Quantitative Chirality and Concentration Sensing of Alcohols, Diols, Hydroxy Acids, Amines and Amino Alcohols using Chlorophosphite Sensors in a Relay Assay

Article abstract of DOI:10.1002/anie.202005324

Analytical methods that allow simultaneous determination of the concentration and enantiomeric composition of small sample amounts and are also compatible with high-throughput multi-well plate technology have received increasing attention in recent years. We now introduce a new class of broadly useful small-molecule probes and a relay sensing strategy that together accomplish these tasks with five classes of compounds including the challenging group of mono-alcohols—a scope that stands out among previously reported UV, fluorescence, and CD assays. Several chlorophosphite probes and aniline indicators have been evaluated and used for on-the-fly CD/UV sensing following a continuous workflow. The wide application range of the readily available sensors is highlighted with almost 30 alcohols, diols, hydroxy acids, amines and amino alcohols, and the accuracy of the stereochemical analysis is showcased with samples covering a wide range of concentrations and enantiomeric ratios.

Full text of DOI:10.1002/anie.202005324

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Accepted Article
Title: Quantitative Chirality and Concentration Sensing of Alcohols,
Diols, Hydroxy Acids, Amines and Amino Alcohols using
Chlorophosphite Sensors in a Relay Assay
Authors: Christian Wolf, F. Yushra Thanzeel, and Kaluvu Balaraman
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202005324
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10.1002/anie.202005324
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Quantitative Chirality and Concentration Sensing of Alcohols,
Diols, Hydroxy Acids, Amines and Amino Alcohols using
Chlorophosphite Sensors in a Relay Assay
F. Yushra Thanzeel, Kaluvu Balaraman and Christian Wolf*[a]
[a]
Ms. F. Y. Thanzeel, Prof. Dr. K. Balaraman, Prof. Dr. C. Wolf
Department of Chemistry
Georgetown University
37th and O Streets, Washington, DC 20057, USA
E-mail: cw27@georgetown.edu
Supporting information for this article is given via a link at the end of the document
Abstract: Analytical methods that allow simultaneous determination
of the concentration and enantiomeric composition of small sample
amounts and are also compatible with high-throughput multi-well plate
technology have received increasing attention in recent years. We
now introduce a new class of broadly useful small-molecule probes
and a relay sensing strategy that together accomplish these tasks with
five classes of compounds including the challenging group of mono-
alcohols - a scope that stands out among previously reported UV,
fluorescence and CD assays. Several chlorophosphite probes and
aniline indicators have been evaluated and used for on-the-fly CD/UV
sensing following a continuous workflow. The wide application range
of the readily available sensors is highlighted with almost 30 alcohols,
diols, hydroxy acids, amines and amino alcohols, and the accuracy of
the stereochemical analysis is showcased with samples covering a
wide range of concentrations and enantiomeric ratios.
composition, altogether. We have studied
a
series of
chromophoric phosphite and amidophosphite probes that
expedite comprehensive stereochemical analysis of chiral mono-
alcohols via simultaneous UV/CD analysis from a single sample.
In addition, we prove the usefulness of this new class of sensors
by demonstrating a wide application spectrum that includes
alcohols but also extends to diols, hydroxy acids, amines and
amino alcohols. The unique molecular recognition and chiroptical
sensing features are based on irreversible formation of alkyl
(amido)phosphite products exhibiting characteristic UV and
circular dichroism (CD) signals that allow combined concentration
and er analysis. While most chirality sensors introduced to date
have a narrow substrate scope we show that our phosphite
probes are very broadly useful.
To date, a wide range of optical chirality assays operating
on the principles of dynamic covalent chemistry, in particular
systems involving reversible Schiff base formation, or metal
The
introduction
of
high-throughput
experimentation
coordination,
multicomponent
assemblies,
host-guest
methodologies has greatly accelerated scientific discoveries and
streamlined efforts in numerous academic and industrial
laboratories aimed at solving complex chemical and biological
tasks under strict time constraints.1 Despite the undisputable
need for quantitative screening methods that can take full
advantage of generally available multiwell plate technology with
parallel data acquisition and processing capabilities it is still
routine to analyze one sample at a time with serial techniques.2,3
The shortage of experimental advance with these endeavors can
at least be partially attributed to the difficulty with simultaneous
determination of the concentration and enantiomeric ratio (er) of
chiral samples, and these tasks are commonly accomplished
separately by gravimetric analysis and chiral chromatography,
respectively. Recent progress with chiroptical sensing
technologies has shown that high-throughput screening of an
increasing variety of chiral compounds is now possible.4 The most
impressive examples have been achieved with chiral amines,
amino alcohols, amino acids, hydroxy acids and diols.5 But with
regard to other compound classes, for example mono-alcohols,
chiroptical on-the-fly concentration and er determination needs to
be demonstrated. In fact, stereochemical analysis of chiral mono-
alcohols by NMR spectroscopy,6 mass spectrometry7 or optical
methods8 has remained challenging.9
complexation, hydrogen bonding interactions and irreversible
substrate binding have surfaced.4c To the best of our knowledge,
stereochemical analysis with a phosphite or amidophosphite
probe has not been demonstrated. We envisioned that fast
binding of alcohol substrates with aromatic chlorophosphite
probes 1-5 should be possible and enable the complicated
stereochemical sensing tasks outlined above, Figure 1. Although
initial focus was placed on the challenging group of alcohols 6-18,
the possibility of optical sensing of diols and hydroxy acids 19-22,
amino alcohols 23-28 and amines 29-33 was also investigated.
The chlorobenzodioxaphosphite
1
was commercially
available and the analogues 2-4 were prepared in a single step
from the corresponding diols and phosphorous trichloride (see SI).
The N,N’-dibenzyl chlorobenzodiazaphophite 5 was synthesized
in three high-yielding steps as shown in Scheme 1. The ability of
the sensors 1-5 to differentiate between the enantiomers of 1-
phenylethanol, 6, and 1-phenylbutanol, 7, was then tested under
various conditions.
We now wish to introduce a chiral alcohol relay sensing
strategy that can accomplish three tasks, i.e. determination of the
absolute configuration, sample concentration and enantiomeric
1
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Scheme 1. Synthesis of sensor 5 (top) and CD signatures of the phosphites
derived from probes
1 and 5, respectively, and the enantiomers of 1-
phenylethanol, 6 (bottom). For comparison, the CD spectrum of the free alcohol
(R)-6 under the same conditions is shown in orange. The CD measurements
were conducted in chloroform at 0.75 mM (left) and 0.22 mM (right). See SI for
details.
Encouraged by these initial findings, we decided to test the
suitability of the chloroamidophosphite 5 for enantioselective
recognition and analysis of the enantiomeric ratio of samples with
various amounts of 6, Table 1 and SI. In all cases, the formation
of the amidophosphite 34 allowed correct identification of the
absolute configuration of the major enantiomer by comparison
with a reference sample and determination of the enantiomeric
composition of the nonracemic samples with high accuracy. For
example, the sensing of the samples containing the (R)- and (S)-
6 in a 95.0:5.0 and 35.0: 65.0 ratio gave 94.6:5.4 and 34.9:65.1,
respectively, entries 1 and 4.
Table 1. Chiroptical determination of the enantiomeric ratio and absolute
configuration of samples of 1-phenylethanol, 6, using probe 5.
Figure 1. Structures of chlorophosphite probes 1-5 and chiral alcohol, diol,
hydroxy acid, amino alcohol and amine target compounds 6-33. Only one
enantiomer is shown.
We were pleased to find that all probes generate CD signals
that are easily obtained by a mixing stoichiometric amounts of the
chlorophosphite and the analyte in the presence of
diisopropylethylamine, see Scheme 1 and SI. The phosphite
formation was verified with several substrates by ESI-MS and
NMR analysis. Importantly, the sensing reaction is very fast, it is
complete within 3 minutes and readily occurs in common organic
solvents, including CHCl3, CH2Cl2, THF and ACN, at room
temperature which underscores the potential of this sensing
assay in high-throughput screening applications.
Sample composition
Sensing results
Entry
Conc.
(mM)
Abs.
config.
Abs.
erb
er
config.a
1
2
3
4
5
5
R
S
R
S
R
95.0:5.0
15.0:85.0
75.0:25.0
35.0:65.0
95.0:5.0
R
S
R
S
R
94.6:5.4
18.8:81.2
78.3:21.7
34.9:65.1
95.5:4.5
10
15
18
19
aBased on the sign of the induced Cotton effects. bThe er was calculated based
on the CD signal at 300 nm. See SI for details.
2
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We then continued with the evaluation of the substrate scope
by applying the standard sensing protocol with probes 1 and 5 to
a large variety of alcohols 6-18 including aliphatic substrates and
natural products such as menthol and isopulegol. In all cases, we
found that the alcohol binding induces a characteristic CD signal
that can be utilized for enantioselective analysis. Representative
sensing results with 11, 12, 14 and 18 are shown in Figure 2.
Based on the general usefulness with mono-alcohols, we
expected that the application spectrum of our sensors would
extend to other important target compounds and we therefore
applied our chiroptical assay to the diols, hydroxy acids, amino
alcohols and amines 19-33. Again, we observed smooth
phosphite formation under mild conditions in the presence of base
and simultaneous induction of strong chiroptical signals at
submillimolar concentrations, Figure 2. The successful testing
with a total of 28 structurally diverse analytes demonstrates that
chirality sensing with aromatic chlorophosphites is broadly useful
and a practical tool for the determination of the absolute
configuration and enantiomeric composition of several compound
classes.
To develop a robust optical assay that can accomplish on-the-
fly sensing of the enantiomeric ratio and total concentration of
chiral compounds we explored the tandem use of sensor 1 and
aniline derived UV indicators. The general concept of this relay
assay is shown in Scheme 2. First, the phosphite formation of an
unknown amount of the alcohol substrate that may be present in
up to 25.0 mM with a full equivalent of 1 matching the maximal
possible analyte concentration is used for the determination of the
absolute configuration and enantiomeric ratio via CD analysis as
described above. The remaining excess of unreacted 1 is then
captured with an aniline indicator to generate a quantifiable UV
signal that is correlated to the original analyte concentration.
Several candidates were screened for this purpose and the
shortest reaction time together with a distinct UV change that can
be used for accurate determination of the original alcohol amount
were obtained with para-anisidine 38 (SI).
Figure 2. Representative CD responses of probes 1 and 5 to alcohols, hydroxy
acids, amino alcohols and amines. All measurements were performed at 0.13-
0.40 mM in chloroform. For more details, see SI.
Scheme 2. a) Workflow of the comprehensive UV/CD sensing of 1-phenylethanol using probe 1 and p-anisidine as indicator. b) Chiroptical probe responses. Left:
CD response of probe 1 at 273 nm to scalemic samples of 1-phenylethanol. Right: UV response at 308 nm to different concentrations of 1-phenylethanol in the
presence of 1 and the indicator. See SI for details.
3
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This chiroptical sensing workflow was first tested with
samples containing the alcohol 6 in varying amounts and
enantiomeric ratios to quantitatively correlate the variation in the
corresponding chiroptical readouts to the change of the analyte
concentration and er. Having established the relay flow we then
attempted the comprehensive sensing analysis of several alcohol
mixtures. The samples were subsequently treated with the
chlorophosphite and the aniline probe and the resulting mixtures
containing 37 and 39 in varying quantities were then subjected to
CD/UV analysis, Table 2. The results prove that the optical relay
sensing concept is very reliable and generates accurate
concentration and er data. The analysis of a nonracemic solution
of 6 with an S:R ratio of 83.4:16.6 and a total concentration (both
enantiomers combined) of 6.0 mM gave 84.1:15.9 and 6.2 mM,
see entry 1.The sensing of other mixtures with vastly different
amounts and enantiomeric compositions was also successful and
we obtained relatively small error margins that are acceptable for
high-throughput screening purposes, entries 2-7. The recording
of CD and UV spectra can be accomplished simultaneously by
modern CD spectrophotometers which takes full advantage of the
inherent speed of the underlying reactions and the continuous
relay workflow. The reproducibility and robustness of this
approach were validated with samples containing similar amounts
of acetophenone, see SI. We like to point out that chirality sensing
with chlorophosphites is not restricted to one particular probe
scaffold although the general scope and usefulness were
evaluated in depth using 1 and 5. Overall, these proved equally
valuable. However, 1 is commercially available which is certainly
advantageous.
altogether underscores the value and practicality of the sensing
with 1-5 described herein, Scheme 3.
Scheme 3. Sensing of the enantiomers and a racemic mixture of alcohol 6 with
(P)-40. The reactions were conducted at 20.0 mM and the CD spectra were
collected at 0.20 mM in chloroform.
In summary, we have introduced a new class of chiroptical
probes and a relay assay sensing strategy that enable accurate
on-the-fly stereochemical analysis of mono-alcohols, diols,
hydroxy acids, amines and amino alcohols – a variety that stands
out among previously reported UV, fluorescence and CD assays.
The chlorophosphite CD probes 1-5 and several aniline derived
UV indicators were evaluated and the commercially available
chlorobenzodioxaphosphite and para-anisidine were combined
into a continuous sensing workflow. The practicality of this
approach and the accuracy of the chiroptical concentration and
enantiomeric ratio analysis were demonstrated with seven 1-
phenylethanol samples. It is envisioned that this assay can be
adapted to high-throughput equipment and multiwell plate
technology which would allow fully automated operation and
simultaneous screening of hundreds of samples in parallel.
Table 2. Simultaneous chiroptical sensing of the concentration, absolute
configuration and enantiomeric ratio of nonracemic samples of 6.
Acknowledgements
We gratefully acknowledge financial support from the U.S.
National Science Foundation (CHE-1764135).
Sample composition
Sensing results
Entry
Conc.
Abs.
Conc.a Abs.
(mM) config.b
er
erc
(mM) config.
1
2
3
4
5
6
7
6.0
S
S
R
S
R
S
S
83.4:16.6
85.0:15.0
6.2
9.9
S
S
R
S
R
S
S
84.1:15.9
82.9:17.1
22.3:77.7
67.5:32.5
34.1:65.9
61.8:38.2
96.9:3.1
Keywords: Optical sensing • chirality • circular dichroism • UV
spectroscopy • chlorophosphites
10.0
13.0
16.0
18.0
20.0
24.0
25.0:75.0 13.8
71.9:28.1 15.6
33.3:66.7 18.6
62.5:37.5 20.5
[1]
[2]
[3]
a) A. McNally, C. K. Prier, D. W. C. MacMillan, Science 2011, 334, 1114-
1117. b) A. B. Santanilla, E. L. Regalado, T. Pereira, M. Shevlin, K.
Bateman, L.-C. Campeau, J. Schneeweis, S. Berritt, Z. -C. Shi, P.
Nantermet, Y. Liu, R. Helmy, C. J. Welch, P. Vachal, I. W. Davies, T.
Cernak, S. D. Dreher, Science 2015, 347, 49-53. c) K. D. Collins, T.
Gensch, F. Glorius, Nat. Chem. 2014, 6, 859-871.
97.9:2.1
22.5
For examples of fast chiral HPLC enantioseparations: a) D. Kotoni, A.
Ciogli, C. Molinaro, I. D’Acquarica, J. Kocergin, T. Szczerba, H. Ritchie,
C. Villani, F. Gasparrini, Anal. Chem. 2012, 84, 6805-6813. (b) C. L.
Barhate, L. A. Joyce, A. A. Makarov, K. Zawatzky, F. Bernardoni, W. A.
Schafer, D. W. Armstrong, C. J. Welch, E. L. Regalado, Chem. Commun.
2017, 53, 509-512.
aBased on the UV change at 308 nm. bBased on the sign of the induced Cotton
effects. cThe er was calculated based on the CD amplitude measured at 273
nm. See SI for details.
Finally, we examined if one could use an atropisomeric
BINOL derived chlorophosphite probe for sensing, too. We
expected that (P)-40 would have a strong CD signal originating
from the BINOL moiety that may be selectively altered upon
binding of either (R)- or (S)-1-phenylethanol, 6, because
diastereomeric products 41 are formed. This would be
fundamentally different from the use of the CD-silent
chlorophosphites 1-5 which are achiral or tropos-type agents that
give enantiomeric products with the chiral target compounds 6-33
thus generating induced CD effects. Interestingly, we found that
sensing of enantiopure 6 and its racemate with (P)-40 yields
essentially the same CD spectrum and therefore does not allow
chiroptical enantiodifferentiation and ee determination which
For recent advances with NMR chiral solvating agents, see a) L. Yang,
T. Wenzel, R. T. Williamson, M. Christensen, W. Schafer, C. J. Welch,
ACS Cent. Sci. 2016, 2, 332-340. b) G. Storch, M. Haas, O. Trapp, Chem.
Eur. J. 2017, 23, 5414-5418. c) Q. H. Luu, K. G. Lewis, A. Banerjee, N.
Bhuvanesh, J. A. Gladysz, Chem. Sci. 2018, 9, 5087-5099 and
references therein.
[4]
[5]
a) D. Leung, S. O. Kang, E. V. Anslyn, Chem. Soc. Rev. 2012, 41, 448-
479. b) C. Wolf, K. W. Bentley, Chem. Soc. Rev. 2013, 42, 5408-5424.
For a recent Perspective on this topic: c) B. T. Herrera, S. L. Pilicer, E.
V. Anslyn, L. A. Joyce, C. Wolf, J. Am. Chem. Soc. 2018, 140, 10385-
10401.
a) S. H. Shabbir, J. R. Clinton, E. V. Anslyn, Proc. Natl. Acad. Sci. USA
2009, 106, 10487-10492. b) S. Nieto, J. M. Dragna, E. V. Anslyn, Chem.
4
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10.1002/anie.202005324
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COMMUNICATION
Eur. J. 2010, 16, 227-232. c) K. W. Bentley, D. Proano, C. Wolf, Nat.
Commun. 2016, 7, 12539. d) E. G. Shcherbakova, E. G.; V. Brega, V. M.
Lynch, T. D. James, P. Anzenbacher, Chem. Eur. J. 2017, 23, 10222-
10229. e) K. W. Bentley, P. Zhang, C. Wolf, C. Science Advances 2016,
2, e1501162. f) F. Biedermann, W. M. Angew. Chem. Int. Ed. 2014, 53,
5694-5699. g) T. A. Feagin, D. P. Olsen, Z. C. Headman, J. M. Heemstra,
J. Am. Chem. Soc. 2015, 137, 4198-4206. h) Z. A. De los Santos, C.
Wolf, J. Am. Chem. Soc. 2016, 138, 13517-13520. i) L. A. Joyce, E. C.
Sherer, C. J. Welch, Chem. Sci. 2014, 5, 2855-2861. j) F. Y. Thanzeel,
K. Balaraman, C. Wolf, Nat. Comm. 2018, 9, 5323. (k) Thanzeel, F. Y.
Sripada, A.; Wolf, C. J. Am. Chem. Soc. 2019, 141, 16382-16387. l) M.
E. Shirbhate, S. Kwon, A. Song, S. Kim, D. Kim, H. Huang, Y. Kim, H.
Lee, S.-J. Kim, M.-H. Baik, J. Yoon, K. M. Kim, J. Am. Chem. Soc. 2020,
142, 4975-4979.
[6]
[7]
a) C. Wolf, A. M. Cook, J. E. Dannatt, Tetrahedron: Asymm. 2014, 25,
163-169. b) L. Yang, T. Wenzel, R. T. Williamson, M. Christensen, W.
Schafer, C. J. Welch, ACS Central Sci. 2016, 3, 332-340. c) G. Bian, S.
Yang, H. Huang, H. Zong, L. Song, H. Fan X. Sun, Chem. Sci. 2016, 7,
932-938. d) M.-S. Seo, S. Jang, H. Kim, Chem. Commun. 2018, 54,
6804-6808.
a) J. Guo, J. Wu, G. Siuzdak, M. G. Finn, Angew. Chem. Int. Ed. 1999,
38, 1755-1758. b) M. T. Reetz, M. H. Becker, H.‐W. Klein, D. Stöckigt,
Angew. Chem. Int. Ed. 1999, 38, 1758-1761. Asymmetric reaction MS
analysis: c) C. Markert, A. Pfaltz, Angew. Chem. Int. Ed. 2004, 43, 2498-
2500. d) C. Markert, A. Pfaltz, J. Am. Chem. Soc. 2008, 130, 3234-3235.
A review on this topic: e) S. Piovesana, R. Samperi, A. Lagana, M. Bella,
Chem. Eur. J. 2013, 19, 11478-11494.
[8]
Examples of optical methods that allow determination of the absolute
configuration of mono-alcohols: a) K. Kobayashi, Y. Asakawa, Y. Kikuchi,
H. Toi, Y. Ayoma, J. Am. Chem. Soc. 1993, 115, 2648-2654. b) J. M.
Lintuluoto, V. V. Borovkov, Y. Inoue J. Am. Chem. Soc. 2002, 124,
13676-13677. c) S. Hayashi, M. Yotsukura, M. Noji, T. Takanami, Chem.
Commun. 2015, 51, 11068-11071. d) S. Shimo, K. Takahashi, N.
Iwasawa, Chem. Eur. J. 2019, 25, 3790-3794. e) T. Madry, A. Czapik, M.
Kwit, ACS Omega 2019, 4, 3244-3256. Methods that allow er analysis of
mono-alcohols: f) L. You, J. S. Berman, E. V. Anslyn, Nat. Chem. 2011,
3, 943–948. g) L. You, J. S. Berman, A. Lucksanawichien, . E. V. Anslyn,
J. Am. Chem. Soc. 2012, 134, 7117-7125. h) L. You, G. Pescitelli, E. V.
Anslyn, L. Di Bari. J. Am. Chem. Soc. 2012, 134, 7126-7134. i) C. Ni, D.
Zha, H. Ye, Y. Hai, Y. Zhou. E. V. Anslyn, L. You, Angew. Chem. Int. Ed.
2018, 57, 1300-1305. j) H. H. Jo, X. Gao, L. You, E. V. Anslyn, M. J.
Krische, Chem. Sci. 2015, 6, 6747-6753.
[9]
For a general overview of analytical methods: C. Wolf in Dynamic
Stereochemistry of Chiral Compounds - Principles and Applications.
RSC Publishing, Cambridge, UK, 2008, pp. 136-179.
5
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Entry for the Table of Contents
Achiral chlorophosphite probes enable accurate optical analysis of the concentration and enantiomeric ratio of chiral alcohols, diols,
hydroxy acids, amino alcohols and amines. The broad utility of these readily available chiroptical sensors stands out among
previously reported UV, fluorescence and CD probes which is highlighted with almost 30 examples including natural compounds.
6
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