An enantioselective fluorescence sensing assay for quantitative analysis of chiral carboxylic acids and amino acid derivatives

Article abstract of DOI:10.1039/b609880k

A chiral 1,8-diacridylnaphthalene-derived fluorosensor exhibiting a C ₂-symmetric cleft designed for stereoselective interactions with hydrogen bond donors has been used for the determination of both concentration and enantiomeric composition of carboxylic acids and amino acid derivatives. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b609880k

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An enantioselective fluorescence sensing assay for quantitative analysis
of chiral carboxylic acids and amino acid derivatives{
Christian Wolf,* Shuanglong Liu and Brian C. Reinhardt
Received (in Cambridge, UK) 11th July 2006, Accepted 9th August 2006
First published as an Advance Article on the web 1st September 2006
DOI: 10.1039/b609880k
A chiral 1,8-diacridylnaphthalene-derived fluorosensor exhibit-
ing a C2-symmetric cleft designed for stereoselective interactions
with hydrogen bond donors has been used for the determina-
tion of both concentration and enantiomeric composition of
carboxylic acids and amino acid derivatives.
The prospect of high-throughput screening of asymmetric reac-
tions has directed increasing attention to the development of
enantioselective UV and fluorescence methods during recent
years.1 Fluorescence spectroscopy offers a variety of advantages
over traditional chromatographic and NMR spectroscopic tech-
niques such as different detection modes (fluorescence quenching,
enhancement, and lifetime measurements), high sensitivity, low
cost of instrumentation, waste reduction, and time-efficiency.2 The
potential of fluorescence sensing for the determination of the
Fig. 1 Structure of fluorosensor 1 and one-dimensional conformational
enantioselectivity of asymmetric reactions has been demonstrated
flexibility of 1,8-diacridylnaphthalenes.
with the titanium tartrate-catalyzed addition of trimethylsilyl
cyanide to an immobilized aldehyde and with the enzymatic kinetic
its use as a practical fluorosensor for the determination of both
resolution of trans-1,2-diaminocyclohexane by Pu et al. and our
group.3 In both cases, fluorescence sensing was found to provide
enantiomeric excess and concentration of mono- and multi-
functional carboxylic acids and amino acids.
accurate ee’s and proved advantageous over laborious and time-
The synthesis of
1 involved regioselective acridine ring
consuming chromatographic methods. Anslyn et al. reported a
practical approach to enantioselective fluorosensing of bifunctional
a-hydroxycarboxylates and diols based on indicator-displacement
assays with chiral boronic acid receptors.4 However, a method
providing the enantiomeric composition and concentration for a
wide range of mono- and multifunctional carboxylic acids and
amino acid derivatives has been elusive to date.
construction from N-3-tert-butylphenylanthranilic acid 5 which
was obtained from 4-tert-butylbromobenzene 2 in three steps.7
Lithiation and subsequent stannylation of 9-bromo-3-tert-butyla-
cridine 6 produced 7, which was then employed in a Stille cross-
coupling with 1,8-dibromonaphthalene to give 1,8-diacridyl-
naphthalene 1, Scheme 1. Slow evaporation of a racemic mixture
of 1 in isopropyl alcohol–hexanes (1 : 1) at room temperature
produced a single crystal suitable for X-ray analysis, Fig. 2.{ The
Previous X-ray and NMR spectroscopic studies conducted in
our laboratories have shown that 1,8-diquinolyl- and 1,8-
diacridylnaphthalenes are highly congested, rigid structures that
possess remarkable one-dimensional flexibility.5 While the two
cofacial heteroaryl rings perpendicular to the naphthalene frame-
work show little splaying, the torsional angle, t, can change over a
range of 50u, in particular upon binding to a hydrogen bond
donor. The usefulness of these fluorescent sensors for the
enantioselective analysis of chiral compounds has been attributed
to this one-dimensional conformational flexibility which facilitates
the accommodation of substrates of varying size in the C2-
symmetric pocket, Fig. 1.6 We wish to report the synthesis of
axially chiral anti-1,8-bis(39-tert-butyl-99-acridyl)naphthalene 1 and
Department of Chemistry, Georgetown University, Washington,
DC 20057, USA. E-mail: cw27@georgetown.edu;
Fax: (+1) 202 687 6209; Tel: (+1) 202 687 3468
{ Electronic supplementary information (ESI) available: Synthesis and
characterization of 1. Stern–Volmer and Benesi–Hildebrand plots and
fluorosensing measurements of the concentration and enantiopurity of
several samples. See DOI: 10.1039/b609880k
Scheme 1 Synthesis of 1,8-diacridylnaphthalene 1.
4242 | Chem. Commun., 2006, 4242–4244
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Fig. 2 Single crystal structure of 1.
splaying and torsional angles between the acridyl rings of 1 were
determined as 5.3u and 23.4u, respectively. Because of the inherent
fluorescence and one-dimensional flexibility of 1,8-diacridyl-
naphthalenes in solution, this C2-symmetric bidentate ligand was
expected to effectively embed hydrogen bonding interactions with
chiral carboxylic acids and amino acids into a highly stereoselective
environment. We anticipated that the formation of diastereomeric
adducts could be conveniently measured and quantified by
fluorescence spectroscopy.
Fig. 4 Stern–Volmer plots showing enantioselective fluorescence
quenching of (2)-1 in the presence of 8 and 9 (top). Sensing of the
enantiomers of 8 with (+)-1 (bottom). Sensor concentration: 3.5 6
1026 M. Excitation (emission) wavelength: 360 nm (535 nm).
After the screening of several chiral HPLC columns, we found
that the enantiomers of anti-1 can be resolved on a Chiralpak AD
column. Enantiopure 1 (excitation at 360 nm, emission maximum
at 535 nm) and carboxylic acids and amino acids 8–19, Fig. 3, were
then employed in fluorescence titration experiments using
acetonitrile as solvent. We were pleased to find that a concentra-
tion of 3.5 6 1026 M of the fluorosensor 1 suffices to effectively
differentiate between minute quantities of the enantiomers of all
analytes studied.
diacridylnaphthalene 1 exhibits a strong fluorescent signal that
effectively responds to chiral interactions with 8–19 at low
concentration, it better exploits the inherent sensitivity of
fluorescence spectroscopy than previously reported sensors and
extends this technique to enantioselective analysis of minute
sample amounts.
With this new efficient sensor in hand, we developed a
fluorescence method that allows accurate measurements of both
the total amount and the enantiomeric excess of a chiral
compound. This was realized by the combination of two assays
using racemic and enantiopure sensor 1. First, the total
concentration of a chiral analyte with unknown enantiomeric
composition was determined by fluorescence sensing with racemic
diacridylnaphthalene 1. As expected, fluorescence titration of
racemic 1 using either enantiomer of 2-chloropropionic acid 8
gave superimposable Stern–Volmer plots, see ESI.{ We
therefore envisioned that (¡)-1 could be used for quantitative
For example, the (R)-enantiomers of a-halogenated carboxylic
acids 8 and 9 showed little quenching whereas the fluorescence of
(2)-1 decreased dramatically even when the (S)-enantiomers were
present at only millimolar concentrations, Fig. 4.
Diacridylnaphthalene 1 has two potential binding sites and can
undergo simultaneous hydrogen bonding with two substrates.
Benesi–Hildebrand plots revealed that (2)-1 forms stronger 1 : 2
complexes with (S)-8 and (S)-9 than with the corresponding (R)-
enantiomers which explains the more pronounced fluorescence
quenching observed with the levorotatory sensor. As expected, the
(+)-sensor affords opposite enantioselectivity, Fig. 4. We were
pleased to find that 1 also differentiates between the enantiomers
of carboxylic acids and amino acids 10–19, see ESI.{ Since
non-stereoselective analysis of
a chiral sample, while the
enantiomeric excess could then be uncovered using the
enantiopure sensor in a succeeding assay. In order to evaluate
the accuracy and reproducibility of our sensing method, we
prepared different samples of chiral acid 8 having concentrations
of 1.5 and 3.0 mM, respectively, and enantiopurities varying from
5 to 95%. Through comparison of the averaged fluorescence
response of racemic 1 with a calibration curve we calculated
concentrations of 8 ranging from 1.47 to 1.51 and 2.97 to 3.06 mM,
respectively. Having determined the individual sample
concentrations, we were then able to uncover the enantiomeric
composition based on fluorescence quenching experiments with
(2)-1, Table 1.
In general, the results obtained for the six samples were
within ¡2% of the actual concentration of 2-chloropropionic
acid 8 and within ¡3% of the actual enantiopurity. For
example, fluorescence analysis of sample C (1.50 mM, 95.0%
(S)-8) gave a concentration of 1.51 mM and 96.8% (S)-8. Excellent
Fig. 3 Structures of chiral carboxylic acids and amino acids.
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Chem. Commun., 2006, 4242–4244 | 4243

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Table 1 Concentration and enantiomeric composition of six samples
of acid 8 determined by fluorescence quenching with (2)-1
Notes and references
{ Crystal structure data for 1?i-PrOH: formula C47H43N2O, M = 652.34,
¯
Actual
concentration/
mM
Calculated
concentration/
mMa
crystal dimensions: 0.1 6 0.1 6 0.1 mm, triclinic, space group P1, a =
˚
˚
˚
Actual
% (S)
Calculated
% (S)a
10.7101(14) A, b = 11.3527(15) A, c = 15.434(2) A, a = 77.800(3)u, b =
87.817(3)u, c = 76.455(3)u, V = 1783.1(4) A , Z = 2, rcalcd = 1.134 g cm23
,
3
˚
Sample
m = 0.070 mm21, range of h for data collection: 1.35 to 28.45u, T =
173(2) K, 8379 independent reflections (Rint = 0.1305), R1 = 0.0828,
A
B
C
D
E
F
a
1.50
1.50
1.50
3.00
3.00
3.00
5.0
55.0
95.0
15.0
55.0
85.0
1.47
1.48
1.51
2.97
3.02
3.06
6.4
56.2
96.8
16.2
56.9
87.8
wR2 = 0.1815 with I . 2s(I), GooF = 0.907, Drmax = 0.379 e A23, Drmin
=
˚
20.322 e A23. The crystal contains a disordered molecule of isopropyl
alcohol in the asymmetric unit. Single crystal X-ray diffractions were
performed at 2100 uC using a Siemens platform diffractometer with
˚
˚
graphite monochromated Mo-Ka radiation (l = 0.71073 A). Data were
integrated with the Siemens SAINT program8a and corrected for the effects
of absorption using SADABS.8b The structures were solved by direct
methods and refined with full-matrix least-square analysis using SHELX-
97-2 software.8c Non-hydrogen atoms were refined with anisotropic
displacement parameters and all hydrogen atoms were placed in calculated
positions and refined with a riding model. CCDC 606070. For crystal-
lographic data in CIF or other electronic format see DOI: 10.1039/
b609880k
Average of three fluorescence measurements at 535 nm.
results were also obtained with the other samples, including A and
D which contained substantial quantities of the (R)-enantiomer.
The data demonstrate the high reproducibility and accuracy of this
method which is suitable for the screening of samples covering a
wide range of enantiopurities and a high excess of either
enantiomer.
1 (a) L. Pu, Chem. Rev., 2004, 104, 1687–1716; (b) L. Zhu and E. V. Anslyn,
J. Am. Chem. Soc., 2004, 126, 3676–3677.
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374, 345–347; (b) Y. Yan and M. L. Myrick, Anal. Chem., 1999, 71,
1958–1962; (c) G. Beer, K. Rurack and J. Daub, Chem. Commun., 2001,
1138–1139; (d) M. Reetz and S. Sostmann, Tetrahedron, 2001, 57,
2515–2520; (e) J. Lin, Q.-S. Hu, M.-H. Xu and L. Pu, J. Am. Chem. Soc.,
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Chem., Int. Ed., 2004, 43, 3461–3464; (g) Z.-B. Li, J. Lin and L. Pu,
Angew. Chem., Int. Ed., 2005, 44, 1690–1693.
3 (a) Z.-B. Li, J. Lin, Y.-C. Qin and L. Pu, Org. Lett., 2005, 7, 3441–3444;
(b) G. E. Tumambac and C. Wolf, Org. Lett., 2005, 7, 4045–4048.
4 L. Zhu, Z. Zhong and E. V. Anslyn, J. Am. Chem. Soc., 2005, 127,
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X. Mei and C. Wolf, J. Org. Chem., 2005, 70, 2299–2305; (d) G.
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6 (a) X. Mei and C. Wolf, Chem. Commun., 2004, 2078–2079; (b) X. Mei
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8 (a) G. M. Sheldrick, SAINT, Bruker AXS Inc., USA, 2003; (b)
G. M. Sheldrick, SADABS, Program for area detector adsorption
correction, Institute for Inorganic Chemistry, University of Go¨ttingen,
Germany, 1996; (c) G. M. Sheldrick, SHELX-97-2, University of
Go¨ttingen, Germany, 1997.
In conclusion, we have prepared anti-1,8-bis(39-tert-butyl-
99-acridyl)naphthalene 1 exhibiting a highly congested C2-sym-
metric pocket for chiral recognition of carboxylic acids and amino
acid derivatives and demonstrated its use for practical stereo-
selective fluorescence analysis. Because 1 affords a strongly
fluorescent signal that effectively responds to enantioselective
interactions with chiral acids, this new sensor is suitable for
quantitative analysis of minute sample amounts. We have
developed a simple method that utilizes diacridylnaphthalene 1
in two facile fluorescence sensing assays. We believe that this
approach combines several attractive features: it allows determina-
tion of both concentration and ee by the use of one sensor (in its
racemic and enantiopure form); it depends on simple assays that
provide accurate values with high reproducibility; it eliminates the
need for substrate derivatization; and it utilizes a cost-effective and
sensitive technique (fluorescence spectroscopy) that minimizes
solvent waste.
Funding from the NSF (CAREER Award, CHE-0347368) and
the PRF administered by the ACS (PRF40897-G4) is gratefully
acknowledged.
4244 | Chem. Commun., 2006, 4242–4244
This journal is ß The Royal Society of Chemistry 2006