Chiral sensing using a blue fluorescent antibody{

Hana Matsushita, Noboru Yamamoto, Michael M. Meijler, Peter Wirsching, Richard A. Lerner,Masayuki Matsushita* and Kim D. Janda*

Received 10th August 2005, Accepted 12th September 2005

First published as an Advance Article on the web 20th September 2005

DOI: 10.1039/b511408j

The chiral sensing of small molecules using a blue fluorescent antibody sensor is described.

Introduction

The development of fluorescence based chemosensing has

generated great excitement in recent years.1,2 Methods based

on fluorescence spectroscopy provide molecular sensing

with high sensitivity, low cost, and wide applicability. Multi-

signaling modes including fluorescence quenching, energy

transfer and excimer formation have yielded information

on the complexation of sensor molecules and analytes.3 In

particular fluorescent sensors for the enantioselective recogni-

tion of chiral molecules are potentially useful for the high

throughput screening of chiral catalyst libraries for asymmetric

synthesis.4 A number of fluorescent chemosensors based on

binaphthyls,1,5 cyclodextrins,6 crown ethers7 and others,8 have

been reported. However, to the best of our knowledge, an

enzyme-bound fluorescent NAD+ analogue, reported by Gafni

in 1980,9 is the only fluorescent biosensor that can perform

chiral discrimination.

Recently, we have reported a series of monoclonal anti-

bodies (mAbs, e.g. mAb 19G2), prepared against a trans-

stilbene hapten 1 (Fig. 1), in which the 19G2–1 complex

produced a powder blue fluorescence with high quantum

yield (lexc 5 327 nm, lem 5 410 nm, Wf 5 0.78).10 The change

in emission occurred over a narrow temperature window,

between 240 K and 260 K, and temperature dependent

excited state dynamics were proposed to explain this unusual

fluorescent emission.11 The structure of the Fab fragment of

19G2 complexed with 1 was solved to 2.4 A resolution at 4 uC(Fig. 2).10 In this structure, the stilbene moiety in 1 was

surrounded by hydrophobic residues; a heavy chain Trp103

(Kabat numbering) was located in a p-stacked position next to

the phenyl ring distal from the linker, and the light chain Tyr36

was located perpendicular within 3.3 A to the central olefinic

carbons. On the other hand, the protein packing in the region

of the glutaric tail was less condensed compared with that of

the stilbene moiety. This constellation of amino acids and its

unique structured combing site have allowed us to demonstrate

novel immunochemistry of 19G2 wherein blue fluorescence is

emitted upon binding stilbene tags attached on DNA analogs12

or to the surface of a cowpea mosaic virus coat protein.13

Even though 19G2 was originally programmed to recognize

the achiral molecule 1, the asymmetric environment of the

antibody combing site should enable recognition of a chiral

chromophore to result in a solvochromic shift. During

subsequent studies to identify alternative ligands for this

antibody, we discovered that each of the chiral trans-stilbene

amino acid esters (R)- and (S)-2 could bind to 19G2, but only

the (S)-2-19G2 complex resulted in blue fluorescent emission.14

The value of the dissociation constant (KD) of 19G2 and

(R)-and (S)-2 were determined to be 52 mM and 4.6 mM

respectively. However, incubation of 19G2 with an excess

equivalent of (R)-2 only resulted in weak fluorescence. Thus

Department of Chemistry and The Skaggs Institute for ChemicalBiology, The Scripps Research Institute, 10550 N, Torrey Pines Road,La Jolla, CA 92037, USA. E-mail: masayuki@scripps.edu;kdjanda@scripps.edu; Fax: +1-858-784-2595; Tel: +1-858-784-2516{ Electronic supplementary information (ESI) available: Synthesis andcharacterization of 4–13, and determination of KD’s of 13 for 19G2.See http://dx.doi.org/10.1039/b511408j

Fig. 1 Structures of the trans-stilbene hapten (1) and chiral stilbenes

(2), and fluorescence intensities of 2 with 19G2.

Fig. 2 Crystal structure of 1 bound to 19G2. Protein databank

accession number 1FL3. The figure was made with VMD15 and

rendered in POV-ray.16

METHOD www.rsc.org/molecularbiosystems | Molecular BioSystems

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the magnitude of the dissociation constant values was not

sufficient to explain the difference in fluorescence emission.

Since a good correlation was observed between fluorescence

intensities and the percentage of the S component of 2, we

demonstrated that 19G2 could serve as a biosensor to

determine the enantiomeric purities of trans-stilbenyl amino

acid esters 2. Using this fluorescent biosensor, a high-

throughput screening of a chiral phase transfer catalyst library

for the synthesis of either of the esters has been performed.14

Based on these findings, we envisioned that 19G2 could act

as a chiral sensor for various other diverse stilbene derivatives.

Herein, we describe the enantioselective fluorescent sensing

of a variety of chiral stilbene derivatives and evaluation of

a pool of Jacobsen’s chiral catalysts in the asymmetric

epoxide opening reaction17 using the blue fluorescent antibody

sensor 19G2.

Results

To initiate our studies, 19G2 was examined for its ability to act

as a fluorescent chiral sensor for various small chiral mole-

cules. Thus, enantiomerically pure (R)- or (S)-phenethyl amine

(3) were attached to trans-stilbene tags, and the fluorescent

properties of the resulting compounds were measured in the

presence of 19G2 (Scheme 1). Four different trans-stilbene tags

were employed, each of the resulting amides ((R)- and (S)-4–7),

(50 mM), was mixed with 19G2 (25 mM) in PBS [10 mM

sodium phosphate, 150 mM NaCl (pH 7.4)] and 5% DMF

co-solvent, and irradiated at lexc 5 327 nm. The excitation

times are in the order of microseconds and the extent of

trans–cis isomerisation in the presence of 19G2 is expected to

be negligible.

Fluorescent intensities at lem 5 410 nm are shown in Fig. 3.

(R)- and (S)-4 resulted in an intense powder blue fluorescence.

The net fluorescence intensity of (S)-4 and 19G2 was 1.2 times

that of by 19G2-(R)-4, i.e., (IS 2 I0)/(IR 2 I0) 5 1.2. It is

noteworthy that even though the asymmetric centers are eight

rotatable bond lengths apart from the stilbene ring, they can

still influence fluorescence intensity.18 Interestingly, com-

pounds with truncated linkers, (R)- and (S)-5–7, resulted in

no observable or very weak fluorescence. We hypothesize that

the phenethyl amine moieties in (R)- and (S)-4 may be located

outside of the binding pocket of 19G2, and may thus affect the

overall depth or conformation of the stilbene moiety, which in

turn could effect the intensities of fluorescence observed. On

the other hand, (R)- and (S)-5–7, present a different scenario

wherein potential steric hindrance of the phenethyl amine

moieties could prevent the stilbene ring to encase itself

within the combining site of 19G2 and therefore a loss in

fluorescent emission.

Based on these findings, (R)- and (S)-isomers of a series

of chiral amines were attached to the trans-stilbene tag, and

examined by measuring the fluorescence emission in the

presence of 19G2. Hence, the trans-stilbene tag was coupled

by reductive N-alkylation of various amino groups with trans-

stilbene carboxaldehyde and NaBH3CN (Scheme 2). The

resulting stilbenylmethylamines (8–13) were mixed with 19G2

and the fluorescence was measured (Fig. 4).

Phenethyl amine derivatives (R)- and (S)-8 gave rise to low

fluorescence. Among the three glycine derivatives (9–11), only

methyl esters (R)- and (S)-10 resulted in moderate blue

fluorescence. The net fluorescence intensity of (S)-10 and

19G2 was 1.6 times that of 19G2-(R)-10. Remarkable chiral

discrimination was observed with amino alcohols (R)- and

(S)-13, and the net fluorescence intensity of (S)-13 and 19G2

was 3.1 times that of the (R)-13-19G2 complex.

Fig. 5 displays the fluorescence spectra of (R)- and (S)-13

(50 mM) in the presence of 19G2 (25 mM), which were mixed in

PBS and 5% DMF co-solvent at lexc 5 327 nm. The specific

binding of (R)- and (S)-13 by the mAb 19G2 was observed,

and the value of the dissociation constant (KD) was determined

to be 31 mM and 6.4 mM respectively. These results suggest that

Scheme 1 Structures of trans-stilbene tagged phenethyl amines (4–7).

Stilbene tag moieties are shown in blue.

Fig. 3 Fluorescence intensities of the (R)- and (S)-isomers of trans-

stilbene tagged compounds (4–7). 4–7 (50 mM) was mixed with

19G2 (25 mM) in PBS and 5% DMF co-solvent, and irradiated at

lexc 5 327 nm. Fluorescence intensities were measured at 410 nm.

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19G2 could function as a sensor in the HTS of chiral

molecules.

Using mAb 19G2, we performed an evaluation of a series of

Jacobsen’s catalysts in the asymmetric ring-opening reaction

detailed in Scheme 3.17 Treatment of racemic 14 with metal

salen catalysts (17–20) and trimethylsilyl azide (TMSN3) gave

azide alcohol 15 in four kinds of enantiomeric purities.

Hydrogenation of the azide group in 15 followed by micro

distillation gave aminoalcohols (R)- and (S)-16. Each of these

mixtures were attached to the trans-stilbene tag to result in

mixtures of (R)- and (S)-13, which were mixed with 19G2, and

the fluorescence intensity was measured by using a 96-well

fluorescence plate reader.

To determine the ee values of the four mixtures of 13 by

fluorescence, we constructed a calibration curve by using

synthetic (R)- and (S)-13, prepared from commercially

available (R)- and (S)-16 (Fig. 6). Both (R)- and (S)-13 were

obtained in .99% ee as analyzed by chiral HPLC. Precise

mixtures of these isomers were prepared (100% ee (R)-13,

50% ee (R)-13, 0% ee, 50% ee (S)-13, 100% ee (S)-13) as solu-

tions in DMF. The data were fitted to a hyperbolic, as required

Scheme 2 Structures of trans-stilbene tagged amines (8–13). Stilbene

tag moieties are shown in blue.

Fig. 4 Fluorescence intensities of (R)- and (S)-isomers of trans-

stilbene tagged amines (8–13). 8–13 (50 mM) was mixed with 19G2

(25 mM) in PBS and 5% DMF co-solvent, and irradiated at lexc 5

327 nm. Fluorescence intensities were measured at 410 nm.

Fig. 5 Fluorescence spectra of (S)- and (R)-13.

Scheme 3 Synthesis of trans-stilbene tagged amino alcohol 13 using

Jacobsen’s catalysts.

Fig. 6 Calibration curve for fluorescence intensity and enantiomeric

excess of 13.

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by the specific binding of (R)- and (S)-13 by the mAb 19G2.

The fluorescence values for each of the four mixtures were then

obtained in the same way as above, and the corresponding ee

values were then calculated (Table 1). To confirm the validity

of the method, four samples were reanalyzed by chiral HPLC

(DAICEL CHIRALCELL AD-HR, 40% CH3CN in H2O,

0.1% Et3N, flow: 1.0 ml min21, retention time: 41 min (R)-13,

46 min (S)-13). The fluorescence sensing method and HPLC

measurements varied, on average, ,10%.

Conclusions

Herein, we have presented the foundation for a chiral sensing

method that employs a blue-fluorescent mAb–ligand complex

to discriminate between different chiralities of small molecules.

A pool of chiral compounds were attached to trans-stilbene

tags, after which fluorescence emission was measured in the

presence of 19G2. Fluorescence changes were observed

between the various enantiomers of stilbene tagged amine 4,

amino acids 2, 10, and amino alcohol 13. Our fluorescent

biosensor approach provides a sensitive and rapid method to

determine enantiomeric excess as compared with HPLC based

ee determination, which has limitations in terms of sensitivity

as well as sample population size. A highly enantioselective

response measured with amino acid 2 and amino alcohol 13

allowed for a practical application in the screening of a series

of chiral catalysts. The structure based mechanism of

differences in fluorescence emission is unclear as of yet,

through crystallographic analysis of (S)-and (R)-2 and 13

complexed with 19G2, as well as dynamic spectroscopic studies

of the fluorescent complexes are underway, and will be

reported in due course.

A variety of fluorescent chemo-sensors have been developed

for the discrimination of chirality in small molecules, however,

the ability to rationally design these sensors is not sufficiently

sophisticated so as to be able to readily synthesize such sensor

molecules. The monoclonal antibody 19G2, a fluorescent bio-

sensor in a mAb format, in the future may allow us to generate

new specific receptors for synthetic molecules, for example, by

means of directed mutation of loop structures or with the help

of immunological diversity.19 We believe that further develop-

ment of these novel fluorescent antibodies will provide a unique

set of biosensors that can recognize a wide variety of structures.

Experimental

Monoclonal antibody production

Details for the preparation of compounds and mAbs have

been described elsewhere. In short, stilbene hapten 1 was

conjugated to the carrier protein keyhole limpet hemocyanin

(KLH). Immunization of the conjugate with balb/B mice

resulted in a panel of 15 mAbs for analysis. The hybridomas

were derived from fusions with an X63-AG8.653 myeloma cell

line. All mAbs were purified from ascites to y95% homo-

geneity as follows: step 1, saturated ammonium sulfate; step 2,

DEAE Sephacel (Pharmacia) chromatography; step 3, protein

G affinity chromatography.

General method for the measurement of fluorescence

A solution of stilbene derivative (50 mM, 25 ml) in PBS (10 mM

sodium phosphate, 150 mM NaCl, pH 7.4) with 10% DMF

co-solvent was mixed with 19G2 (25 mM; 50 mM binding sites,

25 ml) in PBS in a 96-well plate (Costar 3915). The fluorescence

intensities (lexc 5 327 nm, lem 5 410 nm) were recorded by

using a Spectra Max Gemini (Moleculer Devices) plate reader

at 25 uC.

Acknowledgements

This work was supported by funding from The Skaggs

Institute for Chemical Biology.

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Table 1 Enantiomeric excess of 13 prepared by Jacobsen’s catalyst(17–20)

Catalyst Fluorescence ee by fluorescence (%) ee by HPLC (%)

17 2636 79 (R) 99 (R)18 2645 77 (R) 91 (R)19 3587 108 (S) .99 (S)20 3512 93 (S) 92 (S)

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