Fast parallel enantiomeric analysis of unmodified amino acids by sensingwith fluorescent b-cyclodextrins

Roberto Corradini,*a Cristina Paganuzzi,a Rosangela Marchelli,a Sara Pagliari,{a Stefano Sforza,a

Arnaldo Dossena,a Gianni Galavernaa and Alexander Duchateaub

Received 7th December 2004, Accepted 14th February 2005

First published as an Advance Article on the web 28th February 2005

DOI: 10.1039/b418369j

Modified cyclodextrins bearing a metal binding site and a dansyl fluorophore 6-deoxy-6-N-(Na-

[(5-dimethylamino-1-naphthalenesulfonyl)aminoethyl]-aminocylamino-b-cyclodextrin, containing

L-Phe (S-1), L-PhGly (S-2) or L-Pro (S-3) moieties, were used as enantioselective fluorescence

sensors for the discrimination of enantiomers of the amino acids valine and proline, according to a

ligand exchange mechanism. The best conditions to perform enantiomeric analyses were studied

and a fast protocol using fluorescence quenching by the copper(II)/amino acid complexes in a

fluorescence microplate reader was developed, allowing the detection of samples with high

enantiomeric excess. Calibration of the fluorescence response as a function of enantiomeric

composition was obtained using the Stern–Volmer model; the calibration curves showed good

linearity, allowing fast evaluation of enantiomeric excess within 6% error. Two calibration curves

and triplicate analyses of six valine samples were performed in two minutes.

Introduction

The use of combinatorial approaches to chemical synthesis

has been applied in recent years not only to the field of drug

discovery, but also to development of new materials, devices

and catalysts.1 In this context, high throughput techniques

play a central role in the screening of the properties of the

enormous number of new structures which can be developed.

A very important task is to find new enantioselective devices

for fast assessment of the enantiomeric excess, which can be

useful in many fields, including stereoselective synthesis2 and

enantioselective catalyst development.3

High throughput enantioselective analyses have been

described using chiroptical methods,4 UV,5 fluorescence,6

mass spectrometry,7,8 or antibody-based sensors.9 The sensing

process can be based on enantioselective complexation by a

chiral selector, on kinetic resolution with pseudoenantiomers

labelled with different mass tags or fluorophores, or on

amplification of supramolecular chirality in liquid crystals.10 A

very high number of samples can be analyzed using this

approach in combination with emerging technologies (e.g.

microarray systems).2,6

The development of new fast enantioselective methods is

also important in the design of new sensors as components of

‘‘artificial tongue’’ devices able to perform sensory evaluation;

in this type of applications, although good chemoselectivity

has been achieved,11 enantioselectivity is still a crucial point,

since the taste in natural tongue is in most cases strongly

dependent on stereochemistry: for example most D-amino

acids are sweet, while their L-counterparts are bitter.

Enantioselective fluorescent sensors are of great interest in

order to produce optical devices for ee measurement, and most

of them have been recently reviewed.12 Nevertheless, only in a

few cases has the use of this type of sensors been extended to

very fast measuring techniques.2

Cyclodextrins modified with metal binding moieties allow

the coordination of chiral guest molecules around the metal

ion.13 For example, enantiomeric recognition of unmodified

amino acids was performed using the copper complex of a

histamine-modified b-cyclodextrin and related molecules.14,15

In this context, we have developed a model of fluorescent

sensors based on modified b-cyclodextrins containing a

binding site for copper(II) and a dansyl fluorophore.16,17

Similar systems were also designed for enantiomeric

recognition of unmodified amino acids. The design is described

in Fig. 1 and takes into account the following elements: i) a

cyclodextrin as a rigid scaffold; ii) a copper(II) coordinating

side arm similar to those used as selectors in ligand exchange

chromatography;18–22 iii) a dansyl group as fluorophore, able

to interact with both the cyclodextrin cavity and the copper(II)

ion; iv) an additional chiral center in the side arm, which could

enhance enantioselectivity. The ligands are terdentate, with an

amide, an amine and a sulfonamide group as copper(II)

binding sites. The copper(II) ion may in turn act as binding site

for bifunctional moieties, in particular a-amino acids.

The modified cyclodextrins synthesized according to this

model (Fig. 1) were found to produce enantioselective

fluorescence responses in the presence of amino acids and

copper(II).23 The mechanism of enantioselective sensing and

the influence of the structural features of the two cyclodextrins

were investigated using 1D and 2D NMR, circular dichroism,

and steady-state and time-resolved fluorescence measurements.

The cyclodextrin cavity was found to play an important role in

enantioselectivity, since the side arm alone could not perform

efficient enantioselective sensing. Enantioselectivity was found

{ Present address: Callegari S.p.A., Via Adamello 2/A, I-43100 Parma,Italy.*roberto.corradini@unipr.it

PAPER www.rsc.org/materials | Journal of Materials Chemistry

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to be dependent on the stereochemistry and on the conforma-

tional properties of the cyclodextrin derivative, with the

cyclodextrin–fluorophore interaction strongly enhancing the

enantioselectivity of the process.24

In the presence of copper(II) and of amino acids, both binary

Cu2+/cyclodextrin and ternary Cu2+/cyclodextrin/amino acid

complexes can be formed. Time resolved fluorescence measure-

ments showed that both of them are non-fluorescent species,

due to the strong quenching effect of the copper(II) ion. The

chiral discrimination was found to be due to the formation of

diastereomeric ternary complexes of different stabilities.25

Since for some amino acids (such as proline and valine)

enantioselectivity was found to be high, we explored the

possibility of using this chiral sensors for the determination of

enantiomeric excess using fast, simple and low-cost techno-

logy, such as fluorescence microplate readers widely used for

screening tests.

Results and discussion

We chose cyclodextrins S-1–3 as selectors and proline and

valine as analytes, since good enantioselectivities were

observed for these systems.24,25 In our previous studies, we

found that the fluorescence of cyclodextrins S-1–3 was

quenched by copper(II) ions.23,24 However, if the same

experiment was performed in the presence of copper(II) and

amino acids, enantioselective fluorescence quenching was

obtained, according to the following equilibria:

Cu L�AAð Þ2zCD fluorescentð Þ/?KL

L�AAzCu CDð Þ L�AAð Þ non� fluorescentð Þ(1)

Cu D�AAð Þ2zCD fluorescentð Þ/?KD

D�AAzCu CDð Þ D�AAð Þ non� fluorescentð Þ(2)

in which AA represents the amino acid and CD is the

cyclodextrin derivative used as sensor; the equilibria in eqn. (1)

and (2) are the same as those involved in the chiral

discrimination mechanism utilized in ligand exchange chro-

matography (LEC).18 The proper conditions for the enantio-

selective measurements were evaluated by titrating the

cyclodextrins with increasing amounts of the Cu(AA)2

complex (Fig. 2); best enantioselectivity was observed at low

Cu(AA)2/CD molar ratios (box A in Fig. 2), but the

fluorescence intensity in this region is strongly dependent on

the total amount of complex; therefore, small errors in the

concentration of the analytes should result in high variations

in fluorescence intensity and hence in the calculation of the

enantiomeric excess. Under a higher excess of the Cu(AA)2

Fig. 1 General scheme of enantioselective fluorescent cyclodextrins.

Fig. 2 Titration of the fluorescent cyclodextrin S-1 (6 6 1025 M)

with the Cu(Val)2 complex in aqueous sodium borate buffer at pH 7.0.

Box A represents the molar ratio at which a high enantioselectivity is

observed, but also high slope of the curve, while box B is the molar

ratio at which very low dependence of fluorescence intensity on molar

ratio is observed.

(1)

(2)

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complex (box B in Fig. 2), a very small dependence on the

Cu(AA)2 concentration is observed, and fluorescence intensity

is almost entirely dependent on the configuration of the amino

acid used. Similar results were obtained with proline and with

cyclodextrins S-2 and S-3.

Thus a 10 : 1 molar ratio was chosen for the microplate

reader experiment. Eleven calibration solutions of copper(II)/

valine 1 : 2 complex were used with enantiomeric excess

varying from 100% D-Val to 100% L-Val. Two series of

calibration solutions were prepared. On the same microplate

samples of Cu(Val)2 (in triplicate) whose enantiomeric excess

had to be analyzed were added to the fluorescent cyclodextrin,

using the same molar excess. As expected, the intensity of the

fluorescence measured was a function of the enantiomeric

composition of each sample. In Fig. 3, the image of the

microplate reader analysis obtained with cyclodextrin S-2 and

valine is reported: in this representation, the fluorescence

intensities were colour-encoded using a linear algorithm, so

that the differences become more evident.

Good reproducibility was observed for the calibration

points and comparison of colours allows the easy rough

evaluation of the enantiomeric excess of the six samples. Since

in many applications (e.g. in the screening of enantioselective

reaction conditions), it is important that the analytical

method can detect highly enantiopure samples in an easy to

read format, we verified the possibility of showing only these

samples by putting a threshold for visualization at appropriate

fluorescence intensity levels; in this way, only samples with

high optical purity were visualized (Fig. 3 B,C), thus allowing

fast localization by automatic routines. By setting the limit to

higher values, only two of the three samples with 99.5% D-Val

but none of those of 99% of D-Val were detected (results not

shown), indicating that ability to reveal samples with very high

ee can be further improved.

Since these experiments are based on enantioselective

fluorescence quenching, the Stern–Volmer equation can be

used in the following form:26

F0/F 5 1 + KL[Cu(L-AA)2] + KD[Cu(D-AA)2] 5

1 + [KL + (KD 2 KL)xD][Cu(AA)2](3)

where F0 is the fluorescence intensity of the cyclodextrin

solutions without quencher, F is the fluorescence observed

after addition of the Cu(AA)2 complex, xL and xD are the

molar fractions of the two enantiomers and KD and KL are the

Stern–Volmer constants of the two enantiomers in static

quenching (corresponding to the equilibrium constants for the

formation of the two ternary complexes). If the total amino

acid and copper concentration and hence that of the Cu(AA)2

complex is kept constant in the experiment, F0/F varies linearly

with the enantiomer molar fraction xD. Thus quantitative

analysis of the data obtained from the microplate reader

plotted as F0/F versus the percentage of D gave rise to the

calibration curves such as those reported in Fig. 4.

Data obtained with the experiments using different cyclo-

dextrins and valine or proline are summarized in Table 1.

Good correlation coefficients were obtained in all cases. The

highest enantioselectivity (slope of the curve) for valine was

observed with the cyclodextrin S-3, according to previous data

obtained in solution.25 Using cyclodextrin S-1 for proline, a

reversal of the curve slope was observed, in accordance with

the reversal of enantioselectivity observed for proline and

valine for this type of cyclodextrins.25 Although structural data

on the ternary complexes to explain this inversion are not

available, it should be related to the cyclic structure of proline,

which could give a different type of coordination on copper(II),

since with other open-chain amino acids, such as alanine

or phenylalanine, the same enantioselectivity as valine was

observed.

In order to evaluate the accuracy and precision of this

methodology, a series of valine samples of known enantio-

meric excess was analyzed on the same microplate. Therefore,

calibration curve and sample analysis were performed in the

same experiment. Two calibration curves and triplicate

analyses of six valine samples were performed in two minutes

for each cyclodextrin used. The quantitative results are

reported in Table 2. Using a single cyclodextrin the % D-Val

calculated both the standard deviation and the accuracy

within 6%, a value which is comparable with other fast screen-

ing methods previously proposed. However, the combined use

Fig. 3 Microplate reader output (fluorescence intensity is colour-

encoded) obtained with cyclodextrin S-2 (6 6 1025 M) upon addition

of 10-fold excess of Cu(Val)2 complex of different enantiomeric excess.

A: Total output, B: displayed data below a lower threshold corres-

ponding to 95% D; C) displayed data over a threshold corresponding

to 99% L.

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of different cyclodextrins allowed systematic errors to be

compensated for, and the average of the three measurements

showed higher accuracy. Similar results were obtained with

proline samples and cyclodextrin S-1 (Table 2).

Conclusions

The use of fluorescence quenching with highly enantioselective

sensors can greatly improve both the rapidity and simplicity of

screening of enantiomeric excess. Unlike kinetic resolution

methods, the present protocol imply only the mixing of the

selector with appropriate amounts of the copper complex of

the analyte. The dansylated cyclodextrins used in the present

work are particularly suitable for these kind of applications,

since they are highly fluorescent (due to self inclusion) and

highly enantioselective.24,25 Very low amounts of cyclodextrin

(0.77 mmol) and of amino acid (0.54 mmol for each sample)

were used for the entire microplate test. Low-cost instrumen-

tation and apparatus were used, with a system which can be

easily automatized.

Furthermore, in previous systematic studies,24,25 many

other amino acids as well as amino acid derivatives were

found to give enantioselective responses. Fast analysis of other

compounds can therefore be envisaged, provided they are

chemically pure or contaminated with achiral components.

Preparation of microplates with an automatic workstation

can further improve the rapidity and increase the accuracy

and precision of quantitative analysis, making it suitable

for product or process development in industrial chemical or

biotechnological research.

Experimental

General

Starting materials were obtained from commercial suppliers

and used without further purification.

Cyclodextrins S-1, S-2, S-3, were synthesized as described

previously.24,25

Fig. 4 Example of calibration curves obtained using the fluorescence microplate reader. A) Valine samples with S-2; B) proline samples with S-1.

Vertical bars represent ¡ standard deviations.

Table 1 Calibration curves obtained using the fluorescence micro-plate reader with cyclodextrins S-1–3 and plotting F0/F versus % ofD-amino acid

Cyclodextrin Amino acid F0/F raca Slope R2

S-1 Val 9.5128 0.0574 0.9871S-2 Val 3.2054 0.0203 0.9964S-3 Val 13.636 0.1219 0.9911S-1 Pro 7.5921 20.0667 0.9958a F0/F value for the racemic mixtures.

Table 2 Calculated values for valine and proline samples using cyclodextrin S-1–3 (in brackets: standard deviations)

Sample

Cyclodextrin Val 1 Val-2 Val-3 Val-4 Val-5 Val-6

S-1 96.1 (2.5) 95.5 (0.6) 45.3 (3.8) 9.0 (6.2) 2.2 (2.5) n.d.S-2 95.5 (1.1) 87.5 (2.2) 43.5 (5.4) 5.2 (0.5) 1.2 (1.0) 0.4 (0.8)S-3 95.4 (1.2) 85.1 (1.9) 38.5 (2.0) 6.2 (0.5) 1.2 (0.8) 20.2 (2.2)Average S-1–3 95.6 89.4 42.4 6.8 1.5 0.1% D-Val (actual) 94.4 89.9 39.9 5.1 0.9 0.1

Sample

Pro-1 Pro-2 Pro-3 Pro-4 Pro-5

S-1 98.1 (3.4) 95.3 (2.6) 86.1 (2.6) 38.1 (6.8) 6.6 (6.9)% D-Pro (actual) 98.9 94.6 89.3 39.1 4.6

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Stock solutions of D- or L-valine and D- or L-proline

copper(II) 2 : 1 complexes were prepared in water by weighing

the exact amount of the amino acid, adding the corresponding

volume of a CuSO4?5H2O (20 mM) solution, and then diluting

to the final volume (10 mL) with water. Under these conditions,

the 2 : 1 amino acid/copper(II) complex is formed, according to

the stability constants. Solutions of different enantiomeric excess

were prepared by mixing appropriate volumes of these solutions.

Stock solutions of cyclodextrins S-1, S-2, and S-3 (0.7–1.2 mM)

were prepared in doubly distilled water. Measuring solutions

(60 mM) were prepared by dilution of these concentrated

solutions in 0.1 M sodium borate buffer at pH 5 7.0.

The effective enantiomeric compositions of the amino acid

standards were checked by HPLC using a 25 6 0.46 cm I.D.

Chirobiotic T column (Astec, Whippany, NY, USA), operated

at 23 uC, and a mixture of water–ethanol (60 : 40% v/v) as

eluent. The flow rate was 0.75 mL min21 and the injection

volume was 20 mL. Detection was performed at UV 210 nm.

Fluorescence measurements

Fluorescence spectra were recorded on a Perkin Elmer LS50B

instrument in a 1 6 0.2 cm quartz cell thermostatted at 25 uC.

In the quenching experiment reported in Fig. 2, 0.5 mL of the

60 mM solution of each cyclodextrin (S-1–3) were titrated in

the cell by adding 6 6 1023 M Cu(Val)2 or Cu(Pro)2 in water

with a 10 ml syringe. Three measurements were performed for

each solution. Fluorescence intensities at the maximum

emission wavelength were used; correction of the fluorescence

intensity of all the samples was made according to the

expression In 5 I/Ist, where I is the observed fluorescence

intensity and Ist is the intensity of the reference solution of the

free cyclodextrin, both measured at the same excitation and

emission wavelength.

Microplate reader measurements

A FLUOstar* type 0403 (BMG LabTechnologies, Offenburg,

Germany) fluorescence microplate reader was used, with

360 nm (excitation) and 538 nm (emission) filters. In a typical

experiment, small volumes (200 mL for each sample) of a 60 mM

solution of the fluorescent cyclodextrin were put in a 96 well

microplate; to these solutions, a 10 : 1 excess (20 mL) of

Cu(Pro)2 or Cu(Val)2 complex solutions (6 mM) was added.

All volumes were measured with an automatic Eppendorf

EDOS 5222 pipette, with the appropriate Combitip accessory,

and checked by weighing. The microplate was automatically

shaken for 15 s. Each well was read five times (including

unfilled wells) in order to determine the standard deviation of

the fluorescence measurements. The total measurement time

was 2 min (using 1 flash per well) for each of the microplates

prepared. A set of three samples of the fluorescent cyclodextrin

without any added copper complex was used as a reference for

unquenched cyclodextrin (F0). Color codes were generated by

an in-house software application.

Acknowledgements

This work was partially supported by DMS and by grants

from the Ministero dell’Istruzione, Universita e Ricerca

(PRIN2003 project: ‘‘Engineering of separation systems,

sensors and arrays based on chemo- and stereo-

selective molecular recognition’’ and FIRB project

RBNE01KZZM_006: ‘‘Study of multifunctional microsystems

for chemical and biochemical determinations in complex

biological matrixes’’). We gratefully thank Miriam Hillemans

(DSM Research) for technical assistance.

Roberto Corradini,*a Cristina Paganuzzi,a Rosangela Marchelli,a

Sara Pagliari,{a Stefano Sforza,a Arnaldo Dossena,a Gianni Galavernaa

and Alexander Duchateaub

aDipartimento di Chimica Organica e Industriale, Universita di ParmaParco Area delle Scienze 17/A, I-43100 Parma, Italy.E-mail: roberto.corradini@unipr.it; Fax: +39-0521-905472;Tel: +39-0521-905406bDSM Research, P.O. Box 18 - 6160 MD Geleen, The Netherlands

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2746 | J. Mater. Chem., 2005, 15, 2741–2746 This journal is � The Royal Society of Chemistry 2005

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