.-Y.L.); passercloud@gmail.com (C.-Y.C.); coconuthead222@hotmail.com (H.-T.C.)
* Correspondence: cheyhc@ccu.edu.tw; Tel.: +86-5272-9139
† Both authors contributed equally to this work.
Academic Editor: Hua Zhao
Received: 29 August 2016; Accepted: 9 October 2016; Published: 13 October 2016
Abstract: Based on a common structural core of 4,5,6,7-tetrahydro[1,2,3]triazolo[1,5-a]pyridine,
a number of bicyclic triazolium ionic liquids
hands, this optimized synthesis of ionic liquids
Also in this work, ionic liquids were shown to be efficient ionic solvents for fast synthesis of
tryptanthrin natural product. Furthermore, a new affinity ionic liquid was tailor-synthesized and
1
–
3
were designed and successfully prepared. In our
1
and requires no chromatographic separation.
2
1, 2
3
displayed its effectiveness in chemoselective extraction of both Cu(II) ions and, for the first time,
histidine-containing peptides.
Keywords: ionic liquid; Cu-free click reaction; tryptanthrin synthesis; affinity ionic liquid; copper
ion; chemoselective extraction; peptide
1. Introduction
This paper describes the synthesis of bicyclic 1,2,3-triazolium ionic liquids (Figure 1) [
1
–
3
1],
based on an intramolecular Hüisgen [3 + 2] cycloaddition (a Cu-free click reaction) [2,3], with specific
aims to establish a common structural core for molecular diversity development of the ionic liquids and
study their usefulness as reaction media for chemical synthesis and novel materials for biochemical
applications. Ionic liquids are a class of polar solvents entirely made of ions, and many of them are
liquids at room temperature [
They often possess wide liquid state ranges, high conductivity, excellent solubilization of many small
molecules, good thermal stability, and negligible volatility [ ]. The “green” character of ionic liquids
4,5]. Their ionic nature confers desirable properties to these liquids.
4,5
largely refers to their very low vapor pressure, if any, and consequent inability to cause air pollution.
Moreover, from the standpoint of organic synthesis, their blend of positive and negative charges makes
these ionic liquids excellent microwave absorbers, giving rise to their recent emergence as superior
alternatives to conventional molecular solvents for many microwave-assisted organic reactions [6].
N
N
N
N
N
N
N
N
N
N
N
NTf2
[e-4C-tr][NTf2], 2
Figure 1. Structures of [b-4C-tr][NTf2] 1, [e-4C-tr][NTf2] 2 and affinity ionic liquid 3.
NTf2
N
NTf2
[b-4C-tr][NTf2], 1
affinity ionic liquid 3
Molecules 2016, 21, 1355; doi:10.3390/molecules21101355
www.mdpi.com/journal/molecules

Molecules 2016, 21, 1355
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In our research, we are interested in developing new ionic liquids or improving the synthesis of
known ionic liquids and we have an ongoing program to evaluate ionic liquids as stable media for
chemical synthesis [
7–
11] and as functional materials for biochemical applications [12
,13]. We recently
reported the preparation of bicyclic 1,2,3-triazolium ionic liquids in four steps with good yields [
1].
Because of their intriguing chemical stability and our continuing interest in conducting research with
this class of ionic liquids, we decided to develop a more generalized synthesis so that these bicyclic
ionic liquids could be explored and studied systematically.
2. Results and Discussion
Our synthesis of bicyclic 1,2,3-triazolium ionic liquids 1, 2 is illustrated in Scheme 1. We aimed at
demonstrating that these ionic liquids could be reliably prepared by a generalized synthetic route and
therefore amended the previous synthesis by employing 5-hexyn-1-ol as the starting compound and
successfully developed a 5-step synthesis of ionic liquids 1 and 2 in 93% overall isolated yield. First,
the starting alcohol was mesylated using methanesulfonyl chloride in the presence of triethylamine
at ambient temperature, followed by a two-step, one-pot reaction process involved in situ formation
of 6-azidohex-1-yne using sodium azide and subsequently an intramolecular Cu-free, Hüisgen
[3 + 2] cycloaddition in refluxing toluene for 6 h to afford the common 1,2,3-trazole core structure,
4,5,6,7-tetrahydro[1,2,3]triazolo[1,5-a]pyridine, with an excellent 98% isolated yield [14]. The progress
of reactions could be readily monitored by TLC and, once reactions were complete, products
were isolated and purified by straightforward extractions. The next reaction sequence proceeded
with N-alkylation of the 1,2,3-triazole core by bu◦tyl methanesulfonate or ethyl methanesulfonate
◦
under heating conditions (80 C for 18 h and 73 C for 10 h, respectively) to cleanly produce the
corresponding triazolium methanesulfonate salts, followed by metathesis with LiNTf2 in water at
ambient temperature for 13 h to finally produce the desired ionic liquids
yield (95%, in two steps). The overall isolated yield for this 5-step synthesis of both [b-4C-tr][NTf2] (
and [e-4C-tr][NTf2] ( ) ionic liquids was 93% (Scheme 1) [15]. Both ionic liquids obtained are liquids
1 and 2 in excellent isolated
1
)
2
at room temperature. It is of note that this optimized synthesis of ionic liquids
required no chromatographic separation.
1 and 2, in our hands
1. MsCl, Et3N, CH2Cl2, rt, 2 h
OH
N
N
2. NaN3, DMF (1.2 M), rt, 8 h
3. toluene (50 mM), reflux, 6 h
N
98% in 3 steps
1. BuOMs, 80 oC, 18 h
or EtOMs, 73 oC, 10 h
N
NTf2
R
N
N
2. LiNTf2, H2O, rt, 13 h
95% in 2 steps
[R-4C-tr][NTf2]
R = Bu, [b-4C-tr][NTf2], 1
R = Et, [e-4C-tr][NTf2], 2
93% overall yield
Scheme 1. Synthesis of bicyclic 1,2,3-triazolium ionic liquids 1 and 2.
We were interested in incorporating low-melting, hydrophobic [NTf2] anion in
1, 2 primarily
because of its water immiscibility, very low water content and low viscosity [ ]. In addition,
4,5
ionic liquids are known to be excellent microwave absorbers and thus should be useful for
microwave-assisted organic synthesis. Accordingly, this high yielding preparation of ionic liquids
allowed us to expediently investigate their applicability as ionic solvents for synthesis. In this work,
we chose tryptanthrin as the target on which to investigate and support our design that synthesis of
natural products could be promoted in ionic liquids [16].
Tryptanthrin is a biologically active quinazoline alkaloid isolated from a number of fungi
(for instance Schizophyllum commune) and indigo plants (dried roots of Polygonum tinctorium,

Molecules 2016, 21, 1355
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Isatis indigotica and Strobilanthes cusia) commonly known as “Ban Lan Gen” in Traditional
Chinese Medicine [17 18]. In addition to its known antibacterial activity against various
,
pathogenic microbes (e.g., Helicobacter pylori) and recently revealed antitubercular activity against
Mycobacterium tuberculosis [19], tryptanthrin is a potent inhibitor of human cyclooxygenase 2 [20] and
DNA topoisomerase I [21]. All these intriguing biological activities from tryptanthrin justify the study
for its improved synthesis as a useful scaffold for drug discovery and the advanced development as
anti-microbial, anti-inflammatory, and anti-neoplastic agents.
Many syntheses of tryptanthrin were reportedly achieved under basic and refluxing conditions
(e.g., Et3N in refluxing toluene for 16 h) [19,22–30]. As one example, Srinivasan and co-workers
reported the two steps synthesis of tryptanthrin in moderate (54%) isolated yield involving the use of
LDA and starting from anthranilic acid, methyl anthranilate, and ethyl orthoformate [26]. Although
tryptanthrin could also be prepared by following the established Eguchi aza-Wittig reaction protocol,
its overall isolated yield however was low (32%) [31]. In the literature, the most common method for
tryptanthrin synthesis is the reaction of isatoic anhydride with isatin in the presence of equimolar or
excessive amounts of a base [17,18]. Scheme 2 illustrates our efficient microwaves-assisted synthesis of
tryptanthrin carried out in ionic liquids 1 and 2.
O
O
O
base, solvent
N
O
+
O
μ-wave
N
H
N
(100 oC, 30 w)
2 min
N
O
H
O
isatoic anhydride
tryptanthrin
isatin
Scheme 2. Microwave-assisted synthesis of tryptanthrin in ionic liquids 1 and 2.
Table 1 summarizes our results obtained from the synthesis of tryptanthrin of which the middle
two rings were concomitantly assembled from isatoic anhydride reaction with isatin in the presence
or absence of base in both ionic (
work, microwave irradiation was used to promote the double cyclization reactions and the polar,
water-immiscible ionic liquids or were employed to deliberately drive the tryptanthrin-forming
1 and 2) and common organic solvents (DMF and toluene). In this
1
2
reaction to completion since water, once produced, was likely separated from the hydrophobic ionic
liquid phase and rapidly evaporated under heating conditions. After several initial screenings
of experimental conditions, it was quickly revealed that microwave irradiation (30 W) with the
temperature controlled at 100 ◦C produced the best results.
Table 1. Synthesis of tryptanthrin in ionic (1, 2) and molecular (DMF, toluene) solvents a.
Entry
Base
Solvent
Isolated Yield
1
2
3
4
5
6
7
8
9
DIEA
Cs2CO3
DIEA
DIEA
Cs2CO3
–
ionic liquid 1
ionic liquid 1
ionic liquid 2
DMF
DMF
ionic liquid 1
DMF
99%
81%
98%
64%
21%
19% b
6% b
–
DIEA
Cs2CO3
toluene
toluene
no reaction
no reaction
a
Experimental conditions: isatoic anhydride (0.1 mmol), isatin (0.1 mmol), base (0.1 mmol), solvent (50
µL),
microwave at 100 ◦C (30 W) for 2 min. condition: isatoic anhydride (0.1 mmol), isatin (0.1 mmol), solvent
b
(50 µL), microwave at 180 ◦C (30 W) for 10 min.
It was found that with microwaves for a short 2 min, the reaction of isatoic anhydride with
isatin and the base N,N-diisopropylethylamine (DIEA) in ionic liquid
1 cleanly furnished the desired

Molecules 2016, 21, 1355
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natural product tryptanthrin in quantitative isolated yield after chromatography (entry 1, Table 1).
An essentially identical yield (98%) of tryptanthrin synthesis was obtained when performed in ionic
liquid 2 (entry 3, Table 1).
In our hands, the reaction with the inorganic base Cs2CO3 in ionic liquid
1 gave a lower product
yield (81%, entry 2). When carried out in DMF, much lower isolated yields of tryptanthrin were
obtained: 64% (with DIEA as base) and 21% (with Cs2CO3 as base), respectively (entries 4 and 5,
Table 1). These results clearly demonstrated that ionic liquids
1 and 2 are excellent microwave
absorbers, and these ionic solvents outperformed the molecular solvent DMF in the one-pot synthesis
of tryptanthrin. Moreover, under our experimental conditions, toluene, a commonly used solvent for
the synthesis of tryptanthrin, produced no trace of tryptanthrin (entries 8 and 9, Table 1) [19
Although poor isolated yields of tryptanthrin were obtained in the ionic liquid and DMF without
the use of any base, ionic liquid nevertheless produced a higher tryptanthrin yield than DMF: 19%
versus 6%, respectively (entries 6 and 7, Table 1). This result obtained for the synthesis of tryptanthrin
in ionic liquid appears to be consistent with our recent report on the weak Lewis acidic nature of
,22–30].
1
1
1
ionic liquids [32], which likely activates isatoic anhydride to facilitate tryptanthrin synthesis. Figure S1
(Supplementary Materials) illustrates a possible mechanism for the synthesis of tryptanthrin in ionic
liquid without a base. Using thermogravimetric analysis (TGA), Figure S2 (Supplementary Materials)
shows that both ionic liquids
1 and 2
have similar high temperature stability close to 400 ◦C (their
thermal decomposition temperatures at 10% mass loss are 391 ◦C and 382 ◦C, respectively), which
◦
are far above the optimized microwave heating temperature for tryptanthrin synthesis (100 C).
These values are in good agreement with those reported for [NTf2]-based ionic liquids [33].
The success of our general approach to triazolium ionic liquid synthesis and its application for a
one-pot synthesis of the natural product tryptanthrin prompted us further to initiate a preliminary
study to evaluate its use as affinity ionic liquid for both metal ions and peptides. Here, we chose
copper(II) as our initial target ion so that its chemoselective extraction based on molecular recognition
could be facilitated using an affinity ionic liquid
as well as the brain [34], and plays an important role as an endogenous regulator of neuronal activity
in living systems [35 36] and is closely correlated with the pathogenesis of Alzheimer’s disease [37].
3 (AIL 3). The copper ion is abundant in human serum
,
Moreover, exposure to high copper concentrations can cause gastrointestinal disturbances, liver or
kidney damage. Many fluorescence-based Cu(II) chemosensors have been reported and used for
biological applications [38–40]. Scheme 3 illustrates our synthesis of affinity ionic liquid 3.
O
O
Br(CH2)5
O N
N
N
N
N
Zn, 2 N HCl
HONH2●HCl
O
HO
N
HN
85 oC, 2 h
94%
DMF, rt, 1 d
91%
CH2Cl2, rt, 1 h
77%
N
O
N
O
N
N
N
N
N
Br
N
N
N
N
N
70 oC, 1 d
59%
N
Br
N
N
1. TMSCl, CH2Cl2, rt, 15 min
2. LAH, THF, rt, 12 h
LiNTf2, H2O, rt, 12 h
40% in 2 steps
N
N
N
N
NTf2
affinity ionic liquid 3
Scheme 3. Synthesis of affinity ionic liquid 3.

Molecules 2016, 21, 1355
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Our synthesis of AIL 3 began with the Michael addition of hydroxylammonium chloride
to 2-vinylpyridine in DMF at room temperature for 1 d (91% yield), followed by a zinc
reduction under acidic and heating conditions (94% yield) to afford the Cu(II)-recognition element,
N,N-bis(2-(2-pyridyl)ethyl)amine, as a pale yellow oil (85% isolated yield in two steps) [41].
A straightforward amide bond coupling of the resulting bipyridylamine with N-succinimidyl
6-bromohexanoate was then carried out in dichloromethane at room temperature for 1 h (77% yield)
and, after that, the direct alkylation with the bicyclotriazole developed in this work at 70 ◦C for 1 d
(59% yield), a LAH reduction at room temperature for 12 h, and lastly an ion exchange in water at room
temperature for 12 h (40% yield in 2 steps) to ultimately obtain AIL 3. In our hands, the overall isolated
yield in the AIL 3 synthesis was moderate: 16% over a total of six steps starting from 2-vinylpyridine.
This AIL 3 obtained is a viscous, pale yellow liquid at room temperature.
Results of both extraction photos and UV-vis spectra shown in Figure 2 unambiguously
demonstrate that the AIL 3 dissolved in [e-4C-tr][NTf2] ionic liquid
2 chemoselectively recognized
Cu(II) ion in water (the upper layer) and, as a result, effectively extracted it into the ionic liquid
(bottom layer). In these affinity extraction experiments, equal volumes of ionic liquid and water were
first mixed, the resulting mixtures were then vigorously shaken and lastly centrifuged all at ambient
temperature to accomplish the extraction. As shown in Figure 2B,C, Co(II) and Ni(II) ions remained
unaffected in the upper water layer upon extraction, indicating that the partitioning of Co(II) and
Ni(II) into ionic liquid phase containing AIL 3 was negligible as demonstrated in their UV-vis spectra,
and the recognition of AIL 3 toward Cu(II) is clearly chemospecific (Figure 2A).
UV-vis spectra of aqueous phase
before
extraction
after
extraction
(A)
0.5
0.4
0.3
0.2
0.1
0
before
extraction
Cu(OAc)2
after
extraction
400 480 560 640 720 800
wavelength, nm
(B)
0.5
0.4
0.3
0.2
0.1
0
Co(OAc)2
before & after
extractions
400 480 560 640 720 800
wavelength, nm
(C)
0.5
0.4
0.3
0.2
0.1
0
Ni(OAc)2
before & after
extractions
400 480 560 640 720 800
wavelength, nm
Figure 2. Optical photographic images and UV-vis spectra of chemoselective extractions of
) Cu(OAc)2 (0.1 M); ( ) Co(OAc)2 (0.25 M) and ( ) Ni(OAc)2 (0.25 M) in H2O (25 L each) by
AIL 3 (0.25 M) in ionic liquid (25 L), respectively. UV-vis spectra (1 mm path length) of aqueous
layers were obtained by a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA).
(
A
B
C
µ
2
µ

Molecules 2016, 21, 1355
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Moreover, the affinity extraction experiment shown in Figure 3 further proved that, in the presence
of both Cu(OAc)2 (0.1 M) and excessive Co(OAc)2 (0.5 M) in water, the complete transfer of only Cu(II)
ion from the aqueous phase into the ionic liquid was entirely due to affinity recognition by AIL 3
(1.0 M). This chemoselective extraction of Cu(II) ion and total retention of Co(II) were demonstrated in
its UV-vis spectra and also apparent to the naked eyes (Figure 3).
UV-vis spectra of aqueous phase
without
AIL 3
with
AIL 3
0.5
0.4
0.3
0.2
0.1
0
without AIL 3
Cu(OAc)2
+
Co(OAc)2
with AIL 3
400 480 560 640 720 800
wavelength, nm
Figure 3. Optical photographic images and UV-vis spectra of affinity extraction of an aqueous solution
(10 µL) containing Cu(OAc)2 (0.1 M) and Co(OAc)2 (0.5 M) by AIL 3 (1.0 M) in ionic liquid 2 (10 µL).
To determine the stoichiometry of AIL 3-Cu(II) binding interaction, samples having a fixed
concentration of an affinity ligand
various concentrations of Cu(OTf)2 were prepared to perform affinity extractions. As depicted in
Scheme 4, affinity ligand could be readily prepared with good 75% yield by a straightforward Michael
4 (a surrogate of AIL 3) in DMSO/H2O (5:1, v/v) solution and
4
addition of n-hexylamine to 2-vinylpyridine.
N
N
1-hexylamine, HOAc
N
MeOH, reflux, 3 d, 75%
N
4
Cu(II) 4 complex
-
Scheme 4. Synthesis of affinity ligand 4 and the proposed structure of Cu(II)-4 complex.
The results in Figure 4 show that as the ratio of (Cu(II))/(
(i.e., absorbance at 680 nm) of -Cu(II) complex increased until the ratio reached its binding
stoichiometry; beyond this value no more complex could be formed because all has been fully
titrated and associated with Cu(II) ion, and therefore excessive Cu(II) ion remained unbound in the
solution (Figure 4A). This broad band having a max at 680 nm with its growth in intensity was from
the d-d transition of blue Cu(II) complex with affinity ligand 38 40]. The plot of the A680nm vs. the
ratio of (Cu(II))/( ) revealed an abrupt change in the slope, corresponded to the binding stoichiometry
of the system studied; that is, an expected 1:1 stoichiometry of this model -Cu(II) binding interaction
was obtained (Figure 4B). It is likely that the -Cu(II) complex adopt a square-pyramidal coordination
geometry in which affinity ligand binds at the basal plane and two triflates occupy the remaining
sites (Scheme 4) [41 42]. Overall, we were pleased that affinity ionic liquid effectively dissolved and
tightly associated with Cu(II) to form a
4) increased, the concentration
4
4
λ
4
[
–
4
4
4
4
,
3
extracted Cu(II) into ionic liquid
stable 1:1 complex.
2, and its affinity ligand 4

Molecules 2016, 21, 1355
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(A)
2
1.5
1
Absorbance
0.5
0
400 450 500 550 600 650 700 750 800
wavelength, nm
(B)
1.6
1.4
1.2
1
A680nm
0.8
0.6
0.4
0.2
0
0
0.5
1
1.5
2
[Cu(II)] / [4]
Figure 4. Measurement of the stoichiometry of affinity ligand
20, 40, 60, 80, 100, 120, 150, 200 mM) in DMSO/H2O (5:1, v/v) solution. (
length) of samples with various (Cu(II))/( ) molar ratios were scanned, collected and overlapped by a
NanoDrop 2000 spectrophotometer; ( ) A plot of absorbance at 680 nm vs. (Cu(II))/( ) was used
to determine the binding stoichiometry of 4 with Cu(OTf)2.
4
(100 mM) binding to Cu(OTf)2 (0, 10,
A
) UV-vis spectra (1 mm path
4
B
λ
max
4
Figure 5 demonstrates that a complete recovery of Cu(II) ion from ionic liquid phase into the
aqueous layer could be readily achieved by diminishing AIL 3 binding with Cu(II) using a high
concentration (1 M) of HCl. Thus, AIL 3 in ionic liquid 2 could be readily regenerated.
UV-vis spectra of aqueous phase
before
extraction
back extraction 0.1 M CuCl2
using 1 M HCl
solution
0.5
0.4
back
extraction
0.3
Cu(OAc)2
0.2
0.1
0
0.1 M CuCl2
400 480 560 640 720 800
wavelength, nm
Figure 5. Optical photographic images and UV-vis spectra of competitive extraction of Cu(OAc)2
(0.1 M) and AIL 3 (0.25 M) in ionic liquid (25 L) by 1 M HCl in H2O (25 L). A photograph of 0.1 M
CuCl2 aqueous solution (50 µL) was also included for comparison.
2
µ
µ
Since Cu(II)-iminodiacetic acid and related structural motifs are known for their abilities to
bind to histidine residues in peptides and proteins [38 40 43], we envisaged that our AIL 3-Cu(II)
–
,
complex system should be an effective platform for the one-step affinity isolation and purification
of His-containing peptides by AIL 3. Preliminary results of affinity extraction of Cu(II)-chelated
AIL 3 with a dabsylated Dab-RHHHH-NH2 pentapeptide amide shown in Figure 6 clearly indicated

Molecules 2016, 21, 1355
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that AIL 3-Cu(II) complex in ionic liquid
1
quantitatively extracted the peptide from aqueous phase
into the bottom ionic liquid layer. Only with AIL 3 in ionic liquid 1, Dab-RHHHH-NH2 peptide
remained totally unaffected in the upper water layer upon extraction. Furthermore, this peptide could
be conveniently and completely recovered by 1 M HCl (Figure 6).
before
after
extraction
extraction
(a)
without
Cu2+
back extraction
using 1 M HCl
Dab-RHHHH-NH2
peptide
(b)
with
Cu2+
Figure 6. Affinity extraction of the dabsylated His-containing pentapeptide amide Dab-RHHHH-NH2
(1 mM) in 200 mM NaOAc buffer (pH 4.4) containing 10% DMSO (upper layer, 25 L) by AIL 3
(100 mM) solubilized in ionic liquid (bottom layer, 25 L). ( ) are optical photographic images
µ
1
µ
a,b
of peptide extraction into ionic liquid layer without and with chelated Cu2+ (50 mM), respectively.
Back extraction of the peptide in ionic liquid layer into aqueous layer could be conveniently achieved
using 1 M HCl solution (upper layer, 25 µL).
3. Materials and Methods
3.1. General Information
The 1H-NMR and 13C-NMR spectra were recorded at 400 MHz and 100 MHz, respectively on
an Avance DPX 400 spectrometer (Bruker, Billerica, MA, USA) in deuterated solvents (CDCl3 or
1
DMSO-d6) using TMS as the internal standard. The chemical shift (
δ
) for H and 13C are given in
ppm relative to residual signal of the solvent. Coupling constants are given in Hz. The following
abbreviations are used to indicate the multiplicity: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; dd, doublet of doublets; td, triplet of doublets; dt, doublet of triplets; ddd, doublet of
doublet of doublets; brs, broad signal. Microwave irradiation experiments were performed using a
CEM Discover microwave reactor from CEM Corporation (Matthews, NC, USA). The reactions were
monitored using TLC silica gel 60 F254 (Merck, Darmstadt, Germany). Evaporation of solvents was
performed under reduced pressure. Melting points were measured and recorded by the OptiMelt
MPA-100 apparatus (Standford Research Systems, Sunnyvale, CA, USA) and uncorrected. All starting
materials, reagents and solvents were purchased from commercial sources and used as such without
further purification. Unless otherwise stated, all reactions were carried out under inert atmosphere
using anhydrous solvents.
3.2. Synthesis of 4,5,6,7-Tetrahydro[1,2,3]triazolo[1,5-a]pyridine
To a ice chilled solution of 5-hexyn-1-ol (4.77 g, 48.57 mmol) and triethylamine (13.6 mL,
97.14 mmol) in dichloromethane (80 mL) was slowly added solution of methanesulfonyl chloride
(6.68 g, 58.28 mmol) in dichloromethane (40 mL). The resulting solution was aged first for 30 min in
ice bath and then brought to ambient temperature for another 2 h. The reaction solution was washed

Molecules 2016, 21, 1355
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sequentially with citric acid (10 wt %, 3
over Na2SO4. After filtration, the solvent was removed under reduced pressure to afford high purity
product (8.47 g, quantitative yield) with no need for further purification.
×
15 mL), sodium bicarbonate (10 wt %, 3
×
15 mL), then dried
The obtained sulfonate (8.47 g, 48.05 mmol) was dissolved in DMF (40 mL) and then sodium
azide (6.25 g, 96.10 mmol) was added. The substitution reaction was carried out at ambient
temperature for 8 h. The resulting solution was then diluted with toluene (962 mL) and refluxed
for 6 h. After completion of reaction, solvents were removed in vacuo. Dichloromethane (100 mL)
was added and the resulting solution was washed with water (4
dried over Na2SO4, filtered and concentrated under reduced pressure to afford high purity product
×
20 mL). Organic layers was
4,5,6,7-tetrahydro-[1,2,3]triazolo[1,5-a]pyridine as a light brown liquid (5.77 g, 98% yield) with no
1
need for further purification. H-NMR (CDCl3)
δ 1.86–1.97 (m, 2H, CH2), 2.02–2.14 (m, 2H, CH2), 2.84
(t, J = 6.4 Hz, 2H, CH2C=C), 4.37 (t, J = 6.2 Hz, 2H, NCH2), 7.44 (s, 1H, aryl H); 13C-NMR (DMSO-d6)
δ 19.80, 19.82, 22.4, 45.7, 130.3, 133.0.
3.3. Synthesis of Ionic Liquid [b-4C-tr][NTf2] (1)
Solution of methanesulfonyl chloride (5.23 g, 45.66 mmol) in dichloromethane (55 mL) was
slowly added to the ice bathed solution of butanol (2.82 g, 38.05 mmol) and triethylamine (10.6 mL,
76.10 mmol) in dichloromethane (55 mL). The resulting solution was allowed to react in an ice bath for
30 min and then brought to ambient temperature for another 2 h. Sequentially, the reaction solution
was washed with citric acid (10 wt %, 3
×
15 mL), sodium bicarbonate (10 wt %, 3
×
15 mL), then
dried over Na2SO4. After filtration, the solvent was removed under reduced pressure to afford high
purity product (5.77 g, quantitative yield) with no need for further purification. To a round bottle
flask containing 4,5,6,7-tetra-hydro[1,2,3]triazolo[1,5-a]pyridine (4.13 g, 33.52 mmol) was added butyl
◦
methanesulfonate (5.73 g, 37.64 mmol). The alkylation reaction was carried out at 80 C for 18 h.
After completion of reaction, the mixture was allowed to cool and then ether was added to extract and
wash away remaining starting reagents, if any. Ether layer (140 mL in total) was removed and then
the ionic liquid was concentrated under reduced pressure to afford the desired [b-4C-tr][OMs] (8.94 g,
97% yield). To the solution of [b-4C-tr][OMs] (8.94 g, 32.45 mmol) in water (5 mL) was added LiNTf2
(10.59 g, 36.87 mmol). The metathesis reaction was allowed to proceed at ambient temperature for
13 h. After completion of the ion exchange, the solution mixture was extracted with dichloromethane
(3
afford the ionic liquid
0.91 (t, J = 7.4 Hz, 3H, CH2CH3), 1.25–1.37 (m, 2H, CH2CH3), 1.81–1.93 (m, 4H, 2
(m, 2H, CH2), 2.94 (t, J = 6.4 Hz, 2H, CH2C=C), 4.51 (t, J = 6.1 Hz, 2H, NCH2), 4.58 (t, J = 7.1 Hz,
×
5 mL). The combined organic layers were dried over Na2SO4. Evaporation of the solvent in vacuo
, [b-4C-tr][NTf2] (14.54 g, 98% yield). Light brown liquid; 1H-NMR (DMSO-d6)
CH2), 2.03–2.14
1
δ
×
2H, NCH2), 8.68 (s, 1H, aryl H); 13C-NMR (CDCl3)
δ 13.0, 17.8, 19.2, 19.8, 21.2, 30.8, 48.9, 53.6, 119.7
(q, JCF = 319 Hz, CF3), 127.1, 140.4; FAB-HRMS m/z [M]+ calcd for C10H18N3 180.1495, found 180.1501.
3.4. Synthesis of Ionic Liquid [e-4C-tr][NTf2] (2)
Methanesulfonyl chloride (2.59 g, 22.62 mmol) in dichloromethane (25 mL) was slowly added
to the ice bathed solution of ethanol (0.87 g, 18.85 mmol) and triethylamine (5.3 mL, 37.70 mmol)
in dichloromethane (20 mL). The resulting solution was allowed to react in ice bath for 30 min
and then brought to ambient temperature for another 2 h. Sequentially, the reaction solution
was washed with citric acid (10 wt %, 3
×
15 mL), sodium bicarbonate (10 wt %, 3
×
15 mL),
then dried over Na2SO4. After filtration, the solvent was removed under reduced pressure to
afford high purity product (2.03 g, 87% yield) without further purification. To a round bottle
flask containing 4,5,6,7-tetra-hydro[1,2,3]triazolo[1,5-a]pyridine (1.75 g, 14.23 mmol) was added ethyl
methanesulfonate (2.01 g, 16.2 mmol, 1.14 equiv.). The alkylation reaction was performed at 73 ◦C
for 10 h. After completion of reaction, the reaction mixture was allowed to cool and then ether was
added to extract and wash away unreacted starting reagents, if any. Organic layer (80 mL in total)
was then removed and the ionic liquid phase was concentrated under reduced pressure to afford

Molecules 2016, 21, 1355
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[e-4C-tr][OMs] (3.43 g, 98% yield). To the solution of [e-4C-tr][OMs] (3.43 g, 13.88 mmol) in water
(2 mL) was added LiNTf2 (4.38 g, 15.27 mmol). The metathesis reaction was allowed to proceed at
ambient temperature for 13 h. After completion of the ion exchange, the solution mixture was extracted
with dichloromethane. The combined organic layers were dried over Na2SO4. Evaporation of the
solvent in vacuo afford the desired ionic liquid
liquid; 1H-NMR (CDCl3)
1.65 (t, J = 7.5 Hz, 3H, CH3), 1.99–2.09 (m, 2H, CH2), 2.18–2.29 (m, 2H, CH2),
3.03 (t, J = 6.4 Hz, 2H, CH2C=C), 4.51 (t, J = 6.2 Hz, 2H, NCH2), 4.57 (q, J = 7.5 Hz, 2H, CH2CH3), 8.19
2, [e-4C-tr][NTf2] (5.79 g, 97% yield). Light brown
δ
(s, 1H, aryl H); 13C-NMR (CDCl3)
δ 13.9, 17.8, 19.8, 21.1, 48.9, 49.2, 119.6 (q, JCF = 319 Hz, CF3), 126.7,
140.4; EI-HRMS m/z [M]+ calcd for C8H14N3 152.1188, found 152.1183.
3.5. One-Pot Synthesis of Tryptanthrin in [b-4C-tr][NTf2] Ionic Liquid (1)
To a microwave reaction vessel with a magnetic stirring bar was added isatin (14.80 mg,
0.1006 mmol), isatoic anhydride (17.06 mg, 0.1046 mmol), DIEA (13.00 mg, 0.1006 mmol) and
[b-4C-tr][NTf2]
1
(50
µ
L, 2.01 M). The vessel was placed inside a CEM Discover single-mode microwave
◦
synthesizer equipped with a magnetic stirrer, where it was exposed to microwaves at 100 C (30 watts)
for 2 min. The reaction mixture was cooled and then purified by flash column chromatography
(ethyl acetate/hexane = 1:5) to obtain tryptanthrin (24.9 mg, quantitative yield) as yellow crystalline
solid. Yellow crystalline solid; mp 266–268 ◦C; 1H-NMR (CDCl3)
δ 7.43 (t, J = 7.8 Hz, 1H, aryl H), 7.67
(td, J = 7.7, 1.4 Hz, 1H, aryl H), 7.78 (td, J = 7.9, 1.4 Hz, 1H, aryl H), 7.85 (td, J = 7.7, 1.4 Hz, 1H, aryl H),
7.91 (dd, J = 7.0, 1.4 Hz, 1H, aryl H), 7.91 (dd, J = 7.0, 1.4 Hz, 1H, aryl H), 8.03 (dd, J = 8.0, 1.4 Hz, 1H,
aryl H), 8.43 (dd, J = 8.0, 1.4 Hz, 1H, aryl H), 8.62 (d, J = 8.1 Hz, 1H, aryl H); 13C-NMR (CDCl3)
δ 117.9,
121.9, 123.7, 125.4, 127.2, 127.5, 130.2, 130.7, 135.1, 138.2, 144.3, 146.3, 146.6, 158.0, 182.5; EI-HRMS m/z
[M + H]+ calcd for C15H8N2O2 248.0586, found 248.0586.
3.6. Synthesis of Affinity Ionic Liquid 3
To a solution of hydroxylamine hydrochloride (1.0 g, 14.4 mmol) in DMF (2 mL) was added
2-vinylpyridine (2 equiv.). The resulting solution was allowed to react at ambient temperature for
24 h. After the reaction was completed, the reaction mixture was neutralized with sodium bicarbonate
(10 wt %, 20 mL). The product was extracted from the mixture with dichloromethane, and the resulting
organic phase was dried over Na2SO4 and evaporated to dryness. Recrystallization of the residue
from CH2Cl2/hexanes yielded N,N-bis(2-(pyridin-2-yl)ethyl)hydroxylamine (3.10 g, 91% yield) as pale
yellow solid. A mixture of N,N-bis(2-(pyridin-2-yl)ethyl)hydroxylamine (1.2 g, 4.93 mmol) and zinc
powder (1.47 g, 22.4 mol) in 2 N HCl (10 mL) was stirred at 85 ◦C for 2 h. After cooling, the solution
was adjusted to pH 10 using concentrated aqueous ammonia, and the resulting mixture extracted
with CH2Cl2. The organic washings were dried over Na2SO4 and the solvent was removed under
reduced pressure to afford N,N-bis(2-(pyridin-2-yl)ethyl)amine (1.07 g, 94% yield) as a yellow oil. To a
solution of 6-bromohexanoic acid N-hydroxysuccinimide ester (0.72 g, 2.48 mmol) in dichloromethane
was added N,N-bis(2-(pyridin-2-yl)ethyl)amine (2.72 mmol). The resulting solution was allowed to
react at ambient temperature for 1 h, then washed with sodium bicarbonate (10 wt %), brine and
finally dried over Na2SO4. After filtration and solvent evaporation under reduced pressure, the crude
product mixture was purified by flash column chromatography using ethyl acetate as the eluent to
afford the desired amide product (0.432 g, 77% yield) as a pale yellow oil. To a round bottle containing
4,5,6,7-tetrahydro-[1,2,3]triazolo[1,5-a]pyridine (0.77 g, 6.26 mmol) was added the above obtained
amide product (0.72 g, 1.78 mmol). The mixture was stirred at 70 ◦C for 24 h. The resulting slurry
was washed with ether and purified by column chromatography using methanol/dichloromethane
(1:20, v/v) as the solvent to afford the desired ionic salt (0.56 g, 59% yield). To a solution of the
ionic salt (559 mg, 1.06 mmol) in dry CH2Cl2 (2 mL) was added TMSCl (138 mg, 1.27 mmol) in
ice bath. After 15 min, 1 M LAH in THF (5 equiv.) was added slowly to the mixture with syringe.
The resulting solution was allowed to react at ambient temperature for 12 h. After 12 h, the solvent was
removed under reduced pressure to afford solid mixture. Deionized water was added to the resulting

Molecules 2016, 21, 1355
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mixture for quenching LAH in ice bath. After filtration, LiNTf2 (331.5 mg, 1.16 mmol) was added
to filtrate. The reaction was allowed to proceed the ion exchange at ambient temperature for 12 h.
After completion of ion exchange, the mixture was extracted with CH2Cl2, the organic phases were
combined and dried with Na2SO4. After filtration and solution concentration, the mixture purified
using silica column chromatography using methanol/dichloromethane (1:10, v/v) as the solvent to
1
give the targeted affinity ionic liquid
3
(0.28 g, 40% yield in two steps) as a yellow oil. H-NMR (CDCl3)
δ
1.39–1.55 (m, 4H, CH2), 1.80–1.93 (m, 2H, CH2), 1.97–2.10 (m, 4H, CH2), 2.17–2.27 (m, 2H, CH2),
3.01 (t, J = 6.4 Hz, 2H, CH2), 3.21–3.30 (m, 4H, CH2), 3.30–3.37 (m, 2H, CH2), 3.61–3.72 (m, 4H, CH2),
4.48–4.59 (m, 4H, CH2), 4.54 (t, J = 6.2 Hz, 2H, CH2), 7.20–7.21 (m, 2H, aryl H), 7.24 (d, J = 8.0 Hz, 2H,
aryl H), 7.67 (td, J = 7.6, 1.5 Hz, 2H, aryl H), 8.14 (d, J = 4 Hz, 2H, aryl H), 8.22 (s, 1H, aryl H); 13C-NMR
(CDCl3)
δ 17.8, 19.9, 21.2, 22.1,25.1, 25.6, 28.5, 29.3, 48.9, 51.5, 52.2, 53.8, 119.48 (q, JCF = 320 Hz,
CF3), 122.6, 123.8, 127.3, 137.6, 140.5, 148.4, 156.9; ESI-HRMS m/z [M]+ calcd for C26H37N6 433.3080,
found 433.3084.
3.7. Synthesis of 6-Bromohexanoic Acid N-Hydroxysuccinimide Ester
To a round bottom flask with a stir bar, 6-bromohexanoic acid (1.0 g, 5.12 mmol),
N-hydroxysuccinimide (0.89 g, 7.69 mmol), 4-dimethylaminopyridine (0.16 g, 1.02 mol), and CH2Cl2
(12 mL) was added. The solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(1.18 g, 6.14 mmol) in dichloromethane (30 mL) added in small portions solution to the round bottle in
ice bath, then the mixture was allowed to react at ambient temperature for 2 h. When the reaction was
completed, washed sequentially with citric acid (10 wt %), sodium bicarbonate (10 wt %) and brine.
The organic washings were dried over Na2SO4 and the solvent was removed under reduced pressure
to afford 6-bromohexanoic acid N-hydroxysuccinimide ester (1.27 g, 85% yield) as a white solid.
3.8. Synthesis of N,N-Bis(2-(pyridin-2-yl)ethyl)hexan-1-amine (4)
To a solution of n-hexylamine (3.25 g, 3.21 mmol) in MeOH (2 mL) was added 2-vinylpyridine
◦
(2 equiv.) and acetic acid (3 equiv.). The resulting solution was allowed to reflux at 65 C for
72 h. After the reaction was completed, the solvent and 2-vinylpyridine were removed under
reduced pressure. The mixture product was adjusted to pH 10 using concentrated aqueous
NaOH (1 M), and the resulting mixture was extracted with ethyl acetate. The resulting organic
phase was dried over Na2SO4. After filtration and solvent evaporation under reduced pressure,
the crude product mixture was purified by flash column chromatography to afford affinity ligand
1
N,N-bis(2-(pyridin-2-yl)ethyl)hexan-1-amine (
4
, 0.7534 g, 75% yield) as a brown oil. H-NMR (CDCl3)
δ
0.87 (t, CH3, 3H, J = 7.0 Hz), 1.12–1.35 (m, CH2, 6H), 1.35–1.49 (m, CH2, 2H), 2.54 (t, CH2, 2H,
J = 7.6 Hz), 2.93 (bs, CH2, 8H), 7.01–7.15 (m, aryl H, 4H), 7.56 (td, aryl H, 2H, J = 7.2, 1.2 Hz), 8.52
(d, aryl H, 2H, J = 3.2 Hz); 13C-NMR (CDCl3)
δ 14.0, 22.6, 27.1, 27.2, 31.8, 35.9, 54.0, 54.0, 121.0, 123.3,
136.1, 149.1, 160.8; ESI-HRMS m/z [M + H]+ calcd for C20H30N3 312.2440, found 312.2444.
3.9. Binding Stoichiometry Measurement of Cu(OTf)2 with Affinity Ligand 4
Stoichiometric titrations were performed with 200 mM affinity ligand
4
(10
µL) in DMSO/H2O
solution (5:1, v/v). The solution of affinity ligand was titrated with an aqueous solution (10
4
µL)
containing copper triflate (0–400 mM) in DMSO/H2O solution (5:1, v/v). Upon mixing, the resulting
solutions were analyzed by UV-Vis spectrophotometry (NanoDrop 2000, Thermo Scientific, Waltham,
MA, USA). The mixture solutions exhibit a new absorption band at
λ = 680 nm. The absorbance
(680 nm) of resulting solutions were plotted vs. the ratio of (Cu(II))/(affinity ligand 4).
3.10. Mass Spectrometric Analysis of Cu(II)-Affinity Ligand 4 Complex
To a microtube with affinity ligand
triflate (3.1 mg, 8.63 mol). The mixture was allowed to react at ambient temperature for 10 min,
followed by washing with ether three times. The solvent of the resulting mixture was removed
4 (2.7 mg, 8.63 µmol) in methanol (1 mL) was added copper
µ

Molecules 2016, 21, 1355
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under reduced pressure to afford the Cu complex as brown oil. This sample was analyzed by ESI-MS
ESI-HRMS m/z [M + Na]+ calcd for C22H29N3O6F6Na1S2Cu1 695.0596, found 695.0595.
3.11. Affinity Extraction of Cu(OAc)2 by AIL 3 and Its Back Extraction
To a microtube containing AIL 3 (250 mM) in [e-4C-tr][NTf2] ionic liquid
2 (25 µL) was added
copper acetate (100 mM, 25 L). The two phases were vigorously shaken to extract metal ion.
µ
The mixture was then centrifuged to induce phase separation. The upper layer was measured to
determine the concentration of copper acetate remained in aqueous layer by UV-vis spectrophotometry
(NanoDrop 2000). The upper aqueous layer was removed and 1 M HCl solution (25 µL) was added
to the tube for back extraction. The mixture solution was vigorously shaken and then centrifuged to
induce phase separation. The upper layer was then measured to determine the concentration of copper
chloride in the aqueous layer using the NanoDrop 2000 UV-Vis spectrophotometer.
3.12. Affinity Extraction of Cobalt Acetate and Nickel Acetate
To a microtube containing AIL 3 (250 mM) in [e-4C-tr][NTf2] ionic liquid
2 (25 µL) was added
cobalt acetate or nickel acetate (250 mM, 25 L). The two phases were vigorously shaken to extract
µ
metal ion. The mixture was then centrifuged to induce phase separation. The upper layer was
measured to determine the concentration of cobalt acetate or nickel acetate remained in aqueous layer
by UV-vis spectrophotometry (NanoDrop 2000).
3.13. Regeneration of AIL 3
To a microtube with Cu(II)-chelated AIL 3 in ionic liquid
solution (25 L). This solution was vigorously shaken and then centrifuged to induce phase separation.
The upper aqueous solution was removed and an aqueous solution containing 0.1 M Li2CO3 (100 L)
was added to wash the bottom ionic liquid layer five times continuously. After the final Li2CO3
aqueous solution was removed, the AIL 3 in ionic liquid was regenerated and ready for experiments.
2 (25 µL) was added 1 M HCl aqueous
µ
µ
2
3.14. Affinity Extraction of Dabsylated Pentapeptide by Cu(ll)-Chelated AIL 3 and Back Extraction
To a microtube containing AIL 3 (100 mM) in [b-4C-tr][NTf2] ionic liquid (25 L) was added
copper acetate (50 mM) in water (25 L). The two phase were vigorously shaken to extract metal ion,
1
µ
µ
then the mixture was centrifuged to induce phase separation. After removing the upper aqueous
layer, the dabsylated pentapeptide amide solution (1 mM) in 0.2 M NaOAc buffer (pH 4.4) containing
10% DMSO (25 µL) was added to microtube. After extraction and centrifuge, the upper layers were
measured to determine the concentration of dabsylated pentapeptide in aqueous layer by UV-vis
spectrophotometry. The upper aqueous solution was removed and the 1 M HCl solution (25 µL) was
added to the microtube for back extraction. The solution was vigorously shaken and then centrifuged
to induce phase separation. The upper HCl solution was turned red color indicating the peptide being
successfully back-extracted.
4. Conclusions
We have developed a high yielding synthesis of bicyclic 1,2,3-triazolium ionic liquids that appear
to fulfill practical requirement as solvents for chemical reactions. It is noted that this general synthesis
established a common bicyclotriazole core so that derivatives of triazolium ionic liquids could be
prepared, characterized and studied. Ionic liquids
as ionic solvents for the synthesis of tryptanthrin and, likely, other natural and non-natural products.
Lastly, an affinity ionic liquid was synthesized and used to demonstrate its value in affinity extraction
1 and 2 were used to demonstrate their usefulness
3
of both Cu(II) ion and His-containing peptides. Binding interaction study of AIL 3-Cu(II) complex
with various biomolecules as well as experiments on reuse ability of our affinity system are actively
being pursued and the result will be reported in due course.

Molecules 2016, 21, 1355
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Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
10/1355/s1.
Acknowledgments: We gratefully acknowledge support of this work by grants from the Ministry of Science and
Technology of Taiwan, ROC (MOST 103-2113-M-194-002-MY3). We thank Ming-Chung Tseng for useful discussion.
Author Contributions: Y.-H.C. conceived and designed the experiments; H.-Y.L. and C.-Y.C. performed the
experiments; H.-T.C. performed initial experiments; Y.-H.C. analyzed the data and wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
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