Simultaneous Enantiomeric Determination of Dansyl-d,l

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Anal. Chem. 1999, 71, 1958-1962

Simultaneous Enantiomeric Determination of Dansyl-D,L-Phenylalanine by Fluorescence Spectroscopy in the Presence of r-Acid Glycoprotein Yuan Yan* and M. L. Myrick

Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208

Few techniques are amenable to real-time analysis of enantiomers. In this paper, total complexation by r-acid glycoprotein (AGP) is shown to discriminate between enantiomers of dansyl-D,L-phenylalanine (DPs) by changing the local environment of the D and L enantiomers (DDP and DLP, respectively) from hydrophilic to hydrophobic. DDP and DLP show the same native fluorescence at λex/λem ) 200/544 nm in the absence of AGP, but show shifted emissions with a component at λex/λem ) 220/497 nm in the presence of AGP and in lipophilic solutions. The conditions for an analytical determination have been optimized, and the method has been used to measure the enantiomeric composition of DDP/DLP mixtures with concentration ratios varying over 2 orders of magnitude. The mechanism of chiral recognition for DDP and DLP by AGP is discussed and should be equally applicable to other dansyl-derivative amino acid enantiomers. The association constants for AGP with DDP and with DLP have been determined to be 1.33 × 102 L g-1 and 2.29 × 102 L g-1, respectively. Determination of the enantiomeric composition of chiral substances has become important in recent years because chirality is a central factor in biological phenomena. For example, the behavior of the enantiomers of a chiral drug may show striking differences in terms of biological activity, potency, toxicity, transport mechanisms, and routes of metabolism; enantiomeric purity is therefore a desirable quality in a drug candidate.1,2 The methods most commonly employed in the pharmaceutical industry for the determination of enantiomeric purity are separation techniques such as liquid chromatography, gas chromatography, and capillary electrophoresis using chiral stationary phases3,4,5. Other techniques such as circular dichroism,6 absorbance spec(1) Crossley, R. Chirality and the Biological Activity of Drugs; CRC Press: Boca Raton, FL, 1995; Chapter 2. (2) Scott, A. K. Drug Saf. 1993, 8, 149-159. (3) Schreier, P.; Bernreuther, A.; Huffer, M. Analysis of Chiral Organic Molecules. Methodology and Application; Walter de Gruyter: Berlin, 1995; Chapter 3. (4) Koppenhoefer, B.; Epperlein, U.; Schwierskott, M. Fresenius’ J. Anal. Chem. 1997, 359, 107-114. (5) Desiderio, C.; Fanali, S. J. Chromatogr., A 1998, 807, 37-56. (6) Fleischhauer, J.; Harmata, M.; Kahraman, M.; Koslowski, A.; Welch, C. J. Tetrahedron Lett. 1997, 38, 8655-8658.

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trometry,7 infrared transmission spectrometry,8 X-ray anomalous scattering,9 and NMR spectroscopy10 have also been used for enantiomeric characterization. But few techniques11 are amenable to real-time analysis of enantiomers. Separation methods can offer good analytical precision and accuracy, but they are expensive in terms of reagent consumption and cost of instrumentation, while the sensitivity, selectivity, or speed of other methods is inadequate for real-time analysis. Fluorescence techniques have often been used to study the interaction between enantiomers and receptors12,13,14,15 because of their sensitivity, selectivity, and versatility. Receptor systems16,17,11 such as cyclodextrins (CDs) and calixarenes have been observed to selectively bind some enantiomers and produce a complex that can be detected by fluorescence techniques. Unfortunately, this approach is often not sufficiently reliable to serve as a simple and quick analytical method for enantiomer determinations. The centrality of chiral recognition in biology has prompted these investigators to explore the use of biomolecules as receptors for a fluorescence-based determination of enantiomeric purity. In this paper, we investigate bovine R-acid glycoprotein (AGP) as a receptor to discriminate the DP enantiomers by fluorescence spectroscopy and show that differences in the spectroscopy of the enantiomers when totally complexed is sufficient for analytical determination. EXPERIMENTAL SECTION Apparatus and Reagents. All experiments were performed on a Shimadzu PC551 PC spectrofluorometer. Data processing (7) Kubo, Y.; Maeda, S.; Tokita, S.; Kubo, M. Nature (London) 1996, 382, 522524. (8) Chappell, J. S. Analyst (Cambridge, U.K.) 1997, 122, 755-760. (9) Grochowski. J.; Serda, P. Chirality 1993, 5(4), 277-281. (10) Kram, T. C.; Lurie, I. S. Forensic Sci. Int. 1992, 55(2), 131-137. (11) Grady, T.; Harris, S. J.; Smyth, M. R.; Diamond, D. Anal. Chem. 1996, 68, 3775-3782. (12) James, T. D.; Sandanayake, K.; Shinkai, S. Nature (London) 1995, 374, 345-347. (13) Takeuchi, M.; Yoda, S.; Imada, T.; Shinkai, S. Tetrahedron 1997, 53, 83358348. (14) Parker, K. S.; Townshend, A.; Bale, S. J. Anal. Proc. 1995, 32, 329-332. (15) Parker, K. S.; Townshend, A.; Bale, S. J. Anal. Commun. 1996, 33, 265267. (16) Schuette, J. M.; Will, A. Y.; Agbaria, R. A.; Warner, I. M. Appl. Spectrosc. 1994, 48, 581-586. (17) Yang, H.; Bohne, C. J. Photochem. Photobiol., A 1995, 86, 209. 10.1021/ac981281k CCC: $18.00

© 1999 American Chemical Society Published on Web 04/09/1999

was performed using Matlab 5.1 software (Mathworks Inc., Natick, MA) after importing the spectra in ASCII format. DDP and DLP (Sigma Chemical Co., St. Louis, MO) were obtained in analytical purity and were used without further purification. Stock solutions (solutions D and L) with concentrations of 2.51 × 10-4 M were prepared by dissolving 5.0 mg of DDP or DLP in 2.0 mL of ethanol and then diluting to a 50-mL volume in a volumetric flask with distilled water. A 10-fold dilution of each of these solutions (solutions Dd and Ld) was used for preparing the different solutions used in the experiments. A series of 11 mixtures with total volumes of 0.1 mL were prepared from solutions Dd and Ld with volume fractions ranging from 0 to 100% Dd in 10% steps (Mixtures M0-M100, respectively). Bovine AGP (Sigma Chemical Co.) was used without further purification. A stock solution (solution A) of bovine AGP with a concentration of 100 µg/mL was prepared in 50 mM NaCl in distilled water and was used in the experiments directly. Other reagents used in the experiment were of analytical purity or better and were used without further purification. All solutions with volumes of less than 200 µL were transferred with an Edpa electronic digital pipet with a transfer volume between 0 and 250 µL. Fluorescence Measurements. One of the mixtures M0 through M100, 1.3 mL of distilled water, and 0.6 mL of solution A were combined for quantitative fluorescence studies in 5-ml test tubes. One hundred microliters of solution Dd or Ld, 1.5 mL of distilled water, and 0.6 mL of solution A were combined in a 1-cm silica cuvette, and then KI solution (10-2 M) was added to the mixture in increments of 20 µL from 0 to 120 µL to study Iinduced quenching. Luminescence of the solutions from 300 to 600 nm was recorded in 1-nm steps at a resolution of 15 nm with an excitation wavelength of 220 nm in a 1-cm silica cuvette immediately after shaking well. A Newport, Inc. KG3 long-pass filter was used to cut off any second-order diffraction of light into the observed spectral region. The spectral data were analyzed after being smoothed by the 9-point moving-average method. Any effect of this averaging on spectral resolution was neglected because of the large band-pass of the spectrometers. RESULTS AND DISCUSSION Fluorescence Spectra of DP-AGP. The two-dimensional fluorescence spectra of DDP and DLP in the presence and absence of AGP are shown in Figure 1. DDP and DLP exhibit the same fluorescent peak at λex/λem ) 200/544 nm and show identical intensities (Table 1). AGP shows two fluorescence maxima at λex/ λem ) 220/340 and 280/340 nm. The presence of excess AGP alters the fluorescence spectra of DPs considerably, and a new peak emerges at λex/λem ) 220/497 nm. The fluorescence intensity of DDP is much higher than that of DLP (see Table 1) in the presence of excess AGP. At the same time, quenching of native AGP fluorescence at λex/λem ) 220/340 nm or λex/λem ) 280/340 nm can be observed, with a slightly greater effect caused by DDP than DLP. A time-dependence study was performed which showed that these effects occur promptly upon mixing and persist for at least 12 h without change. Mechanism of Fluorescence Change of DPs in the Presence of AGP. The fluorescence spectral change of DDP and DLP in the presence of AGP shows that an interaction occurs between AGP and the DPs. The intense new emission maximum at λem ) 497 nm represents a 47-nm hypsochromic shift of DP

Figure 1. Two-dimensional fluorescence spectra of (a) DDP (1.19 × 10-6 M), (b) DLP (1.19 × 10-6 M), (c) DDP (1.19 × 10-6 M) + AGP (24 µg/mL), (d) DLP (1.19 × 10-6 M) + AGP (24 µg/mL), and (e) AGP (24 µg/mL).

Figure 2. Emission spectra of DPs (1.25 × 10-6 M) in ethanol solution (50% v/v) using an excitation wavelength of 220 nm. Table 1. Fluorescence Characteristics of DDP and DLP in Solution

in water compound DDP DLP

λex/λem (nm)

in ethanol solution (50% v/v)

in AGP solution (24 µg/mL) F

λex/λem (nm)

F

K (L‚g-1)

λex/λem (nm)

F

220/544 566 200/544 196 220/497 514 1.33 × 200/544 201 220/497 247 2.29 × 102 220/544 562 102

fluorescence. Experiments with the less polar solvent ethanol confirm that this is consistent with a more hydrophobic environment for the DPs. As shown in Figure 2 and Table 1, the emission spectra of DPs in ethanol mirror those obtained in the presence of AGP, with a significant increase in DP fluorescence intensity Analytical Chemistry, Vol. 71, No. 10, May 15, 1999

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Figure 3. Dependence of emission spectra of (a) DDP (1.14 × 10-6 M) with AGP (27 µg/mL) and (b) DLP (1.14 × 10-6 M) with AGP (27 µg/mL) on ethanol concentration below 10% v/v using an excitation wavelength of 220 nm. Change of the fluorescent intensities of DPs (1.14 × 10-6 M) and AGP (27 µg/mL) as a function of ethanol concentration at (c) λem ) 497 nm and (d) λem ) 340 nm.

and the emergence of a new emission peak at λem ) 510 nm. The enhanced fluorescence of DPs in the presence of AGP at λex/λem ) 220/497 nm was returned to noncomplexed levels by low concentrations (e 10% v/v) of ethanol (Figure 3), while the fluorescence of AGP at λex/λem ) 220/340 nm increased in intensity back to levels similar to that of the native AGP in the absence of DPs (Figure 4). These data suggest that low concentrations of ethanol do not change the conformation of AGP and that ethanol competes with DPs for binding sites on AGP. This also indicates that the interaction between DPs and AGP is not strong. DPs in the presence of AGP with higher ethanol concentrations (>10% v/v) show a new emission maximum at λem ) 510 nm, a shift similar to that observed with ethanol alone, and the intensity increases for both DDP and DLP and reaches nearly identical values (Figure 4), again similar to the result in ethanol alone. No selectivity between DPs was observed for AGP in the concentrated ethanol solutions. Fluorescence-quenching experiments were employed to study the accessibility of the DP moiety of the DP-AGP complex to the collisional quencher I-.18 Iodide fluorescence-quenching data were analyzed according to eq 1.

I0/I ) Kq[KI]

(1)

Figure 4. Dependence of emission spectra of (a) DDP (1.09 × 10-6 M) with AGP (8.7 µg/mL) and (b) DLP (1.09 × 10-6 M) with AGP (8.7 µg/mL) on ethanol concentration above 10% v/v using an excitation wavelength of 220 nm. (c) Change of the fluorescent intensities of DPs (1.09 × 10-6 M) and AGP (8.7 µg/mL) as a function of ethanol concentration at λem ) 510 nm.

Figure 5. Dependence of emission spectra of (a) DDP (1.14 × 10-6 M) with AGP (27 µg/mL) and (b) DDP (1.14 × 10-6 M) with AGP (27 µg/mL) on KI concentration. Change of the fluorescent intensities of DPs (1.14 × 10-6 M) and AGP (27 µg/mL) as a function of KI concentration at (c) λem ) 497 nm and (d) λem ) 340 nm.

Here, [KI] is the molar concentration of I- (derived from KI), Kq is the quenching constant, and I0 and I are the fluorescence intensity at [KI] ) 0 and the appropriate molar concentration of I-, respectively.19 The DDP - AGP and DLP - AGP complex

emissions were both readily quenched by KI (Figure 5). The quenching constants (Kq) at λex/λem ) 220/497 nm, given by SternVolmer analysis of the data in Figure 6 using eq 1 were 2.01 × 104 ( 380 and 1.25 × 104 ( 338 for DDP-AGP and DLP-AGP, respectively, and indicate a collision-controlled bimolecular inter-

(18) Wang, S. K.; Clemmons, A.; Strader, C.; Bayne, M. Biochemistry 1998, 37, 9528-9535.

(19) Lakowicz, J. Principles of Fluorescence spectroscopy; Lakowicz, J., Ed.; Plenum Press: New York, 1983; pp 258-297.

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Figure 7. Sensitivity of DDP (circle) and DLP (square) with 24 µg/ mL AGP. Slopes are 1012.6 ( 20.2)/µM and (384.3 ( 13.1)/µM for DDP and DLP, respectively, giving a sensitivity ratio of 2.6. Figure 6. Changes in the fluorescent emission intensities of 1.00 µM DPs with AGP concentration at λex/λem ) 220/497 nm. I/I0 represents the enhancement ratio of DDP (circle) and DLP (square) emission as a function of AGP concentration. Intensity (diamond) represents the absolution emission intensity difference between DDP and DLP.

action between DP - AGP and the I- in solution. These relatively large values of Kq also suggest that the DP moiety in the DP AGP complex is not protected from I- by AGP. AGP quenching constants at λem ) 340 nm were 2.61 × 104 ( 1651 and 2.56 × 104 ( 1650 for DDP-AGP and DLP-AGP complexes, respectively, and indicate that most tryptophan residues reside on the surface of AGP. Taken together, these data indicate that DPs bind on a hydrophobic surface region of AGP, and the observed spectral changes in DP fluorescence in the presence of AGP result from this binding. Dependence of DP Fluorescence on AGP Concentration. The emission spectra of DPs as a function of AGP concentration are shown in Figure 6. The results show that the emission intensity at the new fluorescence maximum (λem ) 497 nm) is more sensitive to AGP for DDP than for DLP. Assuming the formation of 1:1 complexes, the association constants (K) were computed from Figure 6 to be 1.33 × 102 L g-1 for DDP and 2.29 × 102 l g-1 for DLP. It was found that the enantiomer with the larger fluorescence increase possesses the smaller K, similar to literature results using saccharide as a receptor for xylose and talose.13 Rather than use the difference in K to discriminate between enantiomers, we chose to use the difference in intensity enhancement as the basis for an analytical method. To do so, we selected 24 µg/mL AGP for our determinations, as this concentration was sufficient to bind almost all of the available DDP and DLP. Standard Curves for DPs in AGP. The relative sensitivity of the DP enantiomers to the presence of AGP is shown in Figure 7. The results show that there is a good linear relationship between fluorescence intensity and enantiomer concentration in the range of 0-0.75 µM for both DDP and DLP. The detection limits (concentration corresponding to 3SD of the background

intensity) of DDP and DLP alone were 1.7 and 4.6 nM, respectively. The ratio of the AGP sensitivity (or slope) for DDP to that for DLP is 2.64, which is the largest value for chiral discrimination using a fluorescence-based technique known to the authors. Simultaneous Determination of DPs. Because the new emission maximum observed for DPs bound to AGP at λex/λem ) 220/497 nm overlaps their native fluorescence spectra, chemometric methods can improve precision in simultaneous determinations of DPs. Principle component regression (PCR) was chosen because it offers quantitation based on patterns of change in sample spectroscopy.20 PCR assumes a linear relation between spectral intensity and concentration. For the data in our measurement set, two principle components were found to describe 99.96% of the data variability. On the basis of this result and of evaluation of prediction errors as a function of the number of factors selected, we concluded that two factors provide a suitable description of our data set. PCR technique implicitly uses many wavelengths, and because the spectral changes are not just in intensity (length of the spectral vector) but in shape (direction of the spectral vector), both the total mass and the ratio of the enantiomers can be determined in theory and in our experiments. Table 2 shows the data for simultaneous determinations of DDP and DLP in AGP solution based on the emission spectra from 300 to 600 nm with λex ) 220 nm and PCR. The relative standard prediction error based on this analysis is (5%, which is greater than that reported in the literature for similar assays. An analysis of our sources of error, however, reveals that (5% is the approximate relative precision with which we were able to make our standards because of imprecision in the pipetting of a volume e 200 µL. Consequently, we believe (5% RSE is considerably larger than the inherent error of prediction based on PCR treatment of these fluorescence spectra. Even with pipetting error, the absolute errors are generally less than 2%. The limits of detection of the method (concentration corresponding to 3SD of the background sample) (20) Malinowski, E. R. Factor Analysis in Chemistry, 2nd ed.; Wiley & Sons: New York, 1991; p 98.

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Table 2. Report on Prediction of Enantiomeric Compositiona DDP

DLP

% DDP

% estim

% rel error

% estim

% rel error

10 20 30 40 50 60 70 80 90

10.81 22.41 31.59 41.23 50.68 61.19 72.63 77.90 88.57

8.07 12.03 5.30 3.07 1.35 1.98 3.76 -2.62 -1.59

89.02 78.12 68.24 58.41 49.14 38.02 27.82 21.98 11.73

-1.09 -2.34 -2.52 -2.54 -1.63 -4.96 -7.28 9.90 17.34

a

All samples were 1.26 × 10-5 M in total DPs.

for DDP in the presence of 0.25 µM DLP and DLP in the presence of 0.25 µM DDP were 5.1 and 20.7 nM, respectively. CONCLUSION Enantiomeric selectivity was observed for DPs based on fluorescence spectroscopy in the presence of excess AGP. A simple, sensitive, and quick assay to simultaneously determine the composition of DPs has been described and its conditions optimized. The mechanism of fluorescence change in DPs caused by the presence of AGP is proposed to be an AGP-binding-induced change in the local environment of DPs from a water-dominated hydrophilic solution sphere to one influenced strongly by a

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hydrophobic binding site on the surface of AGP. The method shows promise as a detection and product-qualification method for chiral drugs on nonchiral stationary phases, where potential interferences can be reduced or eliminated. Our results suggest that ethanol can interfere with the measurement when it is present at a level 4000 greater than AGP and 105 greater than the DPs. Hence, this technique shows promise for measurements when the level of nonspecific hydrophilic interferents is below these values. Data suggest that the proposed binding site is chiral in nature, resulting in enantiomer-dependent perturbations in the fluorescence spectroscopy of DPs. The data reported here are insufficient to completely resolve whether these spectroscopic perturbations are only solvation-sphere dependent or whether energy transfer from AGP or some other mechanism plays a significant role. ACKNOWLEDGMENT The authors would like to acknowledge support for this work from the Office of Naval Research through Grant N00014-97-10806. Y.Y. would like to thank the National Natural Science Foundation of China (39660080) and the Natural Science Foundation of Jiangxi Province for support.

Received for review November 18, 1998. Accepted March 2, 1999. AC981281K