A Method for the Determination of Binding Constants by Electrospray

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Anal. Chem. 2000, 72, 5411-5416

A Method for the Determination of Binding Constants by Electrospray Ionization Mass Spectrometry Esther C. Kempen and Jennifer S. Brodbelt*

Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, Texas 78712

A new method for the determination of binding constants using electrospray ionization mass spectrometry is presented. The intensity of a reference complex with a known log K value is monitored before and after addition of a second host or guest. On the basis of the change in intensity of the reference complex and extrapolation from a calibration curve, the log K value is then derived for the complex of interest using a set of simultaneous equilibrium equations. Binding constants of several crown ether-alkali metal cation complexes that were previously studied were determined to validate this strategy. Log K values for complexes involving dibenzo-16-crown-5 and its sym-oxyacetate derivative with Na+ or K+ were also determined. Molecular recognition plays an essential role in such processes as enzyme catalysis,1-4 antibody action,5 chiral catalysis,6 and ion transport through the membranes of ion-selective electrodes.7 The measurement of binding constants allows the evaluation of the strengths of host-guest interactions and thus gives a quantitative means of comparing the binding properties of different hosts and their selectivities for different guests. Binding constants have been traditionally measured by methods such as potentiometry, NMR titrimetry, or spectrophotometry.8 More recently, electrospray ionization mass spectrometry (ESI-MS) has been used to determine binding constants for various complexes formed in solution.9-15 Electrospray ionization has been successfully utilized to transport a wide variety of noncovalent complexes formed in * Corresponding author: (phone) 512-471-0028; (fax) 512-471-8696; (e-mail) [email protected]. (1) Vogtle, F., Weber, E., Eds. Host-Guest Complex Chemistry: Macrocycles; Springer-Verlag: New York, 1985. (2) Atwood, J. L., Ed. Inclusion Phenomena and Molecular Recognition; Plenium Press: New York, 1988. (3) Cram, D. J. In Applications of Biomedical Systems in Organic Chemistry; Jones, J. B., Sih, C. J., Pearlman, D., Eds.; Techniques of Chemistry Series Vol. 10, Part II; John Wiley: New York, 1976. (4) Reinholt, D. N. J. Coord. Chem. 1988, 18, 21-43. (5) Kuby, J. Immunology; W. H. Freeman and Co.: New York, 1992. (6) Lutz, R. P. Chem. Rev. 1984, 84, 205-244. (7) Korytam, J.; Stulik, K. Ion-Selective Electrodes, 2nd ed.; Cambridge University Press: New York, 1983. (8) Connors; A. K. Binding Constants: The Measurement of Molecular Complex Stability; Wiley: New York, 1997. (9) Loo, J. A.; Hu, P.; McConnell; Mueller, M. T.; Sawyer, T. K.; Thanabal, V. J. Am. Soc. Mass Spectrom. 1997, 8, 234-243. (10) Lim, H.-K.; Hsieh, Y. L.; Ganem, B.; Henion, J. J. Mass Spectrom. 1995, 30, 708-714. 10.1021/ac000540e CCC: $19.00 Published on Web 09/30/2000

© 2000 American Chemical Society

solution into the gas phase for analysis, both for small organic host-guest complexes and for large biological complexes.16-18 In many cases, relative ion abundances of different complexes observed in the ESI-mass spectra correlate well with equilibrium distribution of complexes in solution.9-35 (11) Greig, M. J.; Gaus, H. Cummins, L. L.; Sasmor, H.; Griffey, R. H. J. Am. Chem. Soc. 1995, 117, 10765-10766. (12) Griffey, R. H.; Hofstadler, S. A.; Sannes-Lowery, K. A.; Ecker, D. J.; Crooke, S. T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10129-10133.. (13) Prieto, M. C.; Whittal, R.; Balwin, M.; Burlingame, A. L.; Balhorn, R. Proceedings 47th ASMS Conference on Mass Spectrometry and Allied Topics, Dallas, TX, June 13-17, 1999; pp 614-615. (14) Jorgensen, T. J. D.; Roepstorff, P.; Heck, A. J. R. Anal. Chem. 1998, 70, 4427-4432. (15) Young, D.-S.; Hung, H.-Y.; Liu, L. K. Rapid Commun. Mass Spectrom. 1997, 11, 769-773. (16) Smith, D. L.; Zhang, Z. Mass Spectrom. Rev. 1994, 13, 411-429. (17) Smith, R. D.; Bruce, J. E.; Wu, Q.; Lei, Q. P. Chem. Soc. Rev. 1997, 26, 191-202. (18) Przbylski, M.; Glocker, M. O. Angew. Chem., Int. Ed. Engl. 1996, 35, 806807. (19) Gokel, G. W.; Wang, K. J. Org. Chem. 1996, 61, 4693-4697. (20) Young, D.-S.; Hung, H.-Y.; Liu, L. K. J. Mass Spectrom. 1997, 32, 432437. (21) Young, D.-S.; Hung, H.-Y.; Liu, L. K. Rapid Commun. Mass Spectrom. 1997, 11, 769-773. (22) Loo, J. A.; Holsworth, D. D.; Root-Berstein, R. S. Biol. Mass Spectrom. 1994, 23, 6-12. (23) Cheng, X.; Chen, R.; Bruce, J. E.; Schwartz, B. L.; Anderson, G. A.; Hofstadler, S. A.; Gale, D. C.; Smith, R. D. J. Am. Chem. Soc. 1995, 117, 8859-8860. (24) Li, Y. T.; Hsieh, Y. L.; Henion, J. D.; Ocain, T. D.; Schiehser, G. A.; Ganem, B. J. Am. Chem. Soc. 1994, 116, 7487-7493. (25) Hsieh, Y. L.; Cai, J.; Li, Y. T.; Henion, J. D.; Ganem, B. J. Am. Chem. Soc. 1995, 6, 85-90. (26) Leize, E.; Van Dorsselaer, A.; Kra¨mer, R.; Lehn, J.-M. J. Chem. Soc. Chem. Commun. 1993, 990-993. (27) Goa, J. Cheng, X. Chen, R.; Sigal, G. B.; Bruce, J. E.; Schwartz, B. L.; Hofstadler, S. A.; Anderson, G. A.; Smith, R. D.; Whitesides, G. M. J. Med. Chem. 1996, 39, 1949-1955. (28) Kempen, E.; Reyzer, M.; Brodbelt, J. S. Struct. Chem. 1999, 10, 213219. (29) Leize, E.; Jaffrezic, A.; Van Dorsselaer, A. J. Mass Spectrom. 1996, 31, 537544. (30) Blair, S.; Kempen, E. C.; Brodbelt, J. S. J. Am. Soc. Mass Spectrom. 1998, 9, 1049-1059. (31) Kempen, E. C.; Brodbelt, J. S.; Bartsch, R. A.; Jang, Y.; Kim, J. S. Anal. Chem. 1999, 71, 5493-5500. (32) Goolsby, B.; Hall, B. J.; Brodbelt, J. S.; Adou, E.; Blanda, M. Int. J. Mass Spectrom. 1999, 193, 197-204. (33) Blair, S. M.; Brodbelt, J. S.; Marchand, A. P.; Kumar, K. A.; Chong, H.-S. Anal. Chem. 2000, 72, 2433-2445. (34) Blanda, M. T.; Farmer, D. B.; Brodbelt, J. S.; Goolsby, B. J. Am. Chem. Soc. 2000, 122, 1486-1491. (35) Reyzer, M. L.; Brodbelt, J. S.; Marchand, A. P.; Chen. Z.; Huang, Z. Nanboothiri, I. N. N. N. Int. J. Mass Spectrom., in press.

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The most utilized method for determining binding constants by electrospray ionization mass spectrometry involves monitoring the intensities of both the unbound guest and the bound guest (or bound host and unbound host) during a titrimetric experiment, using these intensities to derive a Scatchard plot.9-13 This method works well for systems in which the host or guest and the hostguest complex are ionic and may be detected by the mass spectrometer. Unfortunately, it is not applicable for systems that involve formation of neutral host-guest complexes or cases where the guests are small ions of low mass-to-charge ratios which are not efficiently transferred by ESI or easily observed in the mass spectrometer (due to mass discrimination, solvent adductions, charge exchange, etc.). Also, this method requires a separate titration for each host-guest system, a time-consuming endeavor. This method has been used to examine dissociation constants for oligonucleotide-serum albumin complexes,11 the binding constants of vancomycin-peptide complexes and ristocetinpeptide complexes,10 the dissociation constants for proteinphosphopetide complexes,9 and the dissociation constants for aminoglycoside-RNA models.12 Another method that has been used to determine binding constants, as illustrated for complexes between bacterial cell wall peptide hosts and vancomycin antibiotic guests, entails monitoring the intensities of the protonated antibiotics and the antibiotic-peptide complexes produced from solutions containing equimolar mixtures of peptides and antibiotics.14 This method makes the assumption that the ESI efficiencies for the complexes (i.e., peptide-antibiotic complexes) are similar to those of the free antibiotics (i.e., protonated vancomycin). Although this assumption is reasonable for cases in which the two ions have similar sizes and have similar solvation energies,36 it does not hold for systems where the host-guest complex has significantly different size or solvation properties relative to the free host or guest ion. Moreover, this method again is not applicable to cases in which the host or host-guest complex is uncharged. It is known that the solvation energies of the ions greatly influence their ESI efficiencies. For instance, Leize et al. showed that for electrospray ionization of alkali metal ions there is a significant correlation between the solvation energy of an ion and its intensity, as observed by ESI-MS, for a solution containing five alkali metal ions of the same concentration.29 Reports from the Brodbelt laboratory have also noted significant differences in the electrospray ionization efficiencies of alkali metal complexes formed for crown ethers of different sizes or significantly different solubilities.30 In many of these cases, “correction” factors (i.e., response factors) of several orders of magnitude were required in order to balance the relative intensities of the complexes in the mass spectra with the known equilibrium distributions in solution. These results clearly show the potential shortcomings of using ESI-MS results to estimate binding constants for cases in which two or more different types of ions are monitored. Liu and co-workers developed a method for the determination of binding constants using one host-guest complex as an internal reference to normalize the relative ESI efficiency of a second complex of interest.45 A requirement of the Liu method is that the unknown complex has a binding constant similar to that of the internal reference complex, a significant limitation. Finally, (36) Van Dorsselaer, A.; Bitsch, F.; Green, B.; Jarvis, S.; Lepage, P.; Bishoff, R.; Kolbe, H. V. J.; Roitsch, C. Biomed. Environ. Mass Spectrom. 1990, 19, 692-704.

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Raffaelli et al. determined the equilibrium concentrations and thus binding constants of cyclodextrin-drug inclusion complexes by comparison with concentrations of either the host or guest in the absence of the complex.37 A “virtual” calibration curve for the complex of interest was constructed by the evaluation of ESI efficiencies for the complex by the determination of the nominal and the equilibrium concentration of the host (i.e., cyclodextrin) or of the guest (i.e., drug).37 Unfortunately, this method also requires observation of the host-guest complex and either the free host or guest. The concept of “competitive” solution equilibria has been successfully used in the past by Gokel et al. to determine binding constants of Ca2+-crown ether complexes using sodium ionselective electrodes38 and by Goff and co-workers to determine the binding constants of Rb+-18-crown-6 analogues in dimethylformamide by cesium NMR.39 Although these methods are not widely used today, they do pave the way for a new versatile ESIMS method for the measurement of binding constants presented in this report. A calibration curve using one host-guest complex as a reference is generated and then a second host or second guest is added to a solution of the reference host-guest system, thus establishing a competitive equilibrium. Since both the binding constant and concentration are known for the reference hostguest complex, the binding constant and concentration of the second host-guest complex of interest may be derived. The method is simple and fast and does not require that either the host-guest complex or host of interest be ionic. The hosts used in this study are shown in Figure 1. EXPERIMENTAL SECTION Instrumentation. The binding experiments were performed with a Finnigan ion trap mass spectrometer operating in the massselective instability mode with modified ITD electronics to allow axial modulation. The electrospray interface is based on a design developed by the ion trap group at Oak Ridge National Laboratory involving differentially pumped regions containing ion focusing lenses.40 This design has neither a heated capillary nor sheath gas and has always given a relatively high upper limit on the dynamic range of useable concentrations for the ESI solutions.30 The experiments were performed using both conventional electrospray technology and nanospray technology at a variety of flow rates with little or no difference in performance. Strategy for Determination of Binding Constants. Because solvation energies play such a significant role in the formation of ions by electrospray ionization, a method involving direct com(37) Raffaelli, A.; Lucarotti, S.; Pucci, S.; Salcadori, P. Proceedings 47th ASMS Conference on Mass Spectrometry and Allied Topics, Dallas, TX, June 13-17, 1999; pp 2797-2798. (38) Gokel, G. W.; Goli, D. M.; Mingati, C.; Echegoyen, L. J. Am. Chem. Soc. 1983, 105, 6784-6790. (39) Goff, C. M.; Matchette, M. A.; Shabestary, N.; Khazaeli, M. A. Polyhedron 1996, 15, 3897-3903. (40) Van Berkel, G. J.; Glish, G. L.; McLuckey, S. A. Anal. Chem. 1990, 62, 1284-1289. (41) Gokel, G. Crown Ethers and Cryptands; The Royal Society of Chemistry: Cambridge, 1991; p 74. (42) Zavada, J.; Pechanec, V.; Zajicek, J.; Stibor, I.; Vitek, A. Collect. Czech. Chem. Commun. 1985, 50, 1184-1193. (43) Okoroafor, N. O.; Popov, A. I. Inorg. Chim. Acta 1988, 148, 91-96. (44) Frensdorff, H. K. J. Am. Chem. Soc. 1971, 93, 600-606. (45) Kay, J. L.; Hales, B. J.; Cunningham, G. P. J. Phys. Chem. 1967, 71, 39253930.

[M]+F ) [M]+T - ([HR + M]+ + [HN + M]+) [H]F ) [H]T - [H + M]+

Figure 1. Compounds studied.

parison of the intensity of a host-guest complex with a known binding constant to the intensity of a complex with an unknown binding constant is not recommended. Instead, a calibration curve for the concentration of the reference host-guest complex versus intensity is constructed based on ESI-MS of a series of solutions prepared at well-defined concentrations. The concentration of the reference complex in each solution is calculated from its known binding constant obtained from the literature and the known amounts of host and guest added to the solution. The host-guest complexes used as references and their reported binding constants are presented in Table 1.41-43 The determination of binding constants for new host-guest complexes is based on the competition of the two different host molecules (reference host and one host of interest) for a common guest or, conversely, the competition of one host for two different guests (reference guest and one guest of interest). Only the intensity of the reference complex is monitored in the ESI mass spectrum. When a second host (or guest) is added to a solution of the reference complex, the second host (or second guest) competes with the reference host for complexation of the guest ion. This competition causes a shift in the equilibrium established for the reference host-guest complex, and thus, the concentration of the reference complex decreases as a function of the binding affinity of the second host (or second guest), thus reflecting the binding constant of the new host-guest complex of interest. For example, the equilibrium for a solution containing known concentrations of reference host (HR), new host (HN), and guest metal ion (M+) is as follows, where KR represents the binding constant of the reference complex, KN represents the binding constant of the complex of interest, and the subscript F signifies the free (unbound) hosts:

KR ) [HR + M]+/([HR]F)([M]+F) KN ) [HN + M]+/([HN]F)([M]+F)

(1)

Using the following mass balance eqs 2, the binding constant of the complex of interest is easily determined by solving the simultaneous equations:

(for each HR and HN)

(2)

In these equations, T is the total concentration present in solution. The unknown parameters present in the simultaneous eqs 1 are the [HN + M]+ (concentration of the (HN + M)+ complex) and KN. KR is a known binding constant of the reference complex, [M]+T and both the [H]T values are known experimental parameters, and the concentration [HR + M]+ for the solution is derived from the calibration curve based on the intensity of the reference complex in the ESI mass spectrum after the second host (or guest) of interest is added. A representative calibration curve for the reference complex (18-crown-6 + K)+ is presented in Figure 2. For this calibration curve, intensities of the (18-crown-6 + K)+ complexes were acquired and averaged over a period of 5 min. The error bars typically represent a (10% variation in the signal intensity, the average standard deviation noted for this ESI assembly. The other parameters in the simultaneous equilibrium eqs 1 are calculated from the mass balance eqs 2 shown above. Although the experimental description provided here describes experiments in which two hosts are in competition for a single guest, this technique is also applicable to cases in which a single host competes for two different guest ions. Moreover, the accurate application of the method requires that the stoichiometry of the host-guest complex of interest is assumed to be 1:1 in these experiments, and the existence of other stoichiometries would lead to the incorrect derivation of log K values. In all cases, the calibration curves used in this study have an R2 value of 0.96 or better. The success of the method requires a stable and reproducible spray throughout the acquisition of the data for the calibration curve and subsequent measurements involving the solutions of interest. Metal cation concentrations are assumed to be equal to the salt concentrations added to the solutions since dissociation in the methanol and acetonitrile solutions can be estimated from ion-pairing equilibrium constants to be 99% or greater dissociated at 1 mM.44,45 The concentrations of metal salts in the solutions used in this work are smaller than 1 mM (0.1 mM); thus, this ion-pairing effect is negligible. The accuracies of the binding constant values derived for previously studied complexes are based on the difference between the observed values and their literature counterparts. Reagents. All reference reagents, 18-crown-6, 15-crown-5, dibenzo-18-crown-6, sodium chloride, potassium chloride, sodium perchlorate, and potassium perchlorate, were purchased from Aldrich, dried under vacuum, and used without further purification. Anhydrous solvents were also purchased from Aldrich and used without further purification. Dibenzo-16-crown-546 (1) and its derivative sym-dibenzo-16-crown-5-oxyacetic acid47-49 (2) were synthesized by Bartsch et al. at Texas Tech University. RESULTS AND DISCUSSION Validation of Method. To determine the accuracy and reproducibility of our electrospray ionization competitive binding (46) Penderson, C. J.; J. Am. Chem. Soc. 1967, 89, 7017. (47) Bartsch, R. A.; Heo, G. S.; Kang, S. I.; Liu, Y.; Strzelbicki, J.; Bills, L. J., J. Org. Chem. 1993, 48, 4864. (48) Ohki, A.; Lu, J. P.; Bartsch, R. A. Anal. Chem. 1994, 66, 651-654. (49) Ohki, A.; Lu, J. P.; Huang, X.; Bartsch, R. A. Anal. Chem. 1994, 66, 43324336.

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Table 1. Binding Constants Derived by Competitive ESI-MS Method complexa

solvent

ESI log KA

MeOH

4.5d

[dibenzo-18-crown-6 + K]+

MeOH

4.9

[dibenzo-18-crown-6 + Na]+

MeOH

4.4

[18-crown-6 + Na]+

H2O

1.4

[18-crown-6 + Na]+

MeCN

4.2

[15-crown-5 + Na]+

MeCN

3.7

[18-crown-6 + K]+

MeCN

4.8

[18-crown-6 +

Na]+

lit.b log KA 41

4.35 (2.1-5.5) 50 5.0 41 (4.8-5.0) 50 4.37 41 (4.18-4.37) 50 1.38 42 (0.63-1.44) 50 4.39 43 (4.2-4.71) 50 4.91 43 (3.6-5.38) 50 5.46 43 (4.5-6.0)50

ref host-guest complexc

lit. log K of ref

K]+

6.08 41

[18-crown-6 + K]+

6.08 41

[18-crown-6 + Na]+

4.35 41

[18-crown-6 + K]+

2.03 42

[18-crown-6 + K]+

5.46 43

[18-crown-6 + Na]+

4.39 43

[18-crown-6 + Na]+

4.39 43

[18-crown-6 +

a This column represents the host-guest complex of interest. b Each log K value on the top represents the value that was reported by the same group that measured the log K values for the references complexes in the last column. The log K values in parentheses indicate the entire range of values reported in the literature by different methods and different groups. c This column indicates the reference host-guest complex for which calibration curves were constructed. d Standard deviation (0.3.

Figure 2. Calibration curve of (18-Crown-6 + K)+ in methanol.

method, the binding constants of several host-guest complexes whose binding constants were already reported in the literature were compared with binding constants derived using our method. Although most of the complexes studied were solvated in methanol, experiments in aqueous media and acetonitrile were also performed to evaluate the versatility of the method in different solvent environments. The first step requires the construction of a calibration curve, as shown in Figure 2. An example of the following key steps is shown in Figure 3. Figure 3A shows the ESI-mass spectrum of the (18-crown-6 + K)+ complex when the initial concentrations of 18-crown-6 and K+ are 1.5 × 10-4 and 1.5 × 10-4 M, respectively, giving a calculated concentration of 1.4 × 10-4 M for the (18-crown-6 + K)+ complex in solution, based on the reference binding constant of log K ) 6.08.41 Figure 3B shows the ESI-mass spectrum that results for a second solution containing the same concentration of 18-crown-6 and K+ as the previous solution but also containing 1.5 × 10-4 M dibenzo-18crown-6. The intensity of the (18-crown-6 + K)+ complex decreases, and extrapolation from the calibration curve indicates that the concentration of the (18-crown-6 + K)+ complex is 1.1 × 10-4 M. Based on this change in concentration, the binding constant of the (dibenzo-18-crown-6 + K)+ is determined to be 4.9, as shown in Table 1, a value that agrees well with the binding constant reported in the literature. The binding constants obtained in this way are summarized in Table 1. It is not necessary for the stoichiometric ratio of the three components comprising the second solution, (i.e., the one contain5414 Analytical Chemistry, Vol. 72, No. 21, November 1, 2000

Figure 3. Example of signal diminishment as a function of the binding constant of the second host.

ing the reference host, the guest, and the host of interest) be 1:1:1. In fact, several experiments reported here involve measurements of solutions where the host of interest was at double or half the concentration of the reference host. For instance, in an experiment analogous to that shown in Figure 3, when the second solution contains 1.5 × 10-4 M each of 18-crown-6 and KCl but 3.0 × 10-4 M of dibenzo-18-crown-6, the resulting (18-crown-6 + K)+ peak intensity is 165 units, corresponding to a concentration of 1.0 × 10-4 M for the (18-crown-6 + K)+ complex. This also yields a value of log K ) 4.9 for the (dibenzo-18-crown-6 + K)+ complex. In cases where the complex of interest has a log K value more than 1 order of magnitude greater than or less than that of the reference complex, it may be necessary to alter the stoichiometry of the mixture by modifying the concentration of the host of interest relative to that of reference complex. Altering the stoichiometry in a simple systematic fashion (such as doubling the concentration of the host of interest) also allows a second independent measurement of the log K value for the host/guest

Table 2. Binding Constants of Dibenzo-16-Crown-5 Derivatives with Sodium and Potassium Ionsa compound

log KNa+

log KK+

dibenzo-16-crown-5 (1) sym-dibenzo-16-crown-5-oxyacetate (2)

3.8 3.7

3.8 4.9

a

Figure 4. Reproducibility of (dibenzo-18-Crown-6 + K)+ binding constants obtained by undertaking multiple measurements of a solution containing (18-crown-6 + K)+ as the reference host (initial solution containing 1.5 × 10-4 M each 18-crown-6, dibenzo-18crown-6 and KCl).

complex of interest without requiring the construction of a second calibration curve. As mentioned previously, the method can be used to characterize the complexation of new hosts or new guests. As shown in Table 1, the binding constant of (18-crown-6 + Na)+ was determined by using (18-crown-6 + K)+ as the reference complex. Addition of the second guest, Na+, to a solution containing known amounts of 18-crown-6 and K+ establishes the competitive equilibria that allow the determination of the binding constant for (18-crown-6 + Na)+. The binding constants derived here for all of the previously studied complexes agree very well with literature values with the exception of the binding constants obtained for (18-crown-6 + K)+ and (15-crown-5 + Na)+ in acetonitrile. The literature values for the (18-crown-6 + K)+ in acetonitrile range from log K ) 4.5 to 6.0.45 Thus, although the ESI-MS-derived value (log K ) 4.8) does not match the log K value obtained by the same research group that derived the log K value used for the reference complex, it is within the range of values obtained from other sources. Likewise, the reported log K values for (15-crown-5 + Na)+ in acetonitrile range from log K ) 3.6 to 5.38.50 In this case also, the ESI-MS value derived here (log K ) 3.8) does not match the binding constant reported from the group that also examined the reference complex, but it does fit within the range reported in the literature. The reproducibility of the competitive ESI-MS method is quite good as is evident from the multiple observations of the binding constants for the (dibenzo-18-crown-6 + K)+ complex (Figure 4), obtained using (18-crown-6 + K)+ as the reference host-guest complex. For the data shown in Figure 4, the average value for the log of the binding constant of (dibenzo-18-crown-6 + K)+ is 4.9 with a standard deviation of (0.3. Although the calibration curves presented here represent only a narrow range of concentrations, linearity has been observed previously in the range of 10-8-3 × 10-4 M.30 The reason that the relatively narrow ranges were used in this work is to enhance the accuracy of the method by better defining the calibration curve for a targeted concentration range of interest. Should a broader range of concentrations be desired, however, it is well within the ability of the instrumentation. (50) Izatt, R. M.; Pawlak, K.; Bradshaw, J. S. Chem. Rev. 1991, 91, 1721-2085.

Average standard deviation, (0.3.

Dibenzo-16-Crown-5 Derivatives. After validation, the competitive ESI-MS method was applied to quantitation of binding constants of complexes of two dibenzo-16-crown-5 derivatives that were synthesized by the Bartsch group at Texas Tech University.47 The binding constants for the complexes of these compounds with Na+ and K+ as determined by the ESI-MS method are located in Table 2. These compounds were synthesized to act as ion-selective reagents in membranes of ion-selective electrodes. The binding constants for dibenzo-16-crown-5 complexes have not been reported in the literature; however, the selectivity of this compound was studied previously by Bartsch and co-workers48,49 by incorporation into a solvent polymeric membrane of an ion-selective electrode (SPME), in which the alkali metal ions of interest were extracted from an aqueous solution. With this electrode, a SPME selectivity of dibenzo-16-crown-5 for Na+ relative to K+ was determined by the characterization of fixed interferences. The selectivity of dibenzo-16-crown-5 has also been determined in our laboratory31 using an electrospray ionization metal selectivity method pioneered by this group.28,30-35 Both the ESI-MS and SPME studies indicated that dibenzo-16-crown-5 is only slightly selective for Na+ relative to K+. This observation is supported by the binding constants for dibenzo-16-crown-5 with Na+ and K+ derived here (log KNa+ ) 3.8, log KK+ ) 3.8). The calculated Na+/ K+ selectivity for a solution containing 1.5 × 10-4 M each of NaCl, KCl, and dibenzo-16-crown-5 in 100% methanol from these binding constants is Na+/K+ ) 1.0. When an ESI metal ion selectivity study using a solution of that same composition was performed, the observed selectivity observed was Na+/K+ ) 1.5. While this does not agree perfectly with the ESI-MS binding constant data, it is within the experimental error of the binding constant experiments. Binding constants have been reported for the ionized oxyacetate derivative of dibenzo-16-crown-5, 2, in 99% methanol/ 1% water (log KNa+ ) 4.02, log KK+ ) 3.71).51 The binding constant of the (2 + Na)+ complex derived by Bartsch and co-workers (log K ) 4.02) agrees reasonably well with the ESI binding constant reported in Table 2 (log K ) 3.7). The ESI binding constant for the (2 + K)+ complex (log K ) 4.9) however, diverges significantly from the number determined by Bartsch and co-workers (log K ) 3.71). The ESI value for (2 + K)+ was repeated several times with two different reference complexes and ranged from log K ) 4.7-5.0. The reason for this difference in values is not known; however, large differences in values determined by different experimental methods are not uncommon. Unfortunately, because the complexes of 2 with Na+ and K+ are neutral and therefore not visible in the mass spectra, it is impossible to perform ESI metal selectivity studies as were performed in the case of dibenzo-16-crown-5 (1). Limitations of the Method. Although the competitive ESIMS method for the determination of binding constants is ex(51) Chang, C. A.; Twu, J.; Bartsch, R. A. Inorg. Chem. 1986, 25, 396-398.

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tremely versatile, some limitations should be noted. Binding constants may only be determined if the log K value for the unknown complex is (2 of the log K value for the reference complex. If the binding constant for the complex of interest is expected to be higher than the log K for the reference complex, then the solution containing all three species should not contain a 1:1:1 ratio of components. The component of the solution necessary for the formation of the complex possessing the greater binding constant should have a diminished concentration relative to the other components, usually 1/5 to 1/10 the concentration. Initial experiments that entail screening solutions containing 1:1:1 ratios of all components may be necessary in order to determine whether the concentration of the reference complex will remain on the calibration curve. If it appear too low to be observed, an adjusted ratio of compounds in the solution may be necessary. CONCLUSIONS The use of competitive binding equilibria coupled with electrospray ionization mass spectrometry provides a new strategy for the quantitation of binding constants. Because this method does not require the need to compare the peak intensities of more than one host-guest complex in a set of experiments, it circumvents the need to make assumptions regarding ESI efficiencies that are inherent in many other ESI-MS methods. Since observation of only the reference complex and not the complex of interest (i.e., the one with an unknown log K value) is necessary, this method may be used to determine binding constants of complexes that are neutral or that have mass-to-charge ratios higher than

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the mass spectrometer is able to analyze. This method also allows the determination of binding constants in mass spectrometers that may suffer from mass discrimination effects, because only one mass to charge is monitored. Binding constants of hosts of interest may be determined if they lie within (2 log K units of that of the reference host-guest complex. Because the host of interest is only involved in one experimental measurement, binding constants for many different complexes may be performed using the same calibration curve, decreasing the time necessary for analysis. Most importantly, as only one solution of relatively low concentration is needed containing the host-guest complex of interest, binding constants for precious samples of which only extremely small quantities are available may be determined because of the minimal sample consumption in this method. ACKNOWLEDGMENT Professor Richard Bartsch from Texas Tech University and his research group are gratefully acknowledged for the donation of dibenzo-16-crown-5 and sym-dibenzo-16-crown-5-oxyacetic acid. The Welch Foundation (F1155), the National Science Foundation (CHE-9820755), and the Texas Advanced Technology Program (003659-0206) are gratefully acknowledged for their generous support.

Received for review May 11, 2000. Accepted August 22, 2000. AC000540E