Anal. Chem. 1988, 60, 2621-2625
2621
Inclusion Complex Formation of Benzo[ a ] pyrene Metabolites with Cyclodextrins Rudyea Woodberry, Sharon Ransom, and Fu-Ming Chen* Department of Chemistry, Tennessee State University, Nashville, Tennessee 37209-1561
Comparative spectroscoplc binding studies of (-)-trans-7,8dlhydroxy-7,8-dlhydrobenzo[a]pyrene (7,8-BPD) with three cyclodextrins (CyDs) suggest that a 2:l guest-host complexation exists In the y-CyD solutlon, whereas a 1:l BPDCyD complex formation and minimal Interaction are observed In the 8- and aCyD solutkns, respectively. These results are consistent with cavlty slzes of the CyDs and the molecular slze estimates of BPD. The interpretatlons are further supported by the klnetics of hydroiysls of trans -7,8-dlhydroxyanf/-9,lO-epoxy-7,8,9,lO-tetrahydrobenzo[a Ipyrene (ant/BPDE), the ultlmate carcinogenic metabolite of benzo[a]pyrene. The rate of ant/-BPDE hydrolysis In the a-CyD solution ls only slightly slower than that In the buffer whereas 3.5- and 100-fold retardations are seen In the 8- and y-CyD soiutlons, respectively. Parallel studles with trans-9,lO-dihydroxy8,1Odihydrobenzo[alpyrene (9,lO-BPD) suggest that R binds to CyDs less strongly than the 7,&Isomer and, unilke 7,8-BPD, the 9,lO-BPD does not form a 2:l complex wlth yCyD. These results suggest that a hlgh-performance ilquld chromatography (HPLC) methodology with CyDs as statlonary or mobile phases can be used to separate 7,8-BPD from its Q,lO-isomer, as demonstrated by our HPLC measurements.
Cyclodextrins (CyDs) are a series of macrocyclic oligosaccharides produced by the action of Bacillus macerans amylase on starch and contain from 6 to 12 a-1,4 linked Dglucose units ( I ) . The most widely used CyDs consist of six, seven, and eight glucose monomers arranged in torus shapes and are denoted as a-, 0-, and yCyD, respectively (2). The coupling of the glucose moieties gives the CyD a rigid, conical molecular structure with a hollow interior. The interiors of the cavities are composed of two rings of C-H groups with a ring of glycosidic oxygen in between, allowing them to be hydrophobic in nature. The internal diameters of these cavities are approximately 5.7,7.8, and 9.5 A, respectively, and the depths are roughly 7.8 (3). The hydrophobic nature of the cavities enables the CyDs to trap compounds such as aromatic, alkyl halides, and gases as guest molecules in their interior, resulting in the formation of inclusion or “host-guest” complexes (2, 4 , 5 ) . The stability of an inclusion complex depends on the size of the CyD cavity and on intermolecular forces such as hydrogen bonding, van der Waals attraction, and hydrophobic interactions. These macrocyclic carbohydrate molecules can be discriminating in their inclusion complexing tendencies toward different structural, positional, or stereomeric molecules. High-performance liquid chromatography (HPLC) columns have been packed with these materials and have been used for separating varieties of isomers (6, 7). The interest in the study of inclusion complex formation between CyDs and other organic or inorganic molecules has been prompted, in part, by the similarities between the inclusion processes of CyD and the enzyme-ubstrate reactions in biological systems (5). Information such as noncovalent 0003-2700/88/0360-262 1$01.50/0
intermolecular forces can be furnished through such studies and can serve as models for the mode of action of enzymes. CyDs have also been used as stabilizers for drugs by pharmacologists and as miracle additives in food processing (2). Another widely used application of CyDs, as already men, tioned, is in the fields of chromatographic separation and purification methods (8). Some polycyclic aromatic hydrocarbons (PAHs) me known to be carcinogenic as well as mutagenic. These relatively inert compounds exert their harmful activities through enzymatic conversion into reactive metabolites which then covalently bind to DNA (9). The most widely studied PAH is benzo[alpyrene whose ultimate carcinogenic metabolite has been identified to be (+)-trans-7,8-dihydroxy-anti-9,lO-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene((+)-anti-BPDE). In vitro studies indicate that when anti-BPDE is added to a DNA solution, less than 10% is covalently bound to DNA while the remaining 90% or so anti-BPDEs are hydrolyzed to tetraols (10). The rate of anti-BPDE hydrolysis is intimately related to the detoxification of this carcinogen. Consequently, elucidation of factors that can alter ita rate of hydrolysis will be of particular value. Our interests in (-)-7,8-BPD stems from the fact that this isomer converts to (+)-anti-BPDE with high stereospecificity (9). The purpose of this work is to investigate the binding characteristics of cyclodextrins with (-)-7,8-BPD and its geometric isomer trans-9,10-dihydroxy-9,lO-dihydrobenzo[alpyrene (9,lO-BPD) to assess the feasibility of using cyclodextrin-bonded HPLC columns for separating geometric and stereoisomers of benzo[a]pyrene metabolites. The effect of inclusion complex formation on the rate of anti-BPDE hydrolysis will also be investigated.
EXPERIMENTAL SECTION a-, @-, and y-CyD were purchased from Advanced Separation
Technologies, Inc. The benzo[a]pyrene metabolites, anti-BPDE, 7,8-BPD and 9,10-BPD, were provided by the National Cancer Institute (NCI) Chemical Carcinogen Reference Standard Repository, a function of the division of Cancer Cause and Prevention, NCI, NIH, Bethesda, MD. Stock solutions of anti-BPDE, 7,8-BPD, and 9,lO-BPD were prepared in tetrahydrofuran and their concentrations determined by dilution in ethanol using extinction coefficients of 48 600 cm-’ M-’ at 344 nm, 43 000 cm-’ M-’ at 365 nm, and 38000 cm-’ M-’ at 345 nm, respectively. Stock solutions for the cyclodextrins were all 10 mM in concentration and prepared in a 10 mM sodium phosphate pH 7 buffer containing 0.01 M NaCl and 1mM ethylenediaminetetraaceticacid (EDTA). Absorption spectra were measured at 23-24 “C with a Cary 210 spectrophotometric system using cuvettes of 1-cm path length. Circular dichroism (CD) spectra were obtained with a Jasco J-500A recording spectropolarimeter at appropriate temperatures, using water-jacketed cylindrical cells of 2-cm path length. The temperatures were maintained by a Neslab RTE-8 refrigerated circulating bath. Fluorescence spectra were measured with a Jasco PF-550 fluorometer at room temperature, using the slit widths of 3 and 5 nm for the excitation and emission, respectively. All experiments were carried out in the pH 7 buffer. HPLC studies were carried out with 250 X 4.6 mm Cyclobond I and I1 columns of Astec packed with 5-pm spherical particles 0 1988 American Chemical Society
2622
ANALYTICAL CHEMISTRY, VOL. 60, NO. 23, DECEMBER 1, 1988
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Figure 1. Absorption spectral titration of (-b7,8-BPD with varying concentrations of p-CyD (A) and yCyD (B) at 23 "C. Cuvette of l t m path length was used and the concentration of (-)-7,8-BPD was kept constant at 12 pM during the titration. of bonded /3- and y-CyD, respectively. The various ratios of MeOH/water or MeCN/water were used as the mobile phase. These measurements were made in an ISCO HPLC system using a guard column and a flow rate of 1 mL/min.
RESULTS AND DISCUSSION Spectroscopic Characterization. Absorption Spectral Characteristics of (-)-7,8-BPD i n Cyclodextrins. The effects of 0-and y-CyD on the absorption spectra of (-)-7,8-BPD are shown in Figure 1. As can be seen, absorbance maxima at 345 and 365 nm are exhibited by (-)-7,8-BPD in a pH 7 buffer. A progressive increase in the concentration of (3-CyD, while keeping the BPD concentration constant, however, results in a corresponding intensity enhancement with a concomitant gradual spectral red shift (Figure 1A). It is apparent that despite a 4-nm spectral red shift and a more than 2-fold intensity enhancement, the spectral characteristics of 7,&BPD in the presence of high concentrations of 0-CyD are very similar to those in the absence of CyD. In contrast, the presence of y-CyD in the solution dramatically alters the spectral characteristics of 7,8-BPD. A progressive increase of y-CyD in the solution results in a gradual disappearance of the 345- and 365- nm maxima with a concomitant appearance of a new maximum at 355 nm with a shoulder around 377 nm (Figure 1B). The distinctly different spectral effects in these two solutions suggest that (-)-7,8-BPD binds to 8and y-CyD in quite different manners. The lack of spectral alteration in the a-CyD solution indicates that the interaction of 7,8-BPD with a-CyD is minimal. The absence of isosbestic points in spectra associated with titrations for both the p- and y C y D titrations is most likely the consequence of enhanced solubility of (-)-7,8-BPD resulting from CyD binding. In a 12 p M BPD solution without CyD, a significant fraction of BPD probably adheres to the
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Figure 2. (A) Fluorescence emission spectra of 12 pM (-)-7,8-BPD in 10 mM cyclodextrin solutions resulting from excaing at 358 nm. (8) Corresponding fluorescence emission spectra of 12 pM (-)-9,lO-BPD in 10 mM cyclodextrln solutions. a-,p-, and y-CyD are represented by dots, solid line, and squares, respectively. cuvette walls or exists in the microcrystal form as a consequence of low BPD solubility in aqueous solutions. Indeed, titrations with a much lower BPD concentration resulted in the appearance of isosbestic points. Since only qualitative spectral characteristics are of interest here, solutions of higher BPD concentrations have been used throughout. Also, a 30-min interval between titrations was maintained (for apparent practical reason) even though there were indications that excimer formation of pyrene in y-CyD may take several days to reach complete equilibration (11). Fluorescence Evidence of a 2:l Inclusion Complex Formation i n y-Cyclodextrin. The unique inclusion complex formation of (-)-7,&BPD with y-CyD is d r a m a t i d y revealed by the fluorescence emission spectral measurements. A characteristic excimer band around 490 nm is clearly apparent for (-)-7,8-BPD in the y C y D solution, but is conspicuously absent in both the 8- and a-CyD solutions (Figure 2A). The appearance of the excimer band suggests that a 2:l ligandCyD inclusion complex is formed in y-CyD solutions. The absence of such an excimer emission in the @CyD solution, despite the absorbance enhancement and spectral red shifts, suggests formation of a 1:l BPD-CyD complex. To see if a 2:l inclusion complex can also be formed with 9,10-BPD, a parallel study with this isomer was also made with cyclodextrins. No evidence of an excimer band is observed even in the y-CyD solution (Figure 2B), suggesting the absence of a 2:l complex with 9,lO-BPD. A fluorescence titration of (-)-7,8-BPD with increasing amounts of y-CyD is shown in Figure 3A. In the absence of y-CyD, the BPD exhibits fluorescence maxima at 400 and 423 nm. The intensities at these two wavelengths are progressively reduced while at the same time the 490-nm excimer intensity
ANALYTICAL CHEMISTRY, VOL. 60, NO. 23, DECEMBER 1, 1988
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Flgure 3. (A) Representative fluorescence emlssion spectra during the titration of (-)-7,8-BPD (kept constant at 12 pM) with varying concentrations of y-CyD. (6)Plot of Z(490 nm)/1(400 nm) vs yCyD concentration. The value of this ratio in a 10 mM y-CyD solution is indicated by crosses.
is correspondingly enhanced as the y-CyD concentration is increased. An isoemissive point is clearly evident at 448 nm, suggesting a two-state equilibrium. Since the emission at 400 nm is characteristic of the monomer fluorescence, a plot of the fluorescence intensity ratio of 490 vs 400 nm can be correlated to the relative dimer and monomer population ratio (Figure 3B). Circular Dichroism (CD) of (-)-7,8-BPD in Cyclodextrin Solutions. The CD spectra of (-)-7,8-BPD in the three cyclodextrin solutions are distinctly different, as can be seen by a comparison of the 20 "C spectra (conneded square) in Figure 4A-C. The (-)-7,&BPD CD spectrum in the a-CyD solution (Figure 4A) is very similar to that in the buffer, suggesting little inclusion complex formation. The broadness of the observed CD spectrum a t 20 OC suggests that molecular aggregation probably plays an important role in the appearance of spectral features in the a-CyD solution. This is supported by the temperature-dependent studies. As the temperature is increased, considerable spectral alteration occurs. At around 80 "C relatively sharp maxima are observed a t 365,346, 290, and 257 nm, corresponding to the observed absorbance maxima in ethanol at room temperature. In the &CyD solution, (-)-7,&BPD exhibits strong negative CD maxima at 369 and 349 nm and positive maxima at the shorter wavelength region (Figure 4B). The locations of the CD maxima correspond well with the red-shifted absorbance maxima (see Figure 1A). Increasing the temperature results in the intensity reduction with concomitant slight blue shifts. At around 100 "C the spectrum is very similar to the hightemperature spectrum in the a-CyD solution (note the scale difference), suggesting dissociation of (-)-7,&BPD from the inclusion complexes.
2623
In conformity with the grossly different absorption spectrum, CD features of (-)-7,8-BPD in y-CyD (Figure 4C) are distinctly different from those of a- and 6-CyD. A strong, negative broad CD band is apparent at 356 nm, corresponding to the absorbance maximum (see Figure 1B). The intensity at this wavelength decreases as the temperature is increased and eventually two maxima at 365 and 346 nm become apparent. Again, the spectrum at the highest temperature measured resembles those of high-temperature a-and P-CyD solutions, indicating the dissociation of inclusion complexes. Since the high-temperature spectra represent those of uncomplexed (-)-7,&BPD monomer, subtraction of these spectra from their low-temperature counterparts should yield the CD spectra induced by complexation with the respective cyclodextrin. The resulting difference spectra in the three cyclodextrin solutions are shown in Figure 4D. It clearly shows that in contrast to the negative CD induced at 370 and 350 nm upon inclusion of (-)-7,&BPD into p-CyD, strong negative CD is induced at 356 nm upon complexation with y-CyD. Induced CD spectra in the shorter wavelength region also differ greatly in these two solutions. As mentioned earlier, the induced CD spectrum in the a-CyD solution, which is similar to that in the buffer, is most likely the consequence of partial (-)-7,8-BPD aggregation in the solution. Conclusion from the Spectral Studies. Comparative spectroscopic binding studies of 7,8-BPD with three cyclodextrins (CyDs) described above suggest that the complex formed with yCyD is distinctly different from those of a-and 8-CyD. The formation of a 2:l guest-host complex in the y-CyD solution is strongly suggested by the prominent appearance of the 7,8-BPD excimer fluorescence. The absence of such an excimer emission despite the absorbance enhancement and spectral red shifts in the p-CyD solution suggests formation of a 1:l BPD-CyD complex formation. The negligible spectral alteration of (-)-7,8-BPD in the presence of a-CyD suggests minimal interactions in this solution. These findings are very similar to those of benzo[alpyrene + CyD systems (Chang and Chen, unpublished results). Our results are also consistent with the findings of cyclodextrin-pyrene inclusion complexation, which indicate the formation of 2:l complexes only in the y-cyclodextrin ( I 1-1 3). Our interpretations appear to conform with the cavity sizes of CyDs and the molecular size estimates of BPD. A molecular width of 8.8 8, along the short axis of benzo[a]pyrene has been estimated, using the standard bond lengths of 1.39 and 1.08 8, for the ring C=C and C-H, respectively, and the atomic radius of 1.2 8, for the hydrogen atom. Thus, (-)-7,8-BPD is too large to be included into an a-CyD, which has an internal diameter of 5.7 8, but can fit snugly into the 7.8-8, cavity size of 0-CyD, considering the dynamic nature of the molecules. The 9.5-8, cavity size of y-CyD should be able to accommodate two stacked BPD molecules. Parallel studies with 9,lO-BPD suggest that 7,&BPD binds to P-CyD more strongly than the 9,lO-isomer and 9,lO-BPD does not form 2:1 complexes with y-CyD. This may be a consequence of the fact that the hydroxyl group at the 10 position (in the bay region of benzo[a]pyrene) of 9,lO-BPD interferes with the inclusion of the pyrenyl ring while the 7-hdyroxyl (somewhat removed from the pyrene ring) of 7,8-BPD does not, assuming the inclusion geometry is such that the long axis of the pyrenyl chromophore lies along the cavity axis. These results suggest that HPLC columns packed with either p- or yCyD can be used to separate 7,8-BPD from its 9,lO-isomer and that the y-CyD is likely to be more effective. Indeed, the feasibility of such a separation was demonstrated with columns of bonded p- and y-CyD as stationary phases and the MeOH/water and MeCN/water
2624
ANALYTICAL CHEMISTRY, VOL. 60, NO. 23, DECEMBER 1, 1988
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Figure 4. Temperaturedependent CD specra of 12 pM (-)-7&BPD in 10 mM a-CyD (A), P-CyD (e), and y-CyD (C) solutions. (D)Comparison of difference CD spectra obtained by subtracting the highest temperature spectra from those of 20 OC in the three cyclodextrin solutions.
mixtures as mobile phases, as to be described in another section. Although our studies do not address the question of BPD excimer formation mechanism in y-CyD, it is reasonable to assume it to be analogous to the pyrene system. Fluorescence lifetime measurements of Yorozu et al. (12)on pyrene suggest that the excimer formation is completed within the duration of an excitation pulse (-0.8 ns). Thus, excimer fluorescence appears to originate from the excitation of the ground-state dimer which is already included in the y C y D rather than the formation of an excited dimer from the complexation of ground and excited monomers. Finally, even though the configuration of the two BPD molecules included in the y-CyD cavity cannot be deduced from the observed CD spectrum without detailed theoretical calculation using accurate transition moments, it is interesting to note that except for some differences in relative intensities, the induced CD spectral pattern for BPD is in general accord with that observed for pyrene in y-CyD (11). The configuration of the two pyrene molecules included had been suggested to be an S-helix. Effects of Inclusion Complexation on the Rate of anti-BPDE Hydrolysis. To see how the inclusion complex formation affects the kinetics of a chemical reaction, hydrolysis rates of anti-BPDE in cyclodextrin solutions were compared with that in the pH 7 buffer. The rate measurements were made possible by the fact that the absorption as well as fluorescent intensities of the hydrolyzed products are much higher than the unhydrolyzed reactant. Single exponential kinetics are observed, as evidenced by the linearity in the semilog plots of Figure 5, and the pseudo-firsborder hydrolysis rate of anti-BPDE in the a-CyD solution (k = 5.6 X s-l) is only slightly slower than in the buffer (k = 9.0 x s-l). On the other hand, the rate of hydrolysis in the p-CyD solution
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Anal. Chem. 1088, 60, 2625-2629
Table I. Comparison of Isocratic Elution Times (in minutes) for BPD Isomers from 8- and y-CyD Columns with Water Mixtures of Methanol or Acetonitrile as Mobile Phases % X/%
water 60140 50150 40160 30170 20180 10/90
X = methanol @-CyD YCYD
7,8
9,lO
7,8
9,lO
X = acetonitrile &CyD Y-CYD 7,8 9,lO 7,8 9,lO
5.3 4.8 6.0 5.2 3.5 3.5 3.6 3.6 7.2 6.1 10.6 7.8 3.7 3.7 4.0 3.9 12.5 9.1 22.5 12.3 4.2 4.1 4.9 4.5 17.7 11.4 57.5 24.6 5.5 5.3 7.4 6.2 58.5 26.0 >60 >60 11.8 9.0 19.2 12.3 >60 59.2 >60 >60 56.2 27.2 >60 44.4
also have application when using the cyclodextrins as vehicles for drug delivery. HPLC Separation with 8- or 7-CyD as Stationary Phase. Our spectroscopic results clearly indicate that the separation of 7,8-BPD from 9,lO-BPD may be accomplished by HPLC methodology using 8- or y-CyD either as a stationary or a mobile phase. Such a feasibility study was carried out and the results of the isocratic runs are shown in Table I. It is apparent from Table I that a base-line separation of 7,8-BPD from its 9,lO-isomer can easily be achieved by using the mobile phase of MeOHlwater with a water content between 40% and 70% or a MeCNfwater mixture with a water content between 70% and 85% for a bonded y-CyD column. A water content between 50% and 80% for the methanol mixture or 80% and 90% for the MeCN mixture is optimal for the bonded @CyD column. It is also noteworthy that
2825
under a given percentage of water content, the elution time with the MeCN mixture is shorter than that of the methanol mixture, consistent with the greater hydrophobicity of the former. As expected from our binding studies, the 7,8-isomer is being retained longer than its 9,lO-counterpart using the same column, and the gamma column is more effective than the beta column in trapping these benzo[a]pyrene metabolites.
ACKNOWLEDGMENT We thank D. Vines for carrying out some of the experiments. LITERATURE CITED (1) French, D. A&. Carbchydr. Chem. 1057, 12, 189. (2) Bender, M. L.; Komlyama, M. Cycbdexfdn Chemistry; Springer-Verlag: Berlin, 1978. (3) Szejtii, J. SterchlStaerke 1078, 30. 427. (4) Cramer, F.; Saenger, W.; Spatz, H.Ch. J . Am. Chem. Soc. 1067, 89, 14-20. ( 5 ) SBenger, W. Angew. Chem.. Int. Ed. Engl. 1080, 19. 344-362. (6) Armstrong. D. W.; DeMond, W. J . Chromatogr. Sci. 1084, 22, 411-415. (7) Armstrong, D. W.; DeMond. W.; Alak, A,; Hinze, W. L.; Riehi, T. E.; Bui, K. H. Anal. Chem. 1085, 57, 234-237. (8) Hinze, W. L. Sep. Purlf. Methods 1081, 10, 159-237. (9) Harvey, R. G. Acc. Chem. Res. 1081, 14, 218-226. (10) Geacintov, N. E.; Yoshkfa, H.; Ibanez, V.; Harvey, R. 0. Blochem. Bbphys. Res. Commun. 1081, 100, 1569-1579. (11) Kobayashi, N.; Saito, R.; Hino. H.; Hino, Y.; Ueno, A.; Osa, T. J . Chem. SOC..Perkin Trans 2 1983, 1031-1035. (12) Yorozu, T.; Hoshino, M.; Imamura, M. J . Phys. Chem. 1082, 86, 4426-4429. (13) Kano, K.; Takenoshita, I.; Ogawa, T. Chem. Lett. 1082. 321-324.
RECEIVED for review July 18,1988. Accepted September 6, 1988. This research was supported by USPHS Grant CA42682 and a subproject of NIH-MBRS Grant S06RR0892.
On-Line Connector for Microcolumns: Application to the On-Column o -Phthaldialdehyde Derivatization of Amino Acids Separated by Capillary Zone Electrophoresis Stephen L. Pentoney, Jr., Xiaohua Huang, Dean S. Bur&,' and Richard N. Zare* Department of Chemistry, Stanford University, Stanford, California 94305
The design of a shrpie, on-column connector for microcolumn Separations is descrlbed. The connector, in the form of a cross or tee, Is fabrlcated from the fused sliica caplliary tubing, Itself, and has sufficlentiy low dead volume to be compatible with the tubing dimensions normally associated with microcolumn separatlon technlques. As a demonstration of one posslbie application, a mixture of amino acids Is separated by capillary zone electrophoresis (CZE), the amino acids are derivatized on-column with o-phthaidiaidehyde (OPA) introduced from a cross connector, and the highly fluorescent adducts are detected downstream by laser-induced fluorescence. Zone broadening by the connector is determined to be approximately 10% for CZE separations performed in 75 pm 1.d. capillary tubes. Detector response is found to be linear over more than 3 orders of magnitude with minimum limits of detection in the subfemtomole range.
* Author to whom correspondence should be addressed.
Current address: Varian Associates, Systems Laboratory, Palo Alto, CA 94303. 0003-2700/88/0360-2625$01.50/0
It is difficult to transfer all of the versatility associated with conventional separation techniques to modern, analytical scale, separations because the latter rely heavily upon the use of microcolumns. Great care must be taken to minimize sources of extracolumn band broadening in order that the advantages of microcolumn techniques may be fully realized (I). This situation becomes especially apparent in the case of microcolumn techniques involving the use of capillary tubes (internal diameter less than 100 pm). Injectors, detectors, and connecting tubing must be designed in such a manner that the increase in system variance caused by each is far less than the fundamental column variance. As a result, the use of connecting devices (tubing, fittings, etc.) for capillary tubes has been avoided whenever possible. Sample introduction is often accomplished in a split fashion with a small sample plug being delivered directly onto the head of the capillary column, while solute detection is commonly made in an on-column configuration. The ability to make extremely low volume connections with capillary tubes would facilitate the extension of various sep0 1988 American Chemical Society
