A Colorimetric Method for Quantifying Cis and Trans Alkenes Using an Indicator Displacement Assay

Article abstract of DOI:10.1002/anie.202101004

A colorimetric indicator displacement assay (IDA) amenable to high-throughput experimentation was developed to determine the percentage of cis and trans alkenes. Using 96-well plates two steps are performed: a reaction plate for dihydroxylation of the alkenes followed by an IDA screening plate consisting of an indicator and a boronic acid. The dihydroxylation generates either erythro or threo vicinal diols from cis or trans alkenes, depending upon their syn- or anti-addition mechanisms. Threo diols preferentially associate with the boronic acid due to the creation of more stable boronate esters, thus displacing the indicator to a greater extent. The generality of the protocol was demonstrated using seven sets of cis and trans alkenes. Blind mixtures of cis and trans alkenes were made, resulting in an average error of ±2 % in the percentage of cis or trans alkenes, and implementing E₂ and Wittig reactions gave errors of ±3 %. Furthermore, we developed variants of the IDA for which the color may be tuned to optimize the response for the human eye.

Full text of DOI:10.1002/anie.202101004

Angewandte
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Hot Paper
A Colorimetric Method for Quantifying Cis and Trans Alkenes Using
an Indicator Displacement Assay
Stephanie A. Valenzuela+, Hannah S. N. Crory+, Chao-Yi Yao+, James R. Howard,
Gabriel Saucedo, A. Prasanna de Silva, and Eric V. Anslyn*
Abstract: A colorimetric indicator displacement assay (IDA)
amenable to high-throughput experimentation was developed
to determine the percentage of cis and trans alkenes. Using 96-
well plates two steps are performed: a reaction plate for
dihydroxylation of the alkenes followed by an IDA screening
plate consisting of an indicator and a boronic acid. The
dihydroxylation generates either erythro or threo vicinal diols
from cis or trans alkenes, depending upon their syn- or anti-
addition mechanisms. Threo diols preferentially associate with
the boronic acid due to the creation of more stable boronate
esters, thus displacing the indicator to a greater extent. The
generality of the protocol was demonstrated using seven sets of
cis and trans alkenes. Blind mixtures of cis and trans alkenes
were made, resulting in an average error of Æ 2% in the
percentage of cis or trans alkenes, and implementing E2 and
Wittig reactions gave errors of Æ 3%. Furthermore, we
developed variants of the IDA for which the color may be
tuned to optimize the response for the human eye.
exploited m-chloroperoxybenzoic acid (m-CPBA) to epox-
idize unsaturated lipids to identify the location of an olefin,
but the method was unable to differentiate stereochemistry.
Osmium tetroxide (OsO4) is a widely used reagent to
dihydroxylate alkenes generating vicinal diols.[7] Under
Upjohn conditions, catalytic amounts of OsO4 can be used
to transform alkenes into their respective diols via syn-
addition.[8] The alkene first undergoes a [3+2] cycloaddition
with OsO4 to form an osmate ester, which is readily hydro-
lyzed in aqueous systems, commonly yielding the vicinal diol
in nearly quantitative yields.[9] In contrast, epoxidation of
alkenes using m-CPBA followed by ring opening results in the
opposite stereochemistry (i.e. anti-addition).[10] Thus, by
choice of oxidant one can control which diastereomeric diol
is created.
While a few host–guest complexes with alkenes as guests
have been reported,[11] we postulated that dihydroxylation of
an alkene would be an attractive in situ derivatization
technique to convert an alkene to a functionality, that is,
a vicinal diol, that is more amenable to molecular recognition
than an alkene. For example, vicinal diols are well-known to
reversibly bind with boronic acids to generate boronate esters,
and many groups have used this equilibrium to create
chemosensors for carbohydrates.[12]
Previously, our group used the equilibrium between diols
and boronic acids to create colorimetric assays that report the
ee of chiral diols and a-hydroxycarboxylic acids.[13] Using
a chiral boronic acid host, these studies exploited an indicator
displacement assay (IDA) to generate different colors for the
enantiomeric diols. An IDA uses a colorimetric or fluorescent
probe, often a pH indicator (I), that has a different optical
signal when bound to a host (H) than when free in solution.[14]
As shown in Figure 1A, addition of a guest (G) leads to
displacement of the indicator, whose optical signal in turn
changes.
We sought to exploit the dihydroxylation of alkenes using
OsO4 or m-CPBA and an IDA to determine the percentage of
cis or trans alkenes in unknown mixtures or from various
reactions. Using 2-pyrrolidinylmethyl-phenylboronic acid as
the host (H) and pyrocatechol violet as the indicator (PV),
the resulting 1,2-diols would displace the indicator from the
host–indicator complex thereby imparting color changes.
Critical to our design was the hypothesis that the diastereo-
meric diols generated in a stereospecific fashion from the cis
and trans alkenes would have different affinities for H.
Dihydroxylation of a cis alkene using OsO4 results in an
erythro diol, which was anticipated to not favor the host–guest
complexation due to steric interactions when the R-groups
are cis to each other in the cyclic boronate ester (Figure 1B).
O
ptical methods for the rapid screening of enantiomeric
excess (ee),[1] and in some cases diastereomeric ratio,[2] of
stereocenters generated by point-chirality have come to the
forefront of high-throughput reaction screening procedures.
These optical assays have the goal of lowering the depend-
ence on serial high-performance liquid chromatography and/
or nuclear magnetic resonance (NMR) spectroscopy analyses
when examining reactions in parallel. However, no optical
methods have yet been created for alkene stereochemistry
determination. The most common approach to determine
alkene stereochemistry is to use 1H NMR coupling constants
or known chemical shifts.[3] In other instances, some form of
chromatography can be utilized when reference samples are
available.[4] In rare cases, reaction-based methods have been
reported. For example, Brown and Moerikofer[5] were able to
differentiate between cis and trans alkenes by their rate of
hydroboration. In a related endeavor, Li and colleagues[6]
[*] S. A. Valenzuela,[+] J. R. Howard, G. Saucedo, Prof. E. V. Anslyn
Department of Chemistry, University of Texas at Austin
100 E 24th Street, Norman Hackerman Building (Room 114A)
Austin, TX 78712 (USA)
E-mail: anslyn@austin.utexas.edu
H. S. N. Crory,[+] C.-Y. Yao,[+] Prof. A. P. de Silva, Prof. E. V. Anslyn
School of Chemistry and Chemical Engineering
Queen’s University Belfast
Stranmillis Road, Belfast BT9 5AG (UK)
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202101004.
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ical shift at 10.44 ppm was found, with no signals at 10.95 or
8.07 ppm for the erythro diol:H complex and H, respectively.
Additional studies using G3TH and G3ER (Table 1; 1:1
guest:H, 40 mM H, 1:1 MeOH-d4/MeCN-d3) further sup-
ported that H preferentially binds with threo diols in the
presence of erythro diols (Figure S2). These studies support
our hypothesis that due to the steric interactions shown in
Figure 1B, threo diols have a higher affinity for H compared
to erythro diols.
The indicator chosen for the IDA was pyrocatechol violet
(PV) based upon our previous work for determining the ee of
chiral diols.[13] To optimize the IDA protocol for the diols
derived from alkenes, solvent conditions were first screened.
A solvent system of methanol and acetonitrile (1:1, pH 8.4
buffered with para-toluenesulfonic acid and Hꢀnigꢁs base)
yielded the largest shift in absorbance spectra of PV upon
binding with H (Figure S3). The slightly alkaline pH value for
the IDA was selected based upon the pKa values for ortho-
amino substituted boronic acids in analogous protic solvents
because binding is known to be enhanced by pH values at or
above the pKa values.[15] Using these conditions, a UV/Vis
spectrophotometric titration of PV with H was implemented
to determine the binding constant (KH:PV = 17.1 ꢂ 103 MÀ1,
Figure S4) and the optimal concentration of the host and
Figure 1. Indicator displacement assay. A) host–indicator (H:I) com-
plex with guest (G) equilibria. B) Representative threo and erythro diol
IDA using pyrocatechol violet (PV) as the indicator and 2-pyrrolidinyl-
methyl-phenylboronic acid (H) as the host.
In contrast, when using OsO4 a trans alkene would give a threo
diol, which places the R-groups trans to one another in the
formation of the boronate ester. Thus, trans alkenes were
anticipated to have larger affinities
of their corresponding diols than
Table 1: The binding constants, KH:G, determined through UV/Vis titration and Os-reaction scheme for
the threo and erythro diols.
cis alkenes, thereby leading to
greater displacement of the indica-
tor from the host and in turn larger
color changes (Figure 1B). The use
of m-CPBA as the oxidant was
anticipated to result in the opposite
response to diols derived from cis
and trans alkenes.
Set
Alkenes
Diols
trans
cis
threo
erythro
1: R1, R2 =CH3
G1T
–
G1C
–
G1TH
0.348
G1ER
0.057
[a]
KH:G
2: R1, R2 =Ph
G2T
–
G2C
–
G2TH
11.1
G2ER
[a]
[b]
KH:G
Prior to developing the IDA
protocol depicted in Figure 1, we
turned to 11B NMR spectroscopy
3: R1 =(CH2)7CH3
G3T
–
G3C
–
G3TH
1.38
G3ER
R2 =(CH2)7COOCH3
[a]
[b]
KH:G
to verify that
H preferentially
binds with threo diols when in
competition with erythro diols.
Using d,l and meso-2,3-butanediol
as the guests and H (2:1 guest:H,
40 mM H, MeOH-d4), the H boro-
nate center has a chemical shift of
8.07 ppm. When meso-butanediol
4: R1, R2 =(CH2)2CH3
G4T
–
G4C
–
G4TH
1.10
G4ER
0.16
[a]
KH:G
5: R1 =CH3
G5T
–
G5C
–
G5TH
2.64[c]
G5ER
0.29[c]
R2 =(CH2)2CH3
[a]
KH:G
6: R1, R2 =CH(CH3)2
G6T
–
G6C
–
G6TH
3.72
G6ER
(erythro) was introduced to
H
[a]
[b]
KH:G
a new boronate ester resonance
emerged downfield at 10.95 ppm,
however,
the
resonance
at
7:
8.07 ppm was the dominate peak
(Supporting Information, Fig-
ure S1). In contrast, upon addition
of d,l-butanediol (threo) to H the
formation of the boronate ester
shifted downfield to 10.44 ppm
with no remaining peak at
8.07 ppm (Figure S1). When both
d,l- and meso-butanediol were
allowed to compete for H a chem-
G7T
–
G7C
–
G7TH
1.41[c]
G7ER
0.41[c]
[a]
KH:G
[a] 103 MÀ1. [b] Negligible measurement. [c] Binding constants determined using the 96-well plate
(Supporting Information).
2
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Figure 2. A) UV/Vis titration of host, H, into indicator PV (0.15 mM). Inset: the change in absorbance at 520 nm with the addition of H. [Ht]: host
total concentration. B) Representative IDA UV/Vis titration using G2TH. C) Representative IDA UV/Vis titration using G2ER. D) The difference in
absorbance at 520 nm with the addition of G2TH (orange) and G2ER (blue). [Gt]: total guest concentration. The KH:G2ER, binding constant for host
H is negligible and the KH:G2TH, binding constant with H is 11.1 (103 MÀ1). All IDAs were analyzed in methanol and acetonitrile (v/v=1:1, 258C,
pH 8.4 buffered with 10 mM para-toluenesulfonic acid and Hꢀnig’s base).
indicator for a subsequent IDA (Figure 2). The concentra-
tions of PV (0.15 mM) and H (0.4 mM) were chosen because
the indicator is approximately 90% saturated with host, and
thus would give a large dynamic range in the reversal of the
signal upon addition of diols generated from alkenes.
for lipid analysis. All of the analyses were conducted in
triplicates. Prior to the analysis of unknowns and reactions,
calibration curves were generated using varying mixtures of
each set of alkenes, ranging from 0:100 cis/trans alkene to
100:0 cis/trans alkene in increments of 10. For each alkene, the
calibration curves were not linear, but instead had a distinct
curvature (Figure 3A; Figure S12). At high percentages of
threo diols, as created from OsO4 with trans alkenes, the
dynamic range of the IDA was large, while changes in the
percentages of threo diol in an excess of erythro diol showed
small optical changes. This is as expected, because the erythro
diol does not displace the indicator, and not until there is
a large enough fraction of threo diol in the mixture (above
ꢀ 0.5) does a significant optical response occur.
To test the generality of the anticipated preference for
threo diols, seven sets of isomeric alkenes (Table 1) were
subjected to OsO4, under Upjohn conditions, as a synthetic
route to generate both threo and erythro diols. Trans alkenes
(or E) give threo diols whereas cis alkenes (or Z) lead to
erythro diols. Using these diols, UV/Vis titration studies were
conducted using H and PV to determine their binding
constants using fitting procedures appropriate for IDAs
(Figure 2).[16] The erythro diols G2ER, G3ER, and G6ER
gave minimal signal changes in the IDA, and hence their
binding constants were negligible. While G1ER and G4ER
did give signals that were amenable to measuring binding with
H, the binding constants were approximately seven times
smaller than the respective threo diol counterparts. Impor-
tantly, as revealed by examination of the binding constants in
Table 1, the threo diol consistently has a stronger affinity to H
compared to the erythro diol for each starting alkene set,
further confirming our design hypothesis.
To explore the diastereoselectivity of the two-step proto-
col the dihydroxylation of G2Tand G2C was carried out using
m-CPBA followed by ring opening using KOH (Figure 3A,
and 3B). As would be expected from the opposing stereo-
As a specific example, the UV/Vis spectrophotometric
titrations using G2TH and G2ER shown in Figure 2B,C
reveal the preferential binding of G2TH. Figure 2D shows
the dramatic difference in the two IDA isotherms (analogous
plots for the other diol sets are shown in Figures S5–S10).
Further, one visually notices the solution turn from red to
yellow as G2TH displaces the indicator from the host. In
contrast, for G2ER no visible color change occurs (Figure 4).
To prepare for the two-step protocol that would involve
oxidation using OsO4 or m-CPBA (assuming near quantita-
tive yields[9,10]) in reactions plates followed by IDAs in
screening plates, we set out to determine the concentrations
for the different diol sets that would give the largest differ-
ences in colors. For example, using the G2TH/G2ER set, the
absorbances were determined at 520 nm for different con-
centrations (Figure S16). The optimal concentration for this
diol set was determined to be 10 mM, translating to 10 mM
for the G2T/G2C set. For other alkene sets, different
concentrations were used (Figures S17–S22).
Figure 3. A) Representative IDA UV/Vis plate reader calibration curves
(520 nm) from screening plates consisting of G2T and G2C at 10 mM
using OsO4 (black) or m-CPBA (red). B) Comparison of the different
stereospecificities of OsO4 and m-CPBA. C) First column: UV/Vis plate
reader data at 520 nm of the G2T/G2C blinds and Wittig and E2
reactions, as well as G3T/G3C blinds. Second column: predicted %
trans alkene written as % erythro diol from oxidation with OsO4 using
the black calibration curve in part A. Third column: actual percent G2T,
written again as % erythro diol, for the blinds and the Wittig and E2
1
For demonstration purposes we focused our testing of the
two-step protocol on only the G2T/G2C and G3T/G3C sets.
G3 is a fatty acid ester and is therefore of particular interest
reactions (determined by H NMR spectroscopy). See Figure S12 for
the relevant titration curve for G3T/G3C. *See Figure S24 for the
relevant calibration curve.
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specificity of OsO4 and m-CPBA oxidations, the two calibra-
tion curves are mirror images across a vertical line at 0.5
fraction of erythro diol. Thus, to generate a large enough
percentage of threo diols for a significant optical response, the
proper oxidant needs to be chosen based upon the stereo-
chemistry of the alkene.
We next developed a two-step protocol using two 96 well-
plates. We chose OsO4 as the oxidant and G2T/G2C and G3T/
G3C as the alkenes (10 and 30 mM respectively). After OsO4
oxidation, the wells of the reaction plate were quenched with
sodium bisulfite and the solutions transferred to and filtered
through silica containing 96-well fritted plates to purify the
Os-products from NaHSO3 other byproducts. The solvent was
removed using a Genevac, and the residues were re-dissolved
and transferred to the screening plate containing H and PV. A
blank sample that lacked only the presence of an alkene was
subjected to the protocol to serve as a control sample. The
control sample confirmed our procedures lacked an absorb-
ance representative of an alkene in the IDA. Furthermore, the
control verified our procedures properly removed byproducts
during the silica purification steps, showing the absence of
residual Os-products. The absorbance of each sample was
measured at 520 nm in a UV/Vis plate reader and could be
correlated to the percentage of trans alkene (Figure 3B) using
the calibration curve in Figure 3A (black line).
Figure 4. Colorimetric IDA preference. No dye: no inert dye was
added. Green Wilton food coloring: 25 mL of green dye was added.
Teal dye: 25 mL of each dye was added. H:PV host–indicator complex;
H:G’ is the host–threo diol complex; G represents the erythro diol.
However, upon addition of the threo diol (H:PV + G2TH)
the solutions turn from burnt red to yellow, orange to lime
green, and gray to bright green, respectively (Figure 4). The
different colorimetric responses were accomplished with no
additional synthesis and can be tuned to alter the appearance
of the IDA to best suit oneꢁs eye. Interestingly, for the authors,
most felt the teal background dye gave the most obvious
response to the erythro diol, but for others, different colors
were optimal.
In conclusion, we have reported the first optical approach
for determining the percentage of cis and trans alkenes that is
amenable to a high-throughput workflow. We successfully
used two synthetic techniques to dihydroxylate alkenes, with
opposite stereospecificity, in 96-well plates. The resulting
diastereomeric 1,2-diols were analyzed using an IDA via
UV/Vis spectroscopy in 96-well plates to determine the
percent of trans or cis alkene. We were successful at
determining the percentage of blind mixtures of alkenes, as
well as two reactions as unknowns, with an average error
consistently less than Æ 3%. Furthermore, we explored the
general utility of an IDA to impart different colors for the
alkenes with no additional synthetic effort. This study gives
chemists another tool to determine the percentage of cis and
trans alkenes—one that is amenable to a high-throughput
experimentation workflow because of the use of parallel
analysis in plates instead of the use of serial NMR spectros-
copy or chromatography.
Three “blind” mixtures of G2T/G2C, as well as syntheti-
cally derived mixtures of G2T/G2C from a Wittig and E2
reaction were analyzed. The blind mixtures were made by one
of the authors and analyzed by a different author without
prior knowledge of the percentage of G2T. The percentages
of G2T from the Wittig and E2 reaction mixtures were verified
via 1H NMR spectroscopy (Supporting Information). The
calibration curve predicted the G2T content in the unknowns
to be 20%, 77%, and 91% erythro diol, respectively (Fig-
ure 3C), with an average absolute error of Æ 2% from the
actual values. Additionally, the Wittig and E2 reaction
mixtures were predicted to have 19% and ꢀ 0% G2T.
1
When verified via H NMR spectroscopy the absolute error
for the reaction mixtures was Æ 3%. As further verification of
the two-step protocol, three blind mixtures of G3T and G3C
were generated and taken through the two-step protocol.
These were analyzed and determined to be 50%, 26%, and
79% G3T with an absolute error of Æ 2% from the actual
values (Figure 3C).
We also set out to change the visual color of the solutions
containing the host–indicator complex and the liberated free
indicator. This allows one to create several different sets of
color pairings that may be better suited for oneꢁs eye
(Figure 4) such that a quick inspection of the color would
indicate the predominate alkene stereochemistry within
a mixture. Mixing fluorescent indicators have previously
been found to optimize sensitivities to pH changes.[17] It is well
known that each human has a slightly different ability to
distinguish colors.[18] As shown in Figure 4, without an inert
food color dye the host–indicator complex (H:PV) is red.
Addition of a green dye (Wilton food colorings for cakes and
icings) makes the solutions orange and addition of a teal dye
gives a grey solution. Upon addition of an erythro diol
(H:PV + G2ER) the solutions retain their original colors.
Acknowledgements
We would like to thank the Brodbelt research group,
University of Texas at Austin, for inspiring our group to
tackle this challenge. For this project, S.A.V. was funded
primarily through the NSF Graduate Research Fellowship
Program: DGE-1610403. Additionally, we would like to
thank and recognize the University of Texas at Austinꢁs
Mass Spectrometry Facility for their help and the NMR
facilities for Bruker AVANCE III 500: NIH Grant Number
1 S10 OD021508-01. This project was primarily funded
through the NSF GOALI Grant Number: 1665040. Further-
more, E.V.A. recognizes support from the Welch Regents
4
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Chair (1-0046). We would like to acknowledge the Depart-
ment of Employment and Learning of Northern Ireland and
China Scholarship Council. The authors recognize Queenꢁs
University Belfast and Prof. P. Nockemann for their contri-
butions to this collaboration.
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The authors declare no conflict of interest.
Keywords: alkene stereochemistry · colorimetric assays ·
dihydroxylation · high-throughput screening · host–
guest systems
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Manuscript received: January 21, 2021
Revised manuscript received: March 12, 2021
Accepted manuscript online: March 15, 2021
Version of record online: && &&, &&&&
Angew. Chem. Int. Ed. 2021, 60, 1 – 6
ꢀ 2021 Wiley-VCH GmbH
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Chemosensors
A high-throughput method was devel-
oped using a colorimetric indicator dis-
placement assay (IDA) to quantify the
alkene stereochemistry present within
mixtures to an accuracy of 2–3%. Within
a series of 96-well plates, alkenes were
dihydroxylated and the resulting diols
were subjected to an IDA with an indica-
tor and a boronic acid to determine the
amount of threo and/or erythro vicinal
diols present.
S. A. Valenzuela, H. S. N. Crory, C.-Y. Yao,
J. R. Howard, G. Saucedo, A. P. de Silva,
E. V. Anslyn*
&&&& — &&&&
A Colorimetric Method for Quantifying
Cis and Trans Alkenes Using an Indicator
Displacement Assay
6
www.angewandte.org
ꢀ 2021 Wiley-VCH GmbH
Angew. Chem. Int. Ed. 2021, 60, 1 – 6
These are not the final page numbers!