Rapid chiral analysis based on liquid-phase cyclic chemiluminescence

Article abstract of DOI:10.1039/d0sc03496g

Rapid chiral analysis has become one of the important aspects of academic and industrial research. Here we describe a new strategy based on liquid-phase cyclic chemiluminescence (CCL) for rapid resolution of enantiomers and determination of enantiomeric excess (ee). A single CCL measurement can acquire multistage signals that provide a unique way to examine the intermolecular interactions between chiral hosts and chiral guests, because the lifetime (τ) of the multistage signals is a concentration-independent and distinguishable constant for a given chiral host-guest system. According to the τ values, CCL allows discrimination between a wide range of enantiomeric pairs including chiral alcohols, amines and acids by using only one chiral host. Even the chiral systems hardly distinguished by nuclear magnetic resonance and fluorescence methods can be distinguished easily by CCL. Additionally, the τ value of a mixture of two enantiomers is equal to the weighted average of each enantiomer, which can be used for the direct determination of ee without the need to separate the chiral mixture and create calibration curves. This is extremely crucial for the cases without readily available enantiomerically pure samples. This strategy was successfully applied to monitoring of the Walden inversion reaction and analysis of chiral drugs. The results were in good agreement with those obtained by high-performance liquid chromatography, indicating the utility of CCL for routine quick ee analysis. Mechanism study revealed that the τ value is possibly related to the activity of the chiral substance to catalyze a luminol-H2O2 reaction. Our research provides an unprecedented and general protocol for chirality differentiation and ee determination, which is anticipated to be a useful technology that will find wide application in chirality-related fields, particularly in asymmetric synthesis and the pharmaceutical industry.

Full text of DOI:10.1039/d0sc03496g

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PleaseC dh oe mn oi ct a al dS cj ui es nt cme argins
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DOI: 10.1039/D0SC03496G
ARTICLE
Rapid Chiral Analysis Based on Liquid-Phase Cyclic
Chemiluminescence
Runkun Zhang, Yanhui Zhong, Zhenyu Lu, Yanlong Chen, and Gongke Li*
Received 00th June 2020,
Accepted 00th June 2020
Rapid chiral analysis has become one of the important aspects of academic and industrial research. Here we describe a new
strategy based on liquid-phase cyclic chemiluminescence (CCL) for rapid resolution of enantiomers and determination of
enantiomeric excess (ee). A single CCL measurement can acquire multistage signals that provide a unique way to examine
the intermolecular interactions between chiral hosts and chiral guests, due to the lifetime (τ) of the multistage signals is a
concentration-independent and distinguishable constant for a given chiral host-guest system. According to the τ values, CCL
allows discrimination between a wide range of enantiomeric pairs include chiral alcohols, amines and acids by using only
one chiral host. Even the chiral systems hardly distinguished by nuclear magnetic resonance and fluorescence method can
be distinguished easily by CCL. Additionally, the τ value of the mixture of two enantiomers is equal to the weighted average
of each enantiomer, which can be used for direct determination of the ee without the need to separate the chiral mixture
and create calibration curves. It is extremely crucial for the cases without readily available enantiomerically pure samples.
This strategy was successfully applied to monitoring of Walden inversion reaction and analysis of chiral drugs. The results
were in good agreement with those measured by high-performance liquid chromatography, indicating the utility of CCL for
routine quick ee analysis. Mechanism study revealed that the τ value is possible related to the activity of chiral substance to
DOI: 10.1039/x0xx00000x
2 2
catalyze luminol-H O reaction. Our research provides an unprecedented and general protocol for chirality differentiation
and ee determination, which is anticipated to being a useful technology that find wide applications in chirality-related fields,
particularly in asymmetric synthesis and pharmaceutical industry.
Introduction
Chirality is a universal phenomenon in nature, most attracted considerable research interest as a result of their
biological species, such as amino acids, nucleic acid, proteins, advantages of rapidity, simplicity, and low cost. In such a
drugs, are chiral and intermolecular chiral-chiral interactions strategy, the molecules or materials containing the chiral unit
1
-3
occur ubiquitously among biological processes.
The are used as chemosensory hosts that can interact with the chiral
simultaneous determination of the absolute configuration and targets and then induce distinguishable optical signals.2
1
enantiopurity of chiral substrates is highly significant in the Although the great efforts and achievements that have been
4
-6
fields of synthetic, biological and pharmaceutical chemistry.
made in this field, some problems still remain to be solved. For
However, chiral analysis remains a great challenge due to instance, most methods suffer from narrow substrate scope
7
28
enantiomers have nearly identical physical properties.
and limitedly differentiable enantiomers. In addition, the
In the past several decades, many effective methods have output signal for chiral discrimination is strongly influenced by
been developed for the chiral analysis, such as gas the analyte concentration, this would normally need two
8
,9
chromatography, high-performance liquid chromatography independent methods to simultaneously determine both
HPLC),1
0,11
capillary electrophoresis,
(NMR)
18,19]
12-14
nuclear magnetic enantiomeric configuration and enantiopurity.
28,29
Especially,
mass these assays usually require highly pure samples to construct
curves for enantiomeric excess (ee)
It would be particularly significant to
(
7
,15-17
resonance
spectrometry.[
spectroscopy
and
However, these methods are time- calibration
4
,30,31
consuming and technically complex, and the instrumentation determination.
required is expensive, bulky, and requires well-trained acquire a concentration-independent parameter for chiral
2
0,21
operators.
dichroism,2
Optical sensing methodologies, such as circular discrimination and enantiopurity determination, due to such
2-24
25,26
27
fluorescence
and colorimetry
have technique can simplify and speed up the measurement, and
thereby find wide applications in rapid screening of ee for
asymmetric synthesis and pharmaceutical industry etc. In this
paper, we report an unprecedented strategy of liquid-phase
cyclic chemiluminescence (CCL) that does not suffer from the
a. School of Chemistry, Sun Yat-sen University, Guangzhou 510275, People’s
Republic of China；E-mail: cesgkl@mail.sysu.edu.cn
Electronic Supplementary Information (ESI) available: Fabrication and working
principle of liquid-phase CCL system; method for immobilization of luminol; Fig.S1-
S10 and Table S1-S5. See DOI: 10.1039/x0xx00000x
above limitations in chiral analysis. To the best of our
knowledge, this paper is the first to report
a
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Journal Name
chemiluminescence-based sensing methodology that providing by reactive oxygen species to form a 3-aminophthalate dianion
View Article Online
DOI: 10.1039/D0SC03496G
a concentration-independent constant for the simultaneous in an excited state, which emitted light when returning to the
4
0
determination of enantiomeric configuration and ee value.
ground state. Specifically, a circular pipeline was designed for
As an important optical analytical technology, CCL system, and the direction of the carrier in CCL system can
chemiluminescence (CL) have been widely used for be changed automatically at the indicated cycle period. Through
3
2
33-35
pharmaceutical analysis, clinical analysis
and biological automatically changing the carrier direction at the indicated
analysis.3
6,37
However, typical CL kinetic curve lacks the period, the chemical species at different stages flowed through
characteristic features of the analytes. To overcome this the sensor cell repeatedly to induce the CL reactions proceed in
drawback, our group proposed a new concept of cyclic succession. Multistage signals that satisfy the exponential decay
chemiluminescence (CCL) for rapid discrimination of law were measured over time. The design of CCL system
3
8
structurally similar compounds. In contrast to conventional extends the observable time, and thereby the distinctions
flow CL system, the direction of the carrier in the CCL system is between different luminous reactions that usually are not easy
in a state of periodical change, which triggers a succession of CL to observe by conventional CL-based detection, but can be
reactions in a single sample introduction to produce multistage observed by CCL detection. The flow paths of liquid-phase CCL
signals. We found that the multistage signals on CCL kinetic detection at different stages are shown in the ESI (Fig. S1).
curve satisfy the first-order exponential decay law:
For chiral analysis, chiral metal-organic complexes that are
able to enhance the luminescence intensity of luminol-H reaction
were used as hosts, new complexes were formed via chiral–

t
2 2
O

I  Ae  I
(1)
n
0
where I
n
is the signal of n th stage reaction, and I
0
is the chiral interactions between chiral host and chiral guest when
background noise. t is the peak time. A is the pre-exponential chiral guests were mixed with chiral hosts. The τ values of the
factor that stands for the maximum luminescence intensity. τ CCL kinetic curves of luminol-H reaction catalyzed by the
2 2
O
represents the lifetime of the multistage signals, it is the time at new complexes were use for chiral analysis. The schematic
which the intensity of the assembly is reduced to 1/e times its representation of liquid-phase CCL for chiral analysis is shown
initial value, its unit is "second". We demonstrated each given in the Fig. 1.
CCL reaction has a unique constant of τ. The distinguishable τ
values allow facile identification of various analytes include
homologous series and structural isomers. Recently, we further
demonstrated that the multistage signals of multicomponent
mixture also satisfy the first-order exponential decay law with
their unique τ vaues.3 This allows facile identification of mixed
9
samples consisting of complex components, such as liquor,
beer, toner, and baby powder. However, our previous
researches were limited to studying the gaseous-phase CL
emitted on nanosized catalysts, and a wider adaptation for the
application of CCL has not been demonstrated yet. Following
our research interest in demonstrating the generality of CCL and
exploring its further application, here, we extend the CCL
strategy to liquid-phase reactions via immobilizing luminous
2 2
reagent, and metal-organic complex-catalyzed luminol-H O
reaction system was used as basic model to probe the
application of CCL in chiral analysis. The most distinct advantage
of liquid-phase CCL is that it provides a concentration-
independent constant (τ value) to discriminate a wide range of
enantiomers, and allows direct determination of the
enantiopurity without the need to separate the chiral mixture,
and thereby the use of enantiomerically pure samples for
creating calibration curves can be avoided.
Fig. 1. The schematic representation of liquid-phase CCL for chiral analysis.
Results and discussion
Resolution of enantiomers
Experimental section
The fabrication of liquid-phase CCL system can be seen in
the ESI. In the present work, immobilized luminol (5-amino-2,3-
dihydro-1,4-phthalazinedione, immobilized method was
detailed in the ESI) was packed into a homemade flow-through
2 2
reactor to fabricate a sensor cell. H O was used as a carrier and
oxidant, and it was catalyzed by a sample containing a metal-
organic complex to produce reactive oxygen species.
Subsequently, immobilized luminol in a sensor cell was oxidized
To examine the feasibility of our strategy for chiral analysis,
three pairs of commercially available chiral metal-organic complexes
(
R1 and S1, R2 and S2, R3 and S3, the detail information of these
chiral complexes can be seen in Table S1 of the ESI) were used as
hosts. The original CCL kinetic curves of luminol-H system
2 2
O
catalyzed by these chiral metal-organic complexes are shown in the
ESI (Fig. S2). It shows that the two enantiomers of one chiral
substrate of metal-organic complex have the same τ value when
without adding chiral guests. This because the differences between
2
| J. Name., 2012, 00, 1-3
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ARTICLE
enantiomers are difficult to observe under normal circumstance, but constant that characteristic of a given chiral system, wVhieicwhAfrtuicnlecOtinolinnes
these differences become much more marked in a chiral as characteristic labelling for directly deteDrmOIi:n1i0n.g103th9e/D0idSeCn0t3it4y96oGf
environment. Thus, chiral guests were added into the solutions of enantiomers.
chiral hosts. We found that additions of (R)-(+)-1-phenylethanol and
(
(
S)-(-)-1-phenylethanol into R1 outputting τ values of 64.6 and 39.0 s
Fig. 2a), respectively. τ values of 35.2 and 51.6 s were observed
when added (R)-(+)-1-phenylethanol and (S)-(-)-1-phenylethanol into
S1 (Fig. 2b).
We further investigated the effects of the concentration of
guest on response behaviors of host at a much broader
concentration range. We have plotted the τ values of R1 and S1
versus the increasing concentrations of (R)-(+)-1-phenylethanol and
(S)-(-)-1-phenylethanol in Fig. 2c and Fig. 2d, respectively. We found
that the τ value is independent of the concentration of the chiral
guest in the range of 0.005 to 0.5 mmol/L but is only dependent on
the chiral configuration. In addtion, the τ value also is independent
of the H
discussion can be seen in the ESI). These results demonstrate that Fig. 2. (a) The CCL kinetic curves of luminol-H
each chiral system has a unique and constant τ value, and the phenylethanol enantiomers; (b) the CCL kinetic curves of luminol-H
2 2
O concentration within a certain range (Fig. S3, detailed
2
O
2
system catalyzed by R1 with 1-
system
2 2
O
distinction in τ value confirms the chiral discrimination. Quite catalyzed by S1 with 1-phenylethanol enantiomers; (c) Plots of τ value for R1 (0.1
different from other sensing methodologies based on intensity mmol/L) in the presence of varying concentrations of 1-phenylethanol; (d) Plots of
quenching/enhancement or spectral changes, the parameter (τ τ value for S1 (0.1 mmol/L) in the presence of varying concentrations of 1-
value) of CCL for chiral analysis is a concentration-independent phenylethanol. The concentration of H
2 2
O is 0.05 mmol/L.
Fig.3. The τ values of different chiral host-guest systems. The concentration of all hosts and guests is 0.1 mmol/L, the concentration of H
2 2
O is 0.05 mmol/L. The unit of
τ is second.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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Chemical Science
Page 4 of 18
The reproducibility of the immobilized luminol was investigated by
Noteworthy, each chiral host-guest system has a corresponding
measure of (R)-(+)-1-phenylethano using S1. We found that the τ value, that is: different hosts show different values
background noise increases and luminescence intensity (A) guest, and the same host exhibits different values of τ to different
V
o
i
f
ew
τ
A
t
r
o
tic
a
le
g
O
i
n
v
line
e
n
DOI: 10.1039/D0SC03496G
decreases gradually with increasing testing number (ESI, Fig. S4), but guests. The above results demonstrate that each chiral host-guest
the τ could remain stable within 25 replicates with a RSD of 3.7% (ESI, system has a corresponding exponential decay equation with a
Fig. S5a). It means that τ has a good stability, possibly due to τ unique τ value. Thus, we can establish a database of τ values of
depends on the rate of luminescence depletion that related to the various chiral host-guest systems in future, which provides
characteristics of the reaction, but not the apparent intensity. The τ facilitation for quick recognition of unknown chiral compounds. For
values measured by 5 different batches of immobilized luminol example, if the τ values of an analyte were measured to be 64.6 and
vary between 4.2% (ESI, Fig. S5b). The sufficient stability of τ within 35.2 s when using R1 and S1 as hosts, respectively, the identity of the
the same batch and between different batches of immobilized analyte could be confirmed to be (R)-(+)-1-phenylethanol by
luminol make CCL is qualified for chiral differentiation. It is worth searching database.
noting that the A value is easily fluctuant and the τ value depends on
the decay rate of multistage signal, and thereby the multistage signal satisfactory enantio-differentiating ability of our strategy. Usually,
can be normalized for nonlinear curve fitting to obtain τ value. we can discriminate most enantiomeric pairs by only using one chiral
Fig. 3 illustrates the broad substrate applicability, and the
An ideal method for chiral sensing should be capable of host. However, the τ values of some chiral compounds on certain
discrimination of different kinds of chiral molecules accompanied host are close. For example, although host S2 allows discrimination
2
1
with the resolution of the two enantiomers of one chiral substrate.
between enantiomers of 1-phenylethanol or 2-pentanol, it unable to
However, current methods usually suffer from narrow substrate resolve (S)-(-)-1-phenylethanol (τ=40.4 s) and (S)-(+)-2-pentanol
1
5
scope and limitedly differentiable enantiomers. We next turned (τ=40.5 s). Our aim is to realize unambiguous discrimination among
attention to demonstrate the ability of CCL to discriminate a wide a large variety of chiral compounds, not just limits to the two
range of enantiomers with broad substrate applicability. The chiral enantiomers of one chiral substrate. In consideration of the cross-
metal-organic complexes shown in the ESI (Table S1) were used as reactivity of the hosts, we combined all the hosts (R1, S1, R2, S2, R3
hosts to sensing two pairs of chiral alcohols, chiral amines and chiral and S3) to construct a sensor array, expecting to realize
acid, respectively. As expected, each enantiomer can be correlated unambiguously discriminate various chiral compounds. Three
to a distinct τ value when it is mixed with a given chiral host (Fig. 3), concentrations (low: 0.005 mmol/L, medium: 0.05 mmol/L and high:
enabling easy discrimination of chiral compounds. For example, 0.1 mmol/L) of 12 chiral compounds were tested by the sensor array,
when chiral complex R1 was used as a chiral host, τ values of 64.6 the resulting τ values are summarized in the ESI (Table S2). For easier
and 39.0 s were observed for (R)-(+)-1-phenylethanol and (S)-(-)-1- visualization, the τ value of each response is depicted in the form of
phenylethanol; 52.7 and 38.2 s for (R)-(-)-2-pentanol and (S)-(+)-2- a heat map according to the scale bar on the right. As shown in Fig.
pentanol; 58.3 and 46.5 s for (R)-(-)-2-phenylglycinol and (S)-(+)-2- 4a, each of the chiral guests creates a unique multidimensional
phenylglycinol; 12.1 and 21.4 s for (R)-3-amino-3-phenylpropan-1-ol response pattern, and different concentrations of the same chiral
and (S)-3-amino-3-phenylpropan-1-ol; 49.6 and 73.9 s for D-(-)- guest induce similar response pattern. Linear discriminant analysis
tartaric acid and L-(+)-tartaric acid; 31.6 and 23.7 s for D-(-)-mandelic (LDA) was then used to further digitize and visualize the response
acid and L-(+)-mandelic acid, respectively. Satisfactory resolution of patterns (6 host units × 12 chiral guest × 3 concentrations). Fig. 4b
the two enantiomers of one chiral substrate is easily achieved by shows the 3-D discrimination plot constructed by the first three
simply comparing the τ values.
factors. The first three factors account for 85.5% of the total variance.
Fig. 4. (a) Response patterns of 6 chiral hosts to 12 guests at different concentrations. 0.005, 0.05 and 0.1 strand for the concentrations of the guests, the unit is mmol/L;
b) LDA 2D plot derived from the colour response pattern. A: (R)-(+)-1-phenylethanol; B: (S)-(-)-1-phenylethanol; C: (R)-(-)-2-pentanol; D: (S)-(+)-2-pentanol; E: (R)-(-)-2-
phenylglycinol; F: (S)-(+)-2-phenylglycinol; G: (R)-3-amino-3-phenylpropan-1-ol; H: (S)-3-amino-3-phenylpropan-1-ol; I: D-(-)-tartaric acid; J: L-(+)-tartaric acid; K: D-(-)-
(
2 2
mandelic acid; L: L-(+)-mandelic acid. The concentration of all host is 0.1 mmol/L, the concentration of H O is 0.05 mmol/L.
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Page 5 of 18
Chemical Science
The same colored symbol within the LDA graphical output assigns to percentages of 1-phenylethanol enantiomers, and the trends of the
specific chiral guests. These chiral guests are classified into 12 groups τ value of the mixture (τ ) versus the percentagesVie
with 100% accuracy, and small spatial distance are observed within phenylethanol and (S)-(-)-1-phenylethanol were plotted in Fig. 5,
the same group. This demonstrates the satisfactory discrimination respectively. Linear-regression analysis shows that the τ is linearly
capacity of the sensor array based on CCL strategy. related to the percentages of the enantiomers in the mixture.
m
o
w
f
Ar
(
t
R
ic
)
le-(
O
+
n
)-1-
l
in
e
DOI: 10.1039/D0SC03496G
m
NMR spectroscopy and fluorescence spectroscopy are common Further study found that the slope of the linear regression equation
technique used for chiral discrimination. However, we found that the is equal to difference between the τ values of the two enantiomers,
R1 or S1 with 1-phenylethanol enantiomers (ESI, Fig. S6). The same and the intercept equals the τ value of one of the enantiomers. The
chiral systems also cannot be resolved by fluorescent method, due
m
τ of the mixture composed by arbitrary proportions of enantiomers
to R1, S1 and 1-phenylethanol are non-fluorescent substances. These can be expressed as:
results demonstrate that CCL can distinguish some chiral host-guest
m  (R) ((R) (S ) ) (S )     (1(R) )(S )
(2)
(R) (R)
systems, which would be difficult for NMR and fluorescence
spectroscopy. Compared with the method for chiral discrimination
Or can be presented in the following form:
m   ( (R) ) (R)  (1 )   
4
1
using QDs enhanced-CL system, the commercial chiral metal-
organic complexes used in the present work are easy available. In
addition, they allow facile discrimination between a wide range of
enantiomeric pairs. It is well known that static injection CL can
provide the full intensity kinetic curve. However, we found that the
CL kinetic curves obtained by static injection lack enough
discriminability to differentiate chiral compounds, mainly due to the
short lifetime of the CL kinetic curve. The τ values of different kinetic
curve obtained by static injection are in the in the range from 1.0 to
(3)
(S )
(S )
(S )
(R)
(S ) (S )
Where ω(R) and ω(S) are the percentages of the two enantiomers
in the mixture, and τ(R) and τ(S) stand for the τ values of the two
enantiomers, respectively. Eqs 2 and 3 indicate that the τ
m
is the
weighted average of the two enantiomers, which is in accordance
39
with the gaseous-phase CCL detection. According to eqs 2 and 3,
ω
(R) and ω(S) in the mixture can be presented by:
m -(S )
5
.6 s (ESI, Fig. S7), the narrow τ-range means low resolution. The
(R)
(S )


(4)
(5)
detailed discussion also can be seen in the ESI. Thus, CCL will be a
powerfully complementary technology for chiral discrimination.
(R) (S )
(R)
-m
(R)
(S )
Determination of ee
The ee (%) is defined as:46
Determination of ee is performed routinely in many scientific
fields, especially in asymmetric synthesis and pharmaceutical
ee  (R) (S )
(6)
4
1-44
industry.
Chiral separation is the most common method for ee
by integrating eqs 3, 4 and 5, the ee can be calculated by:
determination. However, chiral separation is cost-intensive and
time-consuming process due to the long analysis time and the use of
2 - -(S )
m
(R)
2
1,45
ee 
(7)
expensive chiral column.
Simple and inexpensive techniques for
(R)
(S )
rapid determination of ee is attractive for their potential applications
in academic and industrial fields. In order to probe the application
value of our strategy in rapid determination of ee, R1 and S2 was
used to measure a series of mixtures consisting of different
Eqs 4, 5 and 7 indicate that if the τ values of the enantiomers
are known in advance (through searching database), the percentages
of
Fig. 5. The relationship between τ value and the percentages of 1-phenylethanol enantiomers using R1 (a) and S1 (b) as hosts.
the two enantiomers and the ee value can be directly determined by in the ESI compare the calculated versus the actual ee values,
measuring the τ value of the mixture without the need to use excellent linear relationships (regression coefficients exceed 0.998)
enantiomerically pure samples for creating calibration curves. Fig. S8 with slopes close to 1 were obtained. The absolute errors between
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Journal Name
the calculated and actual ee values ranged from -6.9% to 4.6% (ESI, shown in Fig. 6. The general shape of the curves obtained by the two
View Article Online
DOI: 10.1039/D0SC03496G
Table S3).
methods is surprisingly similar, showing the conversion of (S)-(-)-1-
phenylethanolfollows first order reaction kinetics. The relative
content of (S)-(-)-1-phenylethanol steadily decreases, while the yield
of (R)-(+)-1-phenylethanol steadily increases with the conversion of
Applications
To validate the utility of CCL in enantiopurity determination, an
amberlyst-catalyzed Walden inversion was selected as a prototype
reaction because of its industrial application in production of
pharmaceutical and agrochemical building blocks. 1.0 g of
amberlyst-H was added to 100 mL solution of (S)-(-)-1-phenylethanol
(
S)-(-)-1-phenylethanol. Complete substrate conversion within 240
min was observed. The half-lives calculated by CCL (t1/2(CCL)) and HPLC
(t1/2(HPLC)) are in good agreement. The ee values at different reaction
times measured by the two methods are listed in the ESI (Table S5).
The CCL determined ee values correlated well with chiral HPLC
results with absolute errors ranging from -7.1% to 7.0%%.
4
7
o
at 0.2 mmol/L, duplicate the above solutions were heated to 55 C
o
and 65 C under stirring condition, respectively. At various times, 2
It is worth noting that during the processes of the above assays,
the analytical time of each HPCL measurement was 13 min (ESI, Fig.
S9), the total time spent on HPCL detections was about 377 min (13
min of each detection × 7 standard solutions + 13 min of each
detection × 22 samples). Only about 5 min of each CCL measurement,
moreover, chiral separation and calibration curve are not necessary
for CCL measurement, the total analytical time of CCL detections was
mL of the reaction mixture aliquots were withdrawn and added to
cuvettes containing 2 mL of deionized water or chiral host (S1 at 0.2
mmol/L). A specific chiral separation column was used for HPLC
measurement, the enantiopurities were calculated according to the
calibration curves constructed in advance (the detail can be seen in
the ESI, Table S4, Fig. S9 and Fig. S10). For CCL measurement, the
percentages and the ee values of the 1-phenylethanol enantiomers
were directly calculated by eqs 4, 5 and 7 according to the τ values of
the aliquots. The reaction curves monitored by the two methods are
1
10 min (5 min of each detection × 22 samples). The above results
demonstrate CCL represent a fast and reliable tool that can reduce
the cost and time of chiral analysis.
o
o
Fig. 6. The reaction curves of conversion of (S)-(-)-1-phenylethanol at 55 C (a) and 65 C (b) monitored by CCL and HPLC. ω(S)(CCL) and ω(S)(HPLC) stand for the content of (S)-
(-)-1-phenylethanol measured by HPLC and CCL, respectively.
Analysis of ee of chiral drugs plays an important role in and Fig. S13). For CCL measurement, the ee values were directly
pharmaceutical industry and clinical pharmacy, due to only one of caudated by eq 7 according to the τ values of samples. The results
the two enantiomers is pharmaceutically active ingredient in many obtained by the two methods are in good agreement with absolute
4
8
cases Ibuprofen is one of the most potent anti-inflammatory drugs errors ranging from -4.2% to 4.0%(Table 1). This example illustrates
used for the treatment of acute and chronic pain, fever and the promising application of our strategy in analysis of chiral drugs.
inflammatory diseases. It has been proved that (S)-(+)-ibuprofen is
4
9
almost entirely responsible for the anti-inflammatory activity. In
order to further explore of utility of our strategy in enantiopurity
determination, we used S1 as host to analyze ibuprofen samples.
First, the standard samples of (R)-(-)-ibuprofen and (S)-(+)-ibuprofen
were measured by CCL, the τ values of them are found to be 44.6 and
Table 1 Determination of ee Values of Ibuprofen Samples by CCL and
a
HPLC
ee (%) measured
by CCL
-4.5 ± 2.7
5.6 ± 3.8
-1.6 ± 6.0
-5.6 ± 2.7
-42.1 ± 3.5
ee (%) measured by Absolute error
Samples
HPLC
4.2 ± 1.9
1.6 ± 5
(%)
-0.3
4
1
2
3
4
5
2
6.1 s, respectively (ESI, Fig. S11). Next, samples containing
-1.7 ± 8.2
-1.4 ± 0.32
-45.6 ± 2.3
0.1
-4.2
3.5
ibuprofen were dissolved in ethanol, then was diluted with deionized
water. Finally, the prepared solutions were simultaneously analyzed
by CCL and HPLC. Similarly, a specific chiral column was used and
aSamples 1 and 2 are two commercially available ibuprofen tablets; samples 3 and
calibration curves were constructed in advance for HPLC 4 are two commercially available ibuprofen capsules; sample 5 was prepared by
determination (the detail can be seen in the ESI, Table S6, Fig. S12 mixing analytical grade (R)-(-)-ibuprofen with (S)-(+)-ibuprofen at random.
6
| J. Name., 2012, 00, 1-3
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Mechanism study
One of the major approaches to chirality sensing is based on the
formation of diastereomeric complexes generated by intermolecular
chiral–chiral interactions between chiral host and chiral guest.5,21,50,51
brings the hydroxyl radical close to nitrogen atom in the hosts,
forming hydrogen bond with different spatial structures, and thereby
the new adducts exhibit differential Raman bands (Video S1).
It is well known that different complexes show different
View Article Online
DOI: 10.1039/D0SC03496G
activities to catalyze luminol-H
difference in τ values among different chrial systems may relatied to
their catalytic activitie. Thus, the reaction kinetics of luminol-H
2 2
O reaction, we speculate taht the
In order to explore the work mechanism of CCL for chiral
discrimination, the density functional theory and the B3LYP method
in the Gaussian 09 program were employed to gain insight into the
possible modes of interactions between R1 (or S1) and 1-
phenylethanol enantiomers. Geometry optimizations were
performed using the basis set composed of LANL2DZ on cobalt atom
and 6-31G on the rest of the atoms, followed by frequency analysis
to insure all local minima with real frequencies only. The optimized
geometries for the new complexes are presented in Fig. 7, which
points out that 1-phenylethanol enantiomers can be linked to chiral
cobalt complexes (R1 and S1) via hydrogen bond to form adducts.
The binding geometries in the formative adducts are different, and
their binding energies are higher than -4 kcal/mol, indicating that
2 2
O
reactions catalyzed by different chrial systems were investigated by
using HPLC to monitor the consumption rate of luminol, and the
results are shown in the ESI (Fig. S15). In order to facilitate the
comparison, the reaction rate was expressed by lifetime using τ value.
The results showed that luminol-H
4.7 min) and S1 (τ = 15.2 min) have almost similar τ value. The τ
values of luminol-H reactions catalyzed by R1 with (R)-(+)-1-
2 2
O reactions catalyzed by R1 (τ =
1
2 2
O
phenylethanol, R1 with (S)-(-)-1-phenylethanol, S1 with (R)-(+)-1-
phenylethanol, and S1 with (S)-(-)-1-phenylethanol are 28.9, 19.2,
1
7.8 and 22.4 min. A high level of consistency exists between the τ
5
3
value of reaction kinetics monitored by HPLC and τ value of CCL
kinetic curve, which confirm our hypothesis. According to the above
results, we conclude that new adducts with different spatial
structures can be formed via intermolecular chiral–chiral interaction
between the chiral hosts and chiral guests. These new adducts show
stable complexes with different spatial structures were formed.
2 2
different catalytic activities for luminol-H O reaction, the distinction
in the catalytic activities can be reflected in the decay speeds of the
multistage signals measured by CCL, expressing as distinguishable τ
values.
Compared with the static injection CL, it seems that CCL
detection triggers the whole reaction proceed through a staged
process, which expands the differences relating to reaction kinetics
after multistage reaction. In addtion, additional features, such as
diffusion, convection and interaction between the sample and resin,
can probably do occur in CCL system. The difference in these features
may also affect the observed analytical signal. Therefore, extensive
work is still needed to further explore the mechanism.
Conclusion
In conclusion, we have described a brand-new optical strategy
of liquid-phase CCL for chiral analysis. This strategy provides a
concentration-independent parameter (τ value) to discriminate a
large variety of chiral substrates. Importantly, our strategy allows
rapid determination of ee value without the needs for chiral
separation and calibration curve, thereby paving the way for
rapid ee analysis. Further development can focus on establishing a
Fig. 7. The optimized structures of R1 with (R)-(+)-1-phenylethanol (a), R1 with
S)-(-)-1-phenylethanol (b), S1 with (R)-(+)-1-phenylethanol (c) and S1 with (S)-(-)-
(
1
-phenylethanol (d).
In order to further explore the interaction between R1 (or S1) standard database of τ value to realize the quickly and accurately
and 1-phenylethanol enantiomers, the mixtures of the R1 and (R)-(+)- recognize unknown chiral compounds. Mechanism study revealed
1
-phenylethanol (R1+R), R1 and (S)-(-)-1-phenylethanol (R1+S), S1 that that new adducts with different spatial structures can be formed
via intermolecular chiral–chiral interactions between the chiral hosts
and chiral guests. The new adducts exhibits different catalytic activity
for luminol-H O reaction, resulting in distinguishable τ values of the
2 2
and (R)-(+)-1-phenylethanol (S1+R), as well as S1 and (S)-(-)-1-
phenylethanol (S1+S), were measured by Surface-enhanced Raman
spectroscopy (SERS), density functional theory at the B3LYP/6-
multistage signals measured by CCL. Since CCL allows perception of
intermolecular interactions through outputting distinguishable τ
values. we are currently exploring this strategy to discriminate of
chiral biological substrates, and expecting to find applications in the
study of protein structure, molecular dynamics, drug design etc.
based on host-guest interactions. Further studies on these directions
are currently underway in our laboratory and will be reported in due
course. We believe the combination of our strategy and diversified
3
1++G(d,p) level was employed to calculate the Raman bands of the
new adducts. As Fig. S14 in ESI shows, the Raman spectra of R1+R
-1
and R1+S exhibit features at 1296 and 1278 cm , respectively.
Similarly, characteristic bands at 1281.1 and 1296.7 are observed for
S1+R and S1+S, respectively. This further demonstrates the
formation of different adducts. Molecular modeling revealed that
the stretching vibration of C-O in 1-phenylethanol enantiomers
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J. Name., 2013, 00, 1-3 | 7
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Journal Name
chiral hosts will offer a powerful platform to address some challenges
of chirality analysis.
Int. Ed., 2020, doi.org/10.1002/anie.202001680.View Article Online
2
2
2
3
4
5
T. R. Schulte, J. J. Holstein and G. H. Clever, Angew. Chem. Int.
DOI: 10.1039/D0SC03496G
Ed. 2019, 58, 5562−5566.
F. Y. Thanzeel, A. Sripada and C. Wolf, J. Am. Chem. Soc. 2019,
1
41, 16382−16387.
Conflicts of interest
There are no conflicts to declare.
Y. Sasaki, S. Kojima, V. Hamedpour, R. Kubota, S. Takizawa, I.
Yoshikawa, H. Houjou, Y. Kubo and T. Minami, Chem. Sci. 2020,
1
1, 3790−3796.
2
2
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9
A.Akdeniz, T. Minami, S. Watanabe, M. Yokoyama, T. Ema and
P. Anzenbacher Jr, Chem. Sci. 2016, 7, 2016−2022.
Acknowledgements
F. F. Xie, Q. Bai, X. M. Jiang, X. S. Yu, Z. N. Xia and W. L. Wei, Acs
Appl. Mater. Interfaces 2018, 10, 11872−11879.
This work was supported by the National Natural Science
Foundation of China (Nos. 21605163, 21675178 and 21976213), the
Research and Development Plan for Key Areas of Food Safety in
Guangdong Province of China (No. 2019B020211001), and National
S. S. Yu, W. Plunkett, M. Kim and L. Pu, J. Am. Chem. Soc. 2012,
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K. L. Wen, S. S. Yu, Z. Huang, L. M. Chen, M. Xiao, X. Q. Yu and
L. Pu, J. Am. Chem. Soc. 2015, 137, 4517−4524.
Key Research and Development Program of China (No. 30 P. Zardi, K. Wurst, G. Licini and C. Zonta, Chem. Soc. 2017, 139,
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E. Al-Hetlani, M. O. Amin and M. Madkour, Nanophotonics
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View Article Online
DOI: 10.1039/D0SC03496G
Table of Contents
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Page 10 of 18
View Article Online
DOI: 10.1039/D0SC03496G
ARTICLE
Rapid Chiral Analysis Based on Liquid-Phase Cyclic
Chemiluminescence
Runkun Zhang, Yanhui Zhong, Zhenyu Lu, Yanlong Chen, and Gongke Li*
Received 00th June 2020,
Accepted 00th June 2020
Rapid chiral analysis has become one of the important aspects of academic and industrial research. Here we describe a new
strategy based on liquid-phase cyclic chemiluminescence (CCL) for rapid resolution of enantiomers and determination of
enantiomeric excess (ee). A single CCL measurement can acquire multistage signals that provide a unique way to examine
the intermolecular interactions between chiral hosts and chiral guests, due to the lifetime (τ) of the multistage signals is a
concentration-independent and distinguishable constant for a given chiral host-guest system. According to the τ values, CCL
allows discrimination between a wide range of enantiomeric pairs include chiral alcohols, amines and acids by using only
one chiral host. Even the chiral systems hardly distinguished by nuclear magnetic resonance and fluorescence method can
be distinguished easily by CCL. Additionally, the τ value of the mixture of two enantiomers is equal to the weighted average
of each enantiomer, which can be used for direct determination of the ee without the need to separate the chiral mixture
and create calibration curves. It is extremely crucial for the cases without readily available enantiomerically pure samples.
This strategy was successfully applied to monitoring of Walden inversion reaction and analysis of chiral drugs. The results
were in good agreement with those measured by high-performance liquid chromatography, indicating the utility of CCL for
routine quick ee analysis. Mechanism study revealed that the τ value is possible related to the activity of chiral substance to
DOI: 10.1039/x0xx00000x
catalyze the luminol-H
2
O reaction. Our research provides an unprecedented and general protocol for chirality
2
differentiation and ee determination, which is anticipated to being a useful technology that find wide applications in
chirality-related fields, particularly in asymmetric synthesis and pharmaceutical industry.
Introduction
Chirality is a universal phenomenon in nature, most attracted considerable research interest as a result of their
biological species, such as amino acids, nucleic acid, proteins, advantages of rapidity, simplicity, and low cost. In such a
drugs, are chiral and intermolecular chiral-chiral interactions strategy, the molecules or materials containing the chiral unit
1
-3
occur ubiquitously among biological processes.
The are used as chemosensory hosts that can interact with the chiral
simultaneous determination of the absolute configuration and targets and then induce distinguishable optical signals.21
enantiopurity of chiral substrates is highly significant in the Although the great efforts and achievements that have been
fields of synthetic, biological and pharmaceutical chemistry.
4
-6
made in this field, some problems still remain to be solved. For
However, chiral analysis remains a great challenge due to instance, most methods suffer from narrow substrate scope
7
28
enantiomers have nearly identical physical properties.
and limitedly differentiable enantiomers. In addition, the
In the past several decades, many effective methods have output signal for chiral discrimination is strongly influenced by
been developed for the chiral analysis, such as gas the analyte concentration, this would normally need two
8,9
chromatography, high-performance liquid chromatography independent methods to simultaneously determine both
1
0,11
capillary electrophoresis,12-14 nuclear magnetic enantiomeric configuration and enantiopurity.
28,29
(HPLC),
Especially,
mass these assays usually require highly pure samples to construct
curves for enantiomeric excess (ee)
It would be particularly significant to
resonance
spectrometry.
(NMR)
spectroscopy7,15-17
and
[18,19]
However, these methods are time- calibration
4,30,31
consuming and technically complex, and the instrumentation determination.
required is expensive, bulky, and requires well-trained acquire a concentration-independent parameter for chiral
2
0,21
operators.
dichroism,
Optical sensing methodologies, such as circular discrimination and enantiopurity determination, due to such
2
2-24
25,26
27
fluorescence
and colorimetry
have technique can simplify and speed up the measurement, and
thereby find wide applications in rapid screening of ee for
asymmetric synthesis and pharmaceutical industry etc. In this
paper, we report an unprecedented strategy of liquid-phase
cyclic chemiluminescence (CCL) that does not suffer from the
a.School of Chemistry, Sun Yat-sen University, Guangzhou 510275, People’s
Republic of China E-mail: cesgkl@mail.sysu.edu.cn
Electronic Supplementary Information (ESI) available: Fabrication and working
principle of liquid-phase CCL system; method for immobilization of luminol; Fig.S1-
S10 and Table S1-S5. See DOI: 10.1039/x0xx00000x
；
above limitations in chiral analysis. To the best of our
knowledge, this paper is the first to report
a
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chemiluminescence-based sensing methodology that providing by reactive oxygen species to form a 3-aminophthalate dianion
View Article Online
DOI: 10.1039/D0SC03496G
a concentration-independent constant for the simultaneous in an excited state, which emitted light when returning to the
40
determination of enantiomeric configuration and ee value.
ground state. Specifically, a circular pipeline was designed for
As an important optical analytical technology, CCL system, and the direction of the carrier in CCL system can
chemiluminescence (CL) have been widely used for be changed automatically at the indicated cycle period. Through
3
2
33-35
pharmaceutical analysis, clinical analysis
analysis.
and biological automatically changing the carrier direction at the indicated
36,37
However, typical CL kinetic curve lacks the period, the chemical species at different stages flowed through
characteristic features of the analytes. To overcome this the sensor cell repeatedly to induce the CL reactions proceed in
drawback, our group proposed a new concept of cyclic succession. Multistage signals that satisfy the exponential decay
chemiluminescence (CCL) for rapid discrimination of law were measured over time. The design of CCL system
38
structurally similar compounds. In contrast to conventional extends the observable time, and thereby the distinctions
flow CL system, the direction of the carrier in the CCL system is between different luminous reactions that usually are not easy
in a state of periodical change, which triggers a succession of CL to observe by conventional CL-based detection, but can be
reactions in a single sample introduction to produce multistage observed by CCL detection. The flow paths of liquid-phase CCL
signals. We found that the multistage signals on CCL kinetic detection at different stages are shown in the ESI (Fig. S1).
curve satisfy the first-order exponential decay law:
For chiral analysis, chiral metal-organic complexes that are
able to enhance the luminescence intensity of luminol-H reaction
were used as hosts, new complexes were formed via chiral–


t
2 2
O
I  Ae  I
(1)
n
0
where I
n
is the signal of n th stage reaction, and I
0
is the chiral interactions between chiral host and chiral guest when
background noise. t is the peak time. A is the pre-exponential chiral guests were mixed with chiral hosts. The τ values of the
factor that stands for the maximum luminescence intensity. τ CCL kinetic curves of luminol-H reaction catalyzed by the
2 2
O
represents the lifetime of the multistage signals, it is the time at new complexes were use for chiral analysis. The schematic
which the intensity of the assembly is reduced to 1/e times its representation of liquid-phase CCL for chiral analysis is shown
initial value, its unit is "second". We demonstrated each given in the Fig. 1.
CCL reaction has a unique constant of τ. The distinguishable τ
values allow facile identification of various analytes include
homologous series and structural isomers. Recently, we further
demonstrated that the multistage signals of multicomponent
mixture also satisfy the first-order exponential decay law with
3
9
their unique τ vaues. This allows facile identification of mixed
samples consisting of complex components, such as liquor, beer,
toner, and baby powder. However, our previous researches
were limited to studying the gaseous-phase CL emitted on
nanosized catalysts, and a wider adaptation for the application
of CCL has not been demonstrated yet. Following our research
interest in demonstrating the generality of CCL and exploring its
further application, here, we extend the CCL strategy to liquid-
phase reactions via immobilizing luminous reagent, and metal-
2 2
organic complex-catalyzed luminol-H O reaction system was
used as basic model to probe the application of CCL in chiral
analysis. The most distinct advantage of liquid-phase CCL is that
it provides a concentration-independent constant (τ value) to
discriminate a wide range of enantiomers, and allows direct
determination of the enantiopurity without the need to
separate the chiral mixture, and thereby the use of
enantiomerically pure samples for creating calibration curves
can be avoided.
Fig. 1. The schematic representation of liquid-phase CCL for chiral analysis.
Results and discussion
Resolution of enantiomers
Experimental section
The fabrication of liquid-phase CCL system can be seen in
the ESI. In the present work, immobilized luminol (5-amino-2,3-
dihydro-1,4-phthalazinedione, immobilized method was
detailed in the ESI) was packed into a homemade flow-through
2 2
reactor to fabricate a sensor cell. H O was used as a carrier and
oxidant, and it was catalyzed by a sample containing a metal-
organic complex to produce reactive oxygen species.
Subsequently, immobilized luminol in a sensor cell was oxidized
To examine the feasibility of our strategy for chiral analysis,
three pairs of commercially available chiral metal-organic complexes
(
R1 and S1, R2 and S2, R3 and S3, the detail information of these
chiral complexes can be seen in Table S1 of the ESI) were used as
hosts. The original CCL kinetic curves of luminol-H system
2 2
O
catalyzed by these chiral metal-organic complexes are shown in the
ESI (Fig. S2). It shows that the two enantiomers of one chiral
substrate of metal-organic complex have the same τ value when
without adding chiral guests. This because the differences between
2
| J. Name., 2012, 00, 1-3
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Journal Name
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enantiomers are difficult to observe under normal circumstance, but as characteristic labelling for directly determining the identity of
View Article Online
DOI: 10.1039/D0SC03496G
these differences become much more marked in a chiral enantiomers.
environment. Thus, chiral guests were added into the solutions of
chiral hosts. We found that additions of (R)-(+)-1-phenylethanol and
(
(
S)-(-)-1-phenylethanol into R1 outputting τ values of 64.6 and 39.0 s
Fig. 2a), respectively. τ values of 35.2 and 51.6 s were observed
when added (R)-(+)-1-phenylethanol and (S)-(-)-1-phenylethanol into
S1 (Fig. 2b).
We further investigated the effects of the concentration of
guest on response behaviors of host at a much broader
concentration range. We have plotted the τ values of R1 and S1
versus the increasing concentrations of (R)-(+)-1-phenylethanol and
(
S)-(-)-1-phenylethanol in Fig. 2c and Fig. 2d, respectively. We found
that the τ value is independent of the concentration of the chiral
guest in the range of 0.005 to 0.5 mmol/L but is only dependent on
the chiral configuration. In addtion, the τ value also is independent
2 2
of the H O concentration within a certain range (Fig. S3, detailed
discussion can be seen in the ESI). These results demonstrate that
each chiral system has a unique and constant τ value, and the Fig. 2. (a) The CCL kinetic curves of luminol-H
distinction in τ value confirms the chiral discrimination. Quite phenylethanol enantiomers; (b) the CCL kinetic curves of luminol-H
2
O
2
system catalyzed by R1 with 1-
system
2 2
O
different from other sensing methodologies based on intensity catalyzed by S1 with 1-phenylethanol enantiomers; (c) Plots of τ value for R1 (0.1
quenching/enhancement or spectral changes, the parameter (τ mmol/L) in the presence of varying concentrations of 1-phenylethanol; (d) Plots of
value) of CCL for chiral analysis is a concentration-independent τ value for S1 (0.1 mmol/L) in the presence of varying concentrations of 1-
constant that characteristic of a given chiral system, which functions phenylethanol. The concentration of H
2 2
O is 0.05 mmol/L.
2 2
Fig.3. The τ values of different chiral host-guest systems. The concentration of all hosts and guests is 0.1 mmol/L, the concentration of H O is 0.05 mmol/L. The unit of τ
is second.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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Page 13 of 18
Chemical Science
The reproducibility of the immobilized luminol was investigated acid and L-(+)-mandelic acid, respectively. Satisfactory resolution of
by measure of (R)-(+)-1-phenylethano using S1. We found that the the two enantiomers of one chiral substrate is easil
background noise increases and luminescence intensity (A) simply comparing the τ values.
decreases gradually with increasing test number (ESI, Fig. S4), but the Noteworthy, each chiral host-guest system has a corresponding
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τ could remain stable within 25 replicates with a RSD of 3.7% (ESI, Fig. τ value, that is: different hosts show different values of τ to a given
S5a). It means that τ has a good stability, possibly due to τ depends guest, and the same host exhibits different values of τ to different
on the rate of luminescence depletion that related to the guests. The above results demonstrate that each chiral host-guest
characteristics of the reaction, but not the apparent intensity. The τ system has a corresponding exponential decay equation with a
values measured by 5 different batches of immobilized luminol unique τ value. Thus, we can establish a database of τ values of
vary between 4.2% (ESI, Fig. S5b). The sufficient stability of τ within various chiral host-guest systems in future, which provides facilitation
the same batch and between different batches of immobilized for quick recognition of unknown chiral compounds. For example, if
luminol make CCL is qualified for chiral differentiation. It is worth the τ values of an analyte were measured to be 64.6 and 35.2 s when
noting that the A value is easily fluctuant and the τ value depends on using R1 and S1 as hosts, respectively, the identity of the analyte
the decay rate of multistage signal, and thereby the multistage signal could be confirmed to be (R)-(+)-1-phenylethanol by searching
can be normalized for nonlinear curve fitting to obtain τ value.
database.
Fig. 3 illustrates the broad substrate applicability, and the
An ideal method for chiral sensing should be capable of
discrimination of different kinds of chiral molecules accompanied satisfactory enantio-differentiating ability of our strategy. Usually, we
21
with the resolution of the two enantiomers of one chiral substrate.
can discriminate most enantiomeric pairs by only using one chiral
However, current methods usually suffer from narrow substrate host. However, the τ values of some chiral compounds on certain host
15
scope and limitedly differentiable enantiomers. We next turned are close. For example, although host S2 allows discrimination
attention to demonstrate the ability of CCL to discriminate a wide between enantiomers of 1-phenylethanol or 2-pentanol, it unable to
range of enantiomers with broad substrate applicability. The chiral resolve (S)-(-)-1-phenylethanol (τ=40.4 s) and (S)-(+)-2-pentanol
metal-organic complexes shown in the ESI (Table S1) were used as (τ=40.5 s). Our aim is to realize unambiguous discrimination among
hosts to sensing two pairs of chiral alcohols, chiral amines and chiral a large variety of chiral compounds, not just limits to the two
acid, respectively. As expected, each enantiomer can be correlated enantiomers of one chiral substrate. In consideration of the cross-
to a distinct τ value when it is mixed with a given chiral host (Fig. 3), reactivity of the hosts, we combined all the hosts (R1, S1, R2, S2, R3
enabling easy discrimination of chiral compounds. For example, and S3) to construct a sensor array, expecting to realize
when chiral complex R1 was used as a chiral host, τ values of 64.6 and unambiguously discriminate various chiral compounds. Three
39.0 s were observed for (R)-(+)-1-phenylethanol and (S)-(-)-1- concentrations (low: 0.005 mmol/L, medium: 0.05 mmol/L and high:
phenylethanol; 52.7 and 38.2 s for (R)-(-)-2-pentanol and (S)-(+)-2- 0.1 mmol/L) of 12 chiral compounds were tested by the sensor array,
pentanol; 58.3 and 46.5 s for (R)-(-)-2-phenylglycinol and (S)-(+)-2- the resulting τ values are summarized in the ESI (Table S2). For easier
phenylglycinol; 12.1 and 21.4 s for (R)-3-amino-3-phenylpropan-1-ol visualization, the τ value of each response is depicted in the form of
and (S)-3-amino-3-phenylpropan-1-ol; 49.6 and 73.9 s for D-(-)- a heat map according to the scale bar on the right. As shown in Fig.
tartaric acid and L-(+)-tartaric acid; 31.6 and 23.7 s for D-(-)-mandelic 4a, each of the chiral guests creates a unique multidimensional
Fig. 4. (a) Response patterns of 6 chiral hosts to 12 guests at different concentrations. 0.005, 0.05 and 0.1 strand for the concentrations of the guests, the unit is mmol/L;
(
b) LDA 2D plot derived from the colour response pattern. A: (R)-(+)-1-phenylethanol; B: (S)-(-)-1-phenylethanol; C: (R)-(-)-2-pentanol; D: (S)-(+)-2-pentanol; E: (R)-(-)-2-
phenylglycinol; F: (S)-(+)-2-phenylglycinol; G: (R)-3-amino-3-phenylpropan-1-ol; H: (S)-3-amino-3-phenylpropan-1-ol; I: D-(-)-tartaric acid; J: L-(+)-tartaric acid; K: D-(-)-
mandelic acid; L: L-(+)-mandelic acid. The concentration of all host is 0.1 mmol/L, the concentration of H is 0.05 mmol/L.
2 2
O
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Page 14 of 18
response pattern, and different concentrations of the same chiral in academic and industrial fields. In order to probe the application
guest induce similar response pattern. Linear discriminant analysis value of our strategy in rapid determination of ee, R1 an
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DOI: 10.1039/D0SC03496G
(
LDA) was then used to further digitize and visualize the response to measure a series of mixtures consisting of different percentages of
patterns (6 host units × 12 chiral guest × 3 concentrations). Fig. 4b 1-phenylethanol enantiomers, and the trends of the τ value of the
shows the 3-D discrimination plot constructed by the first three mixture (τ ) versus the percentages of (R)-(+)-1-phenylethanol and
factors. The first three factors account for 85.5% of the total variance. (S)-(-)-1-phenylethanol were plotted in Fig. 5, respectively. Linear-
The same colored symbol within the LDA graphical output assigns to regression analysis shows that the τ is linearly related to the
m
m
specific chiral guests. These chiral guests are classified into 12 groups percentages of the enantiomers in the mixture. Further study found
with 100% accuracy, and small spatial distance are observed within that the slope of the linear regression equation is equal to difference
the same group. This demonstrates the satisfactory discrimination between the τ values of the two enantiomers, and the intercept
capacity of the sensor array based on CCL strategy.
m
equals the τ value of one of the enantiomers. The τ of the mixture
NMR spectroscopy and fluorescence spectroscopy are common composed by arbitrary proportions of enantiomers can be expressed
technique used for chiral discrimination. However, we found that the as:
R1 or S1 with 1-phenylethanol enantiomers (ESI, Fig. S6). The same
m  (R) ((R) (S ) ) (S )     (1 (R) )(S )
(2)
(R) (R)
chiral systems also cannot be resolved by fluorescent method, due to
R1, S1 and 1-phenylethanol are non-fluorescent substances. These
results demonstrate that CCL can distinguish some chiral host-guest
systems, which would be difficult for NMR and fluorescence
spectroscopy. Compared with the method for chiral discrimination
Or can be presented in the following form:
m   ( (R) ) (R)  (1 )  
(3)
(S)
(S)
(S)
(R)
(S) (S)
Where ω(R) and ω(S) are the percentages of the two enantiomers
in the mixture, and τ(R) and τ(S) stand for the τ values of the two
enantiomers, respectively. Eqs 2 and 3 indicate that the τ is the
m
41
based on quantum dots enhanced-CL system, the commercial chiral
metal-organic complexes used in the present work are easy available.
In addition, they allow facile discrimination between a wide range of
enantiomeric pairs. It is well known that static injection CL can
provide the full intensity kinetic curve. However, we found that the
CL kinetic curves obtained by static injection lack enough
discriminability to differentiate chiral compounds, mainly due to the
short lifetime of the CL kinetic curve. The τ values of different kinetic
curve obtained by static injection are in the in the range from 1.0 to
weighted average of the two enantiomers, which is in accordance
39
with the gaseous-phase CCL detection. According to eqs 2 and 3,
ω(R) and ω(S) in the mixture can be presented by:
m -(S)
(R)
(S )

(4)
(5)
(R) (S)
(R)
-m
3.6 s (ESI, Fig. S7), the narrow τ-range means low resolution. The
 (R)
detailed discussion also can be seen in the ESI. Thus, CCL will be a
powerfully complementary technology for chiral discrimination.
(S )
The ee (%) is defined as:47
Determination of ee
ee  (R)  (S )
(6)
Determination of ee is performed routinely in many scientific
by integrating eqs 3, 4 and 5, the ee can be calculated by:
fields, especially in asymmetric synthesis and pharmaceutical
42-45
industry.
Chiral separation is the most common method for ee
2
m -(R) -(S )
ee 
(7)
determination. However, chiral separation is cost-intensive and time-
consuming process due to the long analysis time and the use of
expensive chiral column.
(R)
(S)
21,46
Simple and inexpensive techniques for
Eqs 4, 5 and 7 indicate that if the τ values of the enantiomers are
known in advance (through searching database), the percentages of
rapid determination of ee is attractive for their potential applications
Fig. 5. The relationship between τ value and the percentages of 1-phenylethanol enantiomers using R1 (a) and S1 (b) as hosts.
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Page 15 of 18
Chemical Science
the two enantiomers and the ee value can be directly determined by times measured by the two methods are listed in the ESI (Table S5).
measuring the τ value of the mixture without the need to use The CCL determined ee values correlated well with chiraVi ults
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DOI: 10.1039/D0SC03496G
enantiomerically pure samples for creating calibration curves. Fig. S8 with absolute errors ranging from -7.1% to 7.0%.
in the ESI compare the calculated versus the actual ee values, It is worth noting that during the processes of the above assays,
excellent linear relationships (regression coefficients exceed 0.998) the analytical time of each HPCL measurement was 13 min (ESI, Fig.
with slopes close to 1 were obtained. The absolute errors between S9), the total time spent on HPCL detections was about 377 min (13
the calculated and actual ee values ranged from -6.9% to 4.6% (ESI, min of each detection × 7 standard solutions + 13 min of each
Table S3).
detection × 22 samples). Only about 5 min of each CCL measurement,
moreover, chiral separation and calibration curve are not necessary
for CCL measurement, the total analytical time of CCL detections was
Applications
110 min (5 min of each detection × 22 samples). The above results
To validate the utility of CCL in enantiopurity determination, an
demonstrate CCL represent a fast and reliable tool that can reduce
the cost and time of chiral analysis.
amberlyst-catalyzed Walden inversion was selected as a prototype
reaction because of its industrial application in production of
pharmaceutical and agrochemical building blocks. 1.0 g of
48
Analysis of ee of chiral drugs plays an important role in
pharmaceutical industry and clinical pharmacy, due to only one of the
two enantiomers is pharmaceutically active ingredient in many
amberlyst-H was added to 100 mL solution of (S)-(-)-1-phenylethanol
o
at 0.2 mmol/L, duplicate the above solutions were heated to 55 C
4
9
cases Ibuprofen is one of the most potent anti-inflammatory drugs
used for the treatment of acute and chronic pain, fever and
inflammatory diseases. It has been proved that (S)-(+)-ibuprofen is
o
and 65 C under stirring condition, respectively. At various times, 2
mL of the reaction mixture aliquots were withdrawn and added to
cuvettes containing 2 mL of deionized water or chiral host (S1 at 0.2
mmol/L). A specific chiral separation column was used for HPLC
50
almost entirely responsible for the anti-inflammatory activity. In
order to further explore of utility of our strategy in enantiopurity
measurement, the enantiopurities were calculated according to the determination, we used S1 as host to analyze ibuprofen samples. First,
calibration curves constructed in advance (the detail can be seen in the standard samples of (R)-(-)-ibuprofen and (S)-(+)-ibuprofen were
the ESI, Table S4, Fig. S9 and Fig. S10). For CCL measurement, the measured by CCL, the τ values of them are found to be 44.6 and 26.1
s, respectively (ESI, Fig. S11). Next, samples containing ibuprofen
were dissolved in ethanol, then was diluted with deionized water.
Finally, the prepared solutions were simultaneously analyzed by CCL
and HPLC. Similarly, a specific chiral column was used and calibration
curves were constructed in advance for HPLC determination (the
detail can be seen in the ESI, Table S6, Fig. S12 and Fig. S13). For CCL
measurement, the ee values were directly caudated by eq 7
according to the τ values of samples. The results obtained by the two
methods are in good agreement with absolute errors ranging from -
percentages and the ee values of the 1-phenylethanol enantiomers
were directly calculated by eqs 4, 5 and 7 according to the τ values of
the aliquots. The reaction curves monitored by the two methods are
shown in Fig. 6. The general shape of the curves obtained by the two
methods is surprisingly similar, showing the conversion of (S)-(-)-1-
phenylethanolfollows first order reaction kinetics. The relative
content of (S)-(-)-1-phenylethanol steadily decreases, while the yield
of (R)-(+)-1-phenylethanol steadily increases with the conversion of
(
S)-(-)-1-phenylethanol. Complete substrate conversion within 240 4.2% to 4.0% (Table 1). This example illustrates the promising
min was observed. The half-lives calculated by CCL (t1/2(CCL)) and HPLC application of our strategy in analysis of chiral drugs.
(
t1/2(HPLC)) are in good agreement. The ee values at different reaction
o
o
Fig. 6. The reaction curves of conversion of (S)-(-)-1-phenylethanol at 55 C (a) and 65 C (b) monitored by CCL and HPLC. ω(S)(CCL) and ω(S)(HPLC) stand for the content of (S)-
(-)-1-phenylethanol measured by CCL and HPLC, respectively.
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Table 1 Determination of ee Values of Ibuprofen Samples by CCL and
HPLC
In order to further explore the interaction between R1 (or S1)
a
and 1-phenylethanol enantiomers, the mixtures of the
1-phenylethanol (R1+R), R1 and (S)-(-)-1-phenylethanol (R1+S), S1
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DOI: 10.1039/D0SC03496G
ee (%) measured
by CCL
ee (%) measured by Absolute error
Samples
HPLC
(%)
0.3
4.0
0.1
-4.2
3.5
and (R)-(+)-1-phenylethanol (S1+R), as well as S1 and (S)-(-)-1-
phenylethanol (S1+S), were measured by Surface-enhanced Raman
spectroscopy (SERS), density functional theory at the B3LYP/6-
1
2
3
4
5
4.5 ± 2.7
4.2 ± 1.9
1.6 ± 5
5.6 ± 3.8
3
1++G(d,p) level was employed to calculate the Raman bands of the
-1.6 ± 6.0
-5.6 ± 2.7
-42.1 ± 3.5
-1.7 ± 8.2
-1.4 ± 0.32
-45.6 ± 2.3
new adducts. As Fig. S14 in ESI shows, the Raman spectra of R1+R and
-1
R1+S exhibit features at 1296 and 1278 cm , respectively. Similarly,
characteristic bands at 1281.1 and 1296.7 are observed for S1+R and
S1+S, respectively. This further demonstrates the formation of
different adducts. Molecular modeling revealed that the stretching
vibration of C-O in 1-phenylethanol enantiomers brings the hydroxyl
radical close to nitrogen atom in the hosts, forming hydrogen bond
with different spatial structures, and thereby the new adducts exhibit
differential Raman bands (Video S1).
aSamples 1 and 2 are two commercially available ibuprofen tablets;
samples 3 and 4 are two commercially available ibuprofen capsules;
sample 5 was prepared by mixing analytical grade (R)-(-)-ibuprofen
with (S)-(+)-ibuprofen at random.
Mechanism study
One of the major approaches to chirality sensing is based on the
formation of diastereomeric complexes generated by intermolecular
It is well known that different complexes show different
activities to catalyze luminol-H
difference in τ values among different chrial systems may relatied to
2 2
their catalytic activitie. Thus, the reaction kinetics of luminol-H O
2 2
O reaction, we speculate taht the
5
,21,51,52
chiral–chiral interactions between chiral host and chiral guest.
In order to explore the work mechanism of CCL for chiral
discrimination, the density functional theory and the B3LYP method
in the Gaussian 09 program were employed to gain insight into the
possible modes of interactions between R1 (or S1) and 1-
phenylethanol enantiomers. Geometry optimizations were
performed using the basis set composed of LANL2DZ on cobalt atom
and 6-31G on the rest of the atoms, followed by frequency analysis
to insure all local minima with real frequencies only. The optimized
geometries for the new complexes are presented in Fig. 7, which
points out that 1-phenylethanol enantiomers can be linked to chiral
cobalt complexes (R1 and S1) via hydrogen bond to form adducts. The
binding geometries in the formative adducts are different, and their
binding energies are higher than -4 kcal/mol, indicating that stable
reactions catalyzed by different chrial systems were investigated by
using HPLC to monitor the consumption rate of luminol, and the
results are shown in the ESI (Fig. S15). In order to facilitate the
comparison, the reaction rate was expressed by lifetime using τ value.
The results showed that luminol-H
4.7 min) and S1 (τ = 15.2 min) have almost similar τ value. The τ
values of luminol-H reactions catalyzed by R1 with (R)-(+)-1-
2 2
O reactions catalyzed by R1 (τ =
1
2 2
O
phenylethanol, R1 with (S)-(-)-1-phenylethanol, S1 with (R)-(+)-1-
phenylethanol, and S1 with (S)-(-)-1-phenylethanol are 28.9, 19.2,
7.8 and 22.4 min. A high level of consistency exists between the τ
value of reaction kinetics monitored by HPLC and τ value of CCL
kinetic curve, which confirm our hypothesis. According to the above
results, we conclude that new adducts with different spatial
structures can be formed via intermolecular chiral–chiral interaction
between the chiral hosts and chiral guests. These new adducts show
1
5
3
complexes with different spatial structures were formed.
2 2
different catalytic activities for luminol-H O reaction, the distinction
in the catalytic activities can be reflected in the decay speeds of the
multistage signals measured by CCL, expressing as distinguishable τ
values.
Compared with the static injection CL, it seems that CCL
detection triggers the whole reaction proceed through a staged
process, which expands the differences relating to reaction kinetics
after multistage reaction. In addtion, additional features, such as
diffusion, convection and interaction between the sample and resin,
can probably do occur in CCL system. The difference in these features
may also affect the observed analytical signal. Therefore, extensive
work is still needed to further explore the mechanism.
Conclusion
In conclusion, we have described a brand-new optical strategy
of liquid-phase CCL for chiral analysis. This strategy provides a
concentration-independent parameter (τ value) to discriminate a
large variety of chiral substrates. Importantly, our strategy allows
rapid determination of ee value without the needs for chiral
separation and calibration curve, thereby paving the way for
rapid ee analysis. Further development can focus on establishing a
standard database of τ value to realize the quickly and accurately
Fig. 7. The optimized structures of R1 with (R)-(+)-1-phenylethanol (a), R1 with (S)-
-)-1-phenylethanol (b), S1 with (R)-(+)-1-phenylethanol (c) and S1 with (S)-(-)-1-
phenylethanol (d).
(
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Page 17 of 18
PleaseC dh oe mn oi ct a al dS cj ui es nt cme argins
ARTICLE
Journal Name
recognize unknown chiral compounds. Mechanism study revealed
that that new adducts with different spatial structures can be formed
via intermolecular chiral–chiral interactions between the chiral hosts
and chiral guests. The new adducts exhibits different catalytic activity
Soc. 2015, 137, 15402−15405.
View Article Online
16
17
18
19
G. L. Bian, S. W. Yang, H. Y. Huang, H. Zong, L. Song, H. J. Fan
DOI: 10.1039/D0SC03496G
and X. Q. Sun, Chem. Sci. 2016, 7, 932−938.
M. S. Seo and H. Kim, J. Am. Chem. Soc. 2015, 137,
1
4190−14915.
2 2
for luminol-H O reaction, resulting in distinguishable τ values of the
C. Ebner, C. A. Müller, C. Markert and A. Pfaltz, J. Am. Chem.
Soc. 2011, 133, 4710−4713.
multistage signals measured by CCL. Since CCL allows perception of
intermolecular interactions through outputting distinguishable τ
values. we are currently exploring this strategy to discriminate of
S. T. Phillips, J. N. Dodds, B. M. Ellis, J. C. May and J. A. McLean,
Chem. Commun. 2018, 54, 9398−9401.
chiral biological substrates, and expecting to find applications in the 20 Boussouar, Q. J. Chen, X. Chen, Y. L. Zhang, F. Zhang, D. M. Tian,
H. S. White and H. B. Li, Anal. Chem. 2017, 89, 1110−1116.
X. Zhang, J. Yin and J. Yoon, Chem. Rev. 2014, 114, 4918−4959.
H. T. Z. Zhu, Q. Li, Z. C. Gao, H. L. Wang, B. B. Shi, Y. T. Wu, L. Q.
Shangguan, X. Hong, F. Wang and F. H. Huang, Angew. Chem.
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F. Y. Thanzeel, A. Sripada and C. Wolf, J. Am. Chem. Soc. 2019,
study of protein structure, molecular dynamics, drug design etc.
based on host-guest interactions. Further studies on these directions
are currently underway in our laboratory and will be reported in due
course. We believe the combination of our strategy and diversified
chiral hosts will offer a powerful platform to address some challenges
of chirality analysis.
2
1
2
2
23
24
25
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41, 16382−16387.
Y. Sasaki, S. Kojima, V. Hamedpour, R. Kubota, S. Takizawa, I.
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Conflicts of interest
There are no conflicts to declare.
26
A.Akdeniz, T. Minami, S. Watanabe, M. Yokoyama, T. Ema and
P. Anzenbacher Jr, Chem. Sci. 2016, 7, 2016−2022.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (Nos. 21605163, 21675178 and 21976213), the
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