J. Phys. Chem. 1993,97, 6546-6554
6546
Interactions of Pyrene with Cyclodextrins and Polymeric Cyclodextrins Wenying Xu,? J. N. Demas,”t B. A. DeGraff,’*s and M. Whaleyt Departments of Chemistry, University of Virginia, Charlottesville, Virginia 22901, and James Madison University, Harrisonburg, Virginia 22807 Received: December 7, 1992; In Final Form: March 19, 1993
Interactions of a- and 0-cyclodextrin (CD) and polymerically linked &CDs with pyrene were studied using luminescence intensity and lifetime measurements. For the monomeric CDs, both single and double capping of the pyrene were unambiguously observed. The hydrophobicity of the binding environments was quite different for the two monomeric CDs as judged by luminescence spectra. The binding constants, as well as the hydrophobicity of the binding environment, were rationalized on the basis of the differing structures of the inclusion complexes. The polymeric systems exhibited large differences as a function of the average spacer polymer length. In particular, they can have much higher formation constants than the monomeric CDs. Binding was much tighter for the longer spacers, and the environment was much more hydrophilic. A model involving sequential 1:1 and 1:2 clam shell binding with a second CD on the same polymer chain was developed that successfully accounted for these effects. A minimum spacer length is required to permit intramolecular clam shell binding. Molecular modeling supports our interpretation of the monomeric and polymeric CD results. The properties of the polymeric CD suggest their use in catalysis and drug delivery systems. They have very high water solubility, and their very efficient formation of clam shell binding can be used to solubilize otherwise hydrophobic molecules.
Introduction
Experimental Section
There is continuing interest in understanding intermolecular interactions of species with cyclodextrins (CDs), polymers, and solid supports. Many such systems are microheterogeneous and present severe problems in understanding their organization and influence on chemical and photochemical properties. Of increasing interest are polymeric CDs with their potential applications in areas such as drug delivery’and water clean-up? Recent studies have shown that there can be strong cooperation between different CDs on the same polymer chain; multiple binding to a single substrate can enhance binding to large hydrophobicg u e ~ t s . ~ Our primary work is in the design-applicationsof luminescent metal complexes as sensors and molecular probes4 Their association with CDs is a valuable approach for exploring the interactions, binding properties, and environmental effects on luminescentproperties. Before exploringthe complex interactions of polymeric CDs with metal complexes, we examined polymer CDs with pyrene (Py). Py probes binding site polarity by its pronounced emission vibronic structural changes with solvent polar it^.^ Nonpolymeric CD-Py systems as well as other organic probes have been extensively studied.5 We discovered that there is still considerable confusion even in the Py/@-CDcase, and we report on the interactions of Py and 8-CD examined by fluorescence. We confirm some earlier reports that Py can form a 2: 1 CD:Py complex and also examine the role of the 1:1 complex in fluorescence. For comparison we examined a-CD binding. Once we understood monomeric CDs with Py, we examined polymeric @-CDswith Py. We originally hoped to design systems that would double cap Py to form a nearly completely encaged species. The driving force would be the chelate effect from a high local concentration of CD once the first CD was added to the Py. Efficient double encapsulationresulted over a wide range of polymer spacer lengths. Using vibronic spectroscopy we determined the local hydrophobicity of the Py environment and correlated it with polymer structure. Our results promise to assist in designing highly water soluble CD carriers for hydrophobic drugs.
Materials. Pyrene (99+%, Aldrich) was quite impure. It is red, and the luminescence decays showed a pronounced shortlived component. The Py was twice recrystallized from ethyl alcohol and twice vacuum sublimed (120 “C). The very light yellow product still gave a small short-lived fluorescence component, but the absorption, luminescence lifetime ( T ) , and fluorescence spectra agreed well with the literature. 8-CD (Aldrich) was recrystallized twice from deionized water and dried in a vacuum oven at 70 OC for 24 h. We assume that our materialcontained 13.5wt 5% watersofhydration. Noorganic solvents were used in the @-CDpurification proyss since it is well-known that CDs can tenaciously hold on to cosolvents, which dramatically affects luminescence properties. The danger of recrystallizing CDs from organic solvents has been noted;6 recrystallizationactually degraded the quality of the CD, possibly due to selective inclusion of trace organic impurities or due to cosolvent enhancement of the luminescenceof existing impurities. a-CD (Sigma) was used as received (2.7% water). Sodium hydroxide and HCl were AR reagents. Poly(ethy1ene glycol) (PEG) with a molecular weight (MW) of 3400 and epichlorohydrin (EP) (99+%) were used as received. Deionized distilled water was redistilled over alkaline KMn04. Spectrapor membrane tubing was from Spectrummedical Industries Inc. Procedures. The use of aqueous Py presents difficulties. The solubility is about 6 X lCr7 M, and it adsorbs readily on glass; microcrystals are difficult to eliminate. We used aqueous Py solutions of 2 4 X lo-’ M. After much effort we arrived at the following procedurethat gave reproduciblefluorescence intensities and T’S with a minimal short-lived T : Py solutions were made by stirring an excess of Py in water for a few days. Samples were protected from light to minimize photodecomposition. Solutions were also sonicated for 2 h and filtered through a glass filter. Most critically, immediately before use solutions were filtered through a 45 pm micropore size syringe filter nylon membrane (Alltech Associate Inc.) to eliminate microcrystals. Two filtrations were required to remove the bulk of the microcrystals. Even then a small fluorescence spike was still observed in T measurements. On dilution with water, the fluorescenceemission intensity
University of Virginia. t James Madison University.
f
0022-3654/93/2097-6546%04.00/0 0 1993 American Chemical Society
Interactions of Pyrene with Cyclodextrins decreased with the same ratio as the dilution factor, showing the absence of excess Py in the concentrated solutions. A water blank was taken for all measurements. Care was taken to insure that there were no Py crystals on the cell walls. The absenceof microcrystals was confirmed by visually inspecting the blank and by the low emission counts in the characteristic 500-nm region.' Titrations were performed by adding @-CD (solid or fresh concentrated solutions) or solid @-CDoligomer to aqueous Py solutions. For the polymers, the calculated amounts of oligomers were based on the concentration of @-CDunits. Preparationof &CD Oligomers. A convenient way to prepare @-CDpolymers is to react @-CDwith epichlorohydrin. Thedegree of polymerization depends critically on the concentration of base, the reactant ratios, the reaction time, and the temperature. In obtaining water soluble oligomers, the reaction conditions should be controlled properly. In early preparations, the reactions were carried out in a weak base (ca 4-5 w/v % NaOH) for 3 h at 50 OC. While very water soluble products were obtained, all the product went through dialysis tubing with a MW cutoff of 3500. We suggest that the product was a simple @-CDglyceryl ether mixture with a MW of 1500-1600. Alternatively, insoluble crosslinkedpolymerswere obtained at high NaOH concentrations (140 w/v %), high reactant concentrations ( 1 5 0 wt %), and high EP:CD ratios (>3). Crosslinking was especially severe with high reaction temperatures (60 OC) and long (3 h) reactions. A series of water soluble @-CDoligomers was successfully prepared by reacting @-CDwith epichlorohydrin in an alkaline medium. Different polymers were obtained by changingthe molar ratios of @-CDto EP (l:l, 1:2, 1:3, 1:4, and 1:8), We refer to these oligomers as lE, 2E, 3E, 4E, and 8E, which correspond to the starting ratios of @-CDto EP. The reactions were carried out in 20% aqueous NaOH with around 25-30 wt % of reactants at 50 OC for about 3 h. The reaction mixtures were cooled to room temperature and neutralized with 4 N HCl to pH 7. These solutions were diluted 3 times by pure water and transferred into dialysis tubing (MW cutoff, 3500). The tubing was spun in a dialysate (deionized water) over a magnetic plate. The dialysate was changed with fresh deionized water every 4-5 h for 8-10 times; after about 6 dialysis sessions no C1- was detected using Ag+. This procedure insured removal of inorganic salts,unreacted 8-CD, epichlorohydrin, or lower MW products. After dialysis, a solid oligomer was isolated by freeze drying. A fine white powder was obtained with yields of 30-56%. Characterizationof CD-EP Oligomers. The oligomers were characterized by MW determinations, microanalysis, and NMR. The average MW was measured using a 5500 XR vapor pressure osmometer (WESCOR INC) referenced to 290 and 1000mmol/ kg standards. Samples were measured in triplicate. Molecular weights of CD-EP oligomers were as follows: 1E (3700), 2E (3800), 3E (42OO), 4E (4000), and 8E (6000). Standard deviationswere typically 2-3%. All the products arevery soluble in water (up to 40% w/w). The solutions were not excessively viscous, which is consistent with the low average MW. As a confirmation of these MWs, the 4E sample was dialyzed with a 6000-8000 MW cutoff membrane and reisolated by freeze drying. Less than 10% of the sample still remained inside the tube after a 24-h dialysis. Thus, 90% of the product had a MW of 3500 to -6000-8000. The polymer extension-generation chemistry is shown in Scheme I. @-CD, 1 mol, contains 21 hydroxyls, which could potentially react with epichlorohydrin. However, the primary carbohydrate OH groups on the small open end of the CD are much more reactive than the secondary OHs at the larger open end. We assume that the bulk of the substitution is occurring at the primary alcohols. The opening of an epoxide ring by the carbohydrate OH will occur preferentially at the less substituted terminal carbon atom of the epoxide as shown. After the addition of an epoxide, a new epoxide can be formed, which is hydrolyzed to a diol; the terminal primary alcohol will be much more reactive
The Journal of Physical Chemistry, Vol. 97, No. 24, 1993 6547 SCHEME I
OH CD-CHzOH
NaOH
- HCI
/O\ CH2-CH-CHzCI
-/O\
I
CD-CHZ-O-CH~-CH-CH~CI
CD-CHz-O-CHZ-~~-~,
NaOH HZO
/ O\
CHz-CH-
/O\
CHz-CH-CHzCl
CD D-
CHzCl
CHAIN EXTENSION
to the addition of another epoxide and the chain will extend in a linear fashion. Chain termination occurs when the epoxide reacts with the CHzOH of another CD. Themoreepichlorohydrin present in the reaction mixture, the greater the probability of chain extension versus termination and, thus, the longer the chains. While we show simpleextension with a fixed number of polymer linkers, n, between CDs, the polymers will be heterogeneous due to differing values of n between CDs and the initiation of some chains that fail to terminate on another CD. Our system will also have heterogeneity in the number of CDs. However, all polymers must have a minimum of three to four CDs per chain. While we knew the original reaction stoichiometry, we had to verify the structure of the final polymer. H-NMR spectra in D20 of the oligomers was determined as described earlier;* however, it was necessary to use a 500-MHz General Electric GNSOONMR to have adequateintegration accuracy. Our 8-CD spectrum matched well with the l i t e r a t ~ r e .It~ ~consisted of a doublet from HI at 5.06 ppm, and two clusters of multiplets centered around 3.754.05 ppm (H3, and H5) and 3.5-3.7 ppm (H2 and H4). In the polymers, the spectra were similar but broadened. Identifiable peaks from the CH2CH(OH)CHA linker were not distinguishable,but the integrated area under the 3.754.05 and 3.5-3.7 ppm multiplets increased relative to the HI doublet. Since every linker unit added 5 nonexchangeable protons for the seven HI protons, this increase in area relative to the area under the Hi allowed us to calculate the average number of linkers per CD. Typically, this value agreed with the reaction stoichiometryto better than f0.2. As a further test, we performed C, H, and C1 analyses of all the samples. In general the stoichiometriesagreed well with the reaction stoichiometrywith 2-7 waters of hydration per CD unit. There was no chloride in any of the polymers. Thus, in all analyses we assumed that the stoichiometry of the polymers matched that of the reaction. Absorption and Emission Spectra. Most absorption spectra were measured using a Hewlett-Packard 8452A Diode Array Spectrophotometer. However, for more accurate measurements of the changes in spectra with [CD], we used a Varian Carey 2415 spectrophotometer. Room temperature corrected fluorescence spectra were taken on a Spex Fluorolog 2 spectrofluorometerusing 334-nm excitation. The emission slits were decreaseduntil there was no further change in the emission spectra. In all cases no excimer fluorescence was found, Emission spectra were corrected for solvent background. Spectra were measured from deaerated and aerated solutions. Except for about a 10%decreasein emission intensity, the spectra were identical. As the instrument was used in the single photon counting mode, valid statistical weights are available for all intensities. All binding studies were carried out in aerated solutions. Pyrene has a ratio of two vibronic peak intensities that is strongly influenced by solvent polar it^.^ In particular, the ratio
6548
The Journal of Physical Chemistry, Vol. 97, No. 24, 1993
Xu et al.
ofthe first and thirdvibronicpeaks (373 and 384nm) haveproved particularly diagnostic of environment. The quantity, denoted by R, is given by
A
5500-
-
5000-
where the& are emission intensities at peak I and 111,respectively. Room temperature 7 values were measured using a pulsed N2 laser (337 nm) decay ~ y s t e m .The ~ emission was monitored at 404 nm. Both single and multiexponential luminescence decay curves were observed for different samples. For most 7 measurements a Tektronix 2222 digital oscilloscope was used. The 20-MHz digitizer required numerous shots to obtain a single transient, and shot to shot reproducibility caused noisy data, which made multiexponential fitting difficult. The &CD data was obtained on a Tektronix TDS 540 digital oscilloscope (1 Gsample/s digitization rate); 400 transients were averaged. This higher quality data permitted a more certain assessment of the number of components and their parameters than for the other species. Concentrations of 8-CD and oligomers were generally 0-1 0 mM; exceptions involved oligomers and a-CD where >20 mM was used. For PEG, the concentration was up to 700 mM. Complex decays were fit by nonlinear least squared0 to the sum of up to three exponentials (eq 2). D(t) is the luminescence
intensity at time t and the K and T values are the preexponential weighting factors and the excited-state T values, respectively. The intensity fitting was rather complicated and requires a description of the models. We, therefore, delay describing this until the Results and Discussion section. Molecular Modeling. All molecular modeling was done using Biosym Insight I1 software running on a Silicon Graphics Iris 4D35. The a- and 8-CD structures were entered using the crystallographic coordinates.' These structures were used without further minimization in all subsequent dockings. The Py and the interconnecting polymer chains were constructedusing the molecule builder in Insight. Inclusion complexes for the aand &CDs were constructed by manually docking the Py and the CDs while monitoring the total energy. The docking energy was minimized, and all atoms were kept farther apart than the sum of their van der Waals radii. For the polymeric systems, we looked only at foldback configurations that could yield double capping. This was done by linking two &CDs with different length linkages. The CDs and the chain were moved around to give the best 1:2 binding conformation without large increases in total energy and without atoms being closer than the sum of their van derWalls radii.
I
0 1500
-
'lm0
2
1
3
4
5
7
6
8
9
6 7000
I
Peak Ill 2000-l
0
2
6
4
8
10
12
14
16
I 18
WI,mM Figure 1. Intensity (peak I and 111) titration curves for 8-CD (A) and a-CD (B). The squares and plus signs are the experimental points, and the solid lines are the best global fits to the sequential 1:1, 1:2 binding model IV.
that the R versus [CD] data are accurately matched by the parameters that fit the peak intensity data. In addition, the T data must be consistent with any successful model. To fit the data of the intensity titration curves, we tested all of the existing literature models. These include the following: (I) formation of only a 1:l complex with each form being luminescent and having a different intensity and R value, (11) formationof only a 1:2complexwith each form being luminescent and having a different intensity and R value,14 (111) bimolecular quenching of free Py by CD and formation of only a 1:1 complex that is not quenched by free CD. Each form is luminescent and has a different intensity and R, and (IV) sequential formation of a 1:l and 1:2 complex.I6J7 Each form is luminescent and has a different intensity and R. As we will show all of the models failed except model IV:
Results and Discussion Pyrene with B-CDs. Figure 1A shows the intensity titration curves for P-CD. There have been numerous explanations for the shapes of the titration curves of P-CD with Py. These have included simple 1:1 binding, bimolecular quenching or dark complex formation, and solely or predominantly 1:2 binding.12 Most of these results were based solely on the R values and did not take into account the variations in the absolute changes in the intensitiesof peaks I and I11during the titration. Alternatively, T results were used in earlier studies and suggested a 1: 1 complex with the possible involvement of a 1:2 species.13 We have obtained completeintensity titration curves including knowledge of the absolute intensities of peak I and of peak 111. Further, we have 7 titration curves. Any satisfactory interpretation of the data must accurately account for individual changes of intensitiesversus [CD] in peaks I and 111. These peakintensities are the primary data. As an afterthought, one must also show
/.
60007
py
+ CD
KI
Kz
+ CD * CD-py-CD py + hv or A py-CD + hu or A
py-CD py*
py*-CD CD-py-CD*
-
CD-py-CD
+ hv or A
where py is free Py,py-CD is the 1:1 complex, and CD-py-CD is the 1:2 complex. For simplicity, we have omitted the excitation steps. The shapes of our titration curves are h " m t e n t with all of these models except IV. The initial dip in peak I and to a lesser extent peak I11 intensities before their rise cannot be accounted for by either a pure 1:l or a pure 1:2 binding model. Further, the best fits to the data using these models show significant systematic deviations. While a dip in intensity is qualitatively
The Journal of Physical Chemistry, Vol. 97, No. 24, 1993 6549
Interactions of Pyrene with Cyclodextrins consistent with the quenching of model 111, the best fits were unacceptable in terms of the reduced x2 and the residuals plot (vide infra). Further, if quenching were significant, one might expect to see decreases in the excited state 7 at low [CD], but this is not observed. We will now show that model IV is fully consistent with all of our data.
xZ1= zwi(EiI(calc) - EiI(obs))’
For model IV, the intensities, Es, as a function of [CD] are given by
wi = l/Ei(obs)
where f is the fraction of pyrene existing as py, py-CD, or CDpy-CD. E represents the intensitiesfor thedifferent components, and the subscripts I or I11 denote that value for either peak I or peak 111. In our data treatment we assume that the equilibration between different forms is slow compared to their decay times. For &CD, this assumption is borne out by the experimental observations of discrete concentration-independent T values. Rather than using the Es directly, we recast these equations using the more meaningful ratios of peak intensities and equilibrium constants as well as EIIIvalues, which scale the calculation to the observed data.
=fp$py&I
py +fpy-C$py-C&III
py-CD
fCD-py-C$CD-py-C&III
+ CD-py-CD
(9)
The necessaryf values are given by
fpy-CD
A=1
Kl
[CD1/A
+ Kl[CD] + KlKz[CDIZ= 1 + Kl[CD]
+ rKKlZ[CD]’ (13)
We assume that there are negligible absorption spectral changes for the different species at the excitation wavelength. We find this to be true to better than lo%, which means our f values are off by a small but constant amount resulting in a slight error in the calculated fit parameters. Rather than fitting the E1and E111versus[CD] titration curves separately or fitting R versus [CD], we chose a global fitting approach. E1 and El11were simultaneously fit using a common K1 and K2. Fitting E1 and E111separately gives different best fit parameters that need to be reconciled. Fitting only Rs has the disadvantage of reducing two information-rich coupled data sets to a single set and reducing the available information. In our fits the quantity minimized was the reduced x2,xZr,given by
(17)
where N is the total number of points fit, p is the number of parameters floated in the fit, and v is the number of degrees of freedom. In practice, one likes to have a minimum of twice as many data points as parameters; this condition was satisfied for all of our fits. Since intensity measurements were made on a single photon counting instrument, we assumed Poisson statistics and weighting factors,1° which leads to the indicated w values. We return to the applicability of this assumption later. The fitting parameters for model IV were EIIIpy, EIIIP Y ~ El11CD-~~-CD,Rpy,R ~ ~ - c RcD-~Y-cD, D, K1, and rK* While R,, was directly available from the literature or from our titration data before we added any CD, we chose to use it as a floatingparameter. If a chemically unreasonablevalue resulted, then the entire fitting was suspect. Since we were using a Simplex model, it was simple to hold different ratios at chemically plausible values and determine how the overall fit varied with changes in the different parameters. This would have been much more difficult if we had used expressions in terms of absolute intensities rather than ratios. Since R( [CD]) is readily visualized, we also calculated this quantity from the best fit parameters and compared it to the experimentally derived quantities. Attempts to fit R( [CD]) directly yielded similar results, but the best fit parameters were less meaningful because one was fitting a derived function rather than the more reliable directly measured experimental data. The sequential binding model IV produces excellent fits to the experimental intensity results (Figure 1). Table I summarizes the most important best fit parameters. The xZrfor the fits are about 2.6. For perfect single photon counting data, this number should be about 1.lo However, this is based on the assumption that the only source of noise is photon statistics. In our experiments, the cuvette was removed from the fluorimeter for each addition of CD resulting in additional noise from sample placement. Given this additional noise, the closeness of the xZr to unity indicates excellent conformity of the model to the data. In the table, we also provide a range of values for the different parameters. This is an extremely difficult fitting problem, and to determine the probable range in the different parameters, we fixed K2IK1 at a variety of values, refit the data, and examined the xZrand residuals. When the xZror residuals began to rise noticeably, we concluded that the ratio could not go outside this value. The acceptable range of K2IK1 ratios yielded a spread in K1 and R,,*D. These ranges are indicated in the table and represent our best estimates of the uncertainties in the different parameters. Using the best fit parameters, onecan calculateRversus [CD]. Figure 2 shows the experimental points along with the best fit. The excellent agreement between the model and the data is again shown. We wish to point out that the common practice of fitting only the R data is not a completely satisfactory approach. One is discarding the actual information-rich intensities of the two separate emission peaks. Similarly, if one fits only the total quantum yield, one loses the information concerning the Rs. Further, for satisfactory fitting of such complex systems by nonlinear least squares, one must know the statistical weights of the data points used in the fits. For single photon counting data, the weights are inherent in the data and are given by eq 17. While they could be calculated for the R values from the intensities, this adds one more computational layer with all the additional
~ ,
6550 The Journal of Physical Chemistry, Vol. 97, No. 24, 1993
TABLE I: Parameters for Pyrene Binding with @-CDand a-CD 0-CD
0.14 1.5 (0.124.26) (0.5-2.0) 0.67
1.85 (1.85) 1.88
Xu et al. 2.5
0.83 0.58 (0.68-1.4) (0.54-0.59) 1.69
a-CD (1:l) a-CD 0.44 0.49 1.875 1.69 1.72 ( l : l , 1~2) (0.42-0.45) (0.1-1.0) (1.87-1.88) (1.67-1.70) (1.70-1.72)
uncertainties. We believe that the best approach is to always fit the measured quantities directly and to use proper weights. Our data require a sequential 1:1, 1:2 model with significant concentration of the 1:l species at low [CD]. Consider the structures of the 1:l and 1:2 species. Molecular models show a limited number of plausible configurations. For the 1:1 complex, the Py must go into the CD cavity along its long axis in order to fit well into the cavity and displace the water. Figure 3 shows a molecular model of the 1:1 complex. Slightly greater than 50% of the Py can fit comfortably into the cavity. However, the Py still has significant exposure to water. The only reasonable 2: 1 complex then involves a double capping arrangement where the portion of the Py projecting into the water inserts itself into a second @-CD. This double capped species can completely encapsulate the Py and protect it from any water contact. We do not show the double capped form as only two CD in close contact are seen and no portion of the enclosed Py is showing. For the double capped species, R is 0.6 and varies little for the different fits. This value is similar to that of Py in cyclohexane or an extremely hydrophobic environment. Since the doubly capped species essentially excludes all contact of the Py with the aqueous environment, the Py basically sees only the hydrophobic CD core and a value near that for cyclohexane is reasonable. Indeed, the very low R value is a major point in favor of the existence of the 1:2 complex. K2/K1 is 1.5 with an estimated range of 0.5-2; either extreme is chemically reasonable. There is still a large hydrophobicbinding region free for interaction with a second CD after addition of the first CD. On the basis of this binding region, a ratio of 0.5 would be reasonable since there are half the number of binding sites available for the second binding. A ratio of 1.O or greater is also chemically plausible. The two CDs in the double capped species are close enough to each other to hydrogen bond through their OHs; this extra stabilization could easily cause K2 to be larger than Kl. Unfortunately, our data are not good enough to unambiguously differentiate between the two possibilities. However, our best fits suggest that K2 is greater than K1,and thus, hydrogen bonding favors the second binding.'* The overall association constant, KlK2, is 0.030 mM-2 which is about an order of magnitude lower than the 0.8 mM-2 reported by Wamer14but agrees well with the value of Kusumato of 0.01 mM-2.16However, both groups worked at high [CD] where they felt that they could neglect the 1:1complex in their data analysis, so some differences are to be expected. The Rs of the different species are quite revealing. Our 1.85 for the free Py in water agrees well with the literature value.14 For the 1:1complex, the value of approximately 0.7-0.9 indicates a reasonably nonpolar average environment that is intermediate between aliphatic alcohols and hydrocarbons. Our 1:2 species, of course, agrees well with the limiting value found by others and is comparable to a cyclohexane en~ironment.~~ We consider the 7 titration curves. Our 7 for pure Py in water is about 130 ns, in good agreement with most literature. The decay data with @-CDcan only be fit well by a double exponential decay. One of the 7 values was always very near the 130 ns 7 of pure Py, while the other was substantially longer (=300 ns). As the [CD] was raised, the ratio of the preexponential factors of the long to short 7 values grew monotonically. Figure 4 shows the measured 7 values and the ratio of the Ks. These data are consistent with sequential 1:1,1:2 binding if the 1:1 complex has
I
0.5 0
2
4
6
8
10
[CDI, mh4
Figure2. R versus [D-CD]. The solid line is the best fit using a sequential 1:1, 1:2 model. The dashed line is the best 1:l binding model.
Figure 3. Molecular models (from top to bottom) 1 :1 Py/B-CD complex and 1:2 Py/a-CD complex.
a 7 that is similar to that of the free Py and the long lived 300 ns component arises from the 1:2 species. The hesitation in the rise of the fraction of long- to short-lived components comes from an intermediatethird component that cannot be detected directly because its 7 is similar to that of free Py. That the 1:l species has a 7 similar to that of the free Py is not surprising. The fits to theintensity titrationcurves indicate that theemissionefficiency of the 1:l complex is similar to that of free Py. If the quantum yield does not change appreciably, then the 7 values should be similar. The longer 7 clearly arises from the double capped species. This form has a substantially higher quantum yield than free Py
The Journal of Physical Chemistry, Val. 97, No. 24, 1993 6551
Interactions of Pyrene with Cyclodextrins Long Litetime
x-
o.a
2.0
4.0
CD
(m8fi)
8.0
10.0
+,
Figure 4. Lifetimes versus [@-CD] for Py in water. *, 71; 0 , T ~ ; K I / K , . The solid line for the short lifetime is the average of all values. No theoretical significanceshould be attached to the other two lines;they are provided solely for visualization.
as shown by the increase in both emission peaks. The increase in yield should be reflected in a longer 7, as is observed. In summary, our results unambiguously establish the presence of both 1:l and 1:2 Py/B-CD complexes. The binding strength is similar for both species, which is consistent with the two hydrophobic binding ends of the Py. The 1:l complex has a r-nably polar environmentwhile the 1:2 has a very hydrophobic environment consistent with almost total water exclusion by the clam shell binding of two CDs. Judging from the 7 , Py in the 1:l complex is similar to that in water, but the 1:2 complex is much longer lived. Sincewater and OH of CDs do quench excited states,15the longer 7 may arise from essentially total removal of the quenching OHs of the water and of the open face of the CD by hydrogen bonding with the opposing CD. Pyrene with a-CDs. For a-CD, there is a monotonic decrease in the emission intensity of peaks I and I11 with [CD] (Figure 1). R drops slightly to 1.7. The data cannot be fit at all by a quenching model even if formation of an unquenched 1:1complex is postulated. A 1:2 model with no intermediate 1:l species does a very poor job of fitting the data, and we discount it. A 1:l binding model with a lower yield on the 1:1 complex does a fair job of fitting the data; however, the reduced x2and the residuals plot show that this model is very likely incorrect. For comparison with the sequential 1:1,1:2 results, weinclude the parameters for the 1:l model in Table I. Not surprisingly a sequential 1:1, 1:2 model gives the best overall fit, but there are significant problems with uniqueness as with the 8-CD results. The best fits for the sequential model are shown in Figure 1. As with the 8-CD data, we determined a range of allowable parameters by fixing K2/K1 at different values and then fitting the remaining parameters. Table I shows the range of K ~ / Kvalues I that yielded acceptable fits. These Kz/Kl values produced a range of Kl and Rs,which are also indicated in the table. It is clear that KI and the Rs have a very narrow acceptable range. Kz/K1 has a relatively wide uncertainty, but the range implies that K2 is no greater than K1 and has a best fit value half that of K1. From both our fitting and chemical arguments, we feel that sequential 1:1, 1:2 binding is the only reasonable model. The need for a second binding arises since the first CD covers only a small portion of the Py leaving free a binding region on the other half of the Py that is equivalent to the first binding site. Thus, except for a reduced number of binding sites and a somewhat lower diffusion coefficient, there should be nothing to stop the formationof a 1:2 complex, albeit with a reduced binding constant. Our best fit supports this. The K ~ / K of I 0.5 is essentially what one expects for a I/z-fold reduction in the number of available sites. The Rs are much closer to that of water than to that of 8-CD. This is reasonable since the two a-CDs do not fully enclose
the Py but leave an annular ring around the center of the Py that is fully exposed to water (Figure 3). This is unlike the 8-CD, 1:2 complex, where complete encapsulation is possible. In the a-CD case, we do not expect K2 to be larger than KIsince the two a-CDs cannot touch each other to form a hydrogen bond stabilized form; this is supported by the data. The two surprises in the fitting parameters are the similarity of the Rs for the 1:l and 1:2 complexes. This may just reflect the fact that a substantial portion of the Py is still directly exposed to water, and the central portion may be the piece most susceptible to solvent perturbation. Also, KI for a-CD is somewhat larger than that for B-CD, and (even given the uncertainties of the complex data fitting) we cannot reverse the magnitudes of these K1 values. The reason for this is not obvious, but such large Ks are well-known for a-CD with small ring aromaticsthat are similar to Py. For example, 2,6-dimethyl-4-nitrophenolor the analogous phenolate anion bind by insertion at the nitro end (the methyls form blocks similar to the central rings of Py) and have Ks of 0.57 mM-' and 1.06 mM-I, respecti~e1y.I~ Thus, our Ks for a-CD are reasonable. T is independent of [a-CD]. Apparently, even though there is a change in the quantum yield, the change in environment alters the radiative rate constant in such a way as to keep the 7 essentially constant. The variation in R with environment does indicate a change in the nature of the excited state, and thus, a change in radiative rate constant is certainly possible. Pyrene with Polymeric CDS. We now consider the interaction of Py and polymeric CDs. The polymeric CDs share some common features. All were polydispersed with typically 4-5 CDs in the chain. The major variation among the polymers was the average number of the ether linkages per CD unit. Since not every linker unit is necessarily involved in attaching CDs, the average number of linkage units between CDs will be less than this number. The chemical composition of linkers/CD varied over the range 1-8, which allows for a considerable variation in the flexibility and length of the linkage between the CD units. Our hope was to probe thevariation in double capping that would result from the variation in chain length. Pyrene with Poly(ethy1ene glycol) (PEG). As we will show, Py binds much more strongly to the CD polymers than to 8-CD alone. In the binding of the polymer CDs to Py, there are two potential binding regions: the CD and the hydrophilic connecting CH2CH(OH)CHA. It was necessaryto sort out the differences in potential binding of CD and the connecting linear polymer. To do this we examined binding of Py to PEG. While not a perfect model for our connectingpolymer, we felt that thevery hydrophilic ether regions were a reasonable facsimile of the critical portion. The R, (the limiting R at infinite [PEG]) and K1 for PEG were 1.87 and 0.002 mM-$ we base our PEG concentrations on the monomer unit. Thus, binding is extremely weak compared to the CDs and the CD polymers. Further, the binding environment is very hydrophilic as judged by the large R,. The low binding constant indicates that the connectingpolymer in our CD oligomer is not solely responsible for the enhanced binding constants of the CD polymers. Indeed, the suggestion is that the connecting linkage alone is actually detrimental to binding. Pyrene with Polymeric CDs. Figure 5 shows the results of Py intensity titrations using the different polymers. Clearly the binding increases dramatically with increasing average polymer chain length. The solid lines are the best fits to 1:1binding (model I). The fits are poorest for the short chains. Using the 1:1 model we present the best fit parameters for the different polymers in Table 11. Rather than report a K1, we give an apparent first binding constant, K,,,; the reason for this will become clear when we present our model. The results are most imprecise for the 1E polymer where Kappis reproducible to about 50%. R, is reproducible for the 1E polymer to 0.03. The other systems are probably reproducible to about 25% in Kappand 0.03 in R,. R , increases rapidly with n. The 1E polymer environment is very similar to that of the doubly capped Py with 6-CD, or that
Xu et al.
6552 The Journal of Physical Chemistry, Vol. 97, No. 24, 1993 4o00,
, 8
TABLE LI: P.rrmeters d m m B md B-CD oligomers polymer PEG &CD 1E 2E 3E 4E 8E
-
2500.-
2500-1
-
,'
1 I
i
2000
D 6ooo
I
*i
i
I
1
Figure 5. Intensity (peak I and 111) titration curves for Py with CD polymers. (A) n = 1, (B) n = 2, (C) n = 3, (D) n = 4, (E) n = 8. The solid lines are the best fit using the model of Figure 6.
of cyclohexane. However, for the other polymers the environment is much more hydrophilic; the 8E polymer environment approaches
R, 1.87 0.60 0.63 1.11 1.38 1.55 1.60
KIPp(mM-*) 0.002 0.14 0.12 0.18 0.51 1.1 1 4.2
m WMIPEG,B-CD, (ne) 127 300 290
T,
235 250
Kint
2.4 4 14 31 120
that of cyclohexane. Similarly,Kappdepends strongly on n. While the 1E and 2E polymers have a Kappcomparable to that for 8-CD, for the longer chain polymers Kappincreases rapidly with chain length. The 8E polymer has a K1 that is 20 times higher than for 8-CD. Any model must satisfactorily account for the adequacy of the 1:1 binding model for the polymers that fails for pure 8-CD, the n dependence of Kapp,and the increasingly polar environment of Py for larger n values. First consider the n dependence of KaPp A high affinity for the linkage would aid initial association and enhance CD binding. This is not likely since the PEG studies show very poor association of the ether linkage with Py. For steric reasons, individual CDs should bind more poorly in the polymer than as free monomers. The linkage would shield the CD from the Py. In addition, the restricted motion during an encounter would allow less efficient sampling of different conformations and would yield weaker binding than for monomeric CD. Favorable binding to the interconnectinglinkage could offset steric interference;however, our PEG results indicate that this is not the case. Thus, the large Kappvalues for higher n cannot be due to increased linkage association to the Py but must arise from other sourcea. We suggest that polymeric CDs act as chelate or clam shell binders, as originally hoped (Figure 6 ) . We are observing a mixture of the singly and doubly capped Py with the second CD coming from the same polymer chain. The ratio of these allows the determination Of Kint. The larger Kint,the smaller the amount of singly capped Py. Even if KI remains Fixed, the larger Kapp is, the smaller the fraction of polymer bound singly capped Py. This singly capped form coupled with KIdetermines the amount of free Py. Thus, the larger Kint, the less singly capped Py, and the less free Py. Therefore, even if the primary binding of Py to the polymer has a fixed equilibrium constant, variations in Kint can affect Kawand, thus, the fraction of free Py. The equations describing the fractions of bound and free Py are given by
These expressionsare identical to simple 1: 1 binding, except that we have an apparent 1:l binding constant, Kappgiven by
Thus,if we monitor a composite property of the single and doubly capped Py, especially if the ratio of the single and double capped species remains fixed, we cannot distinguish between a singly capped model and the intramolecularly double capped model. Double capping is very reasonable for larger n. The chelate effect of intermolecular foldback is very much enhanced by the extremely high local CD concentration once one CD binds to the Py. We can estimate Kht for the different polymers. Our K1 for the polymer system is going to be smaller than that for 8-CD. First, steric effects should reduce the chances of sticking on an encounter. Further, our& is based on the total CD concentration; however, the actual concentration of the polymer, which deter-
Interactions of Pyrene with Cyclodextrins
The Journal of Physical Chemistry, Vol. 97, No. 24, 1993 6553
Figure 6. Schematic representation of the polymer clam shell binding model.
mines the encounter rate, is about one-fourth that value. Therefore,disregardingsteric effects, we estimate K,at no larger than 0.035 mM-*. Based on this value, Kin1 was calculated from eq 21. Table I1 lists the Kint values. There is a pronounced impact of n on double capping. The longer chain has a Kin1 that is 50 times larger for the 8E polymer versus the 1E polymer. There is a particularly largejump between the 2E and 3E polymers. We attribute this chain length dependence as arising from the ease of chelate or clam shell binding. Too short a chain prevents double binding of adjacent CDs on the polymer; one must go to third (1,3) or fourth (1,4) CDs to get good wrap around. Molecular modeling supports this interpretation. Figure 7 shows three polymer CD subunits, each with two CDs and a different length tether. We show n = 1,3,and 4 linker subunits. The chains are adjusted so as to give optimal opportunity for clam shell binding. It is clear that thereis noconfigurationpossible with one linker that allows adjacent binding. Starting at n = 3, good double capping can occur with adjacent CD binding, although the polymer chain is at full extension. The concomitant rise in Kin, is attributable to this new mode of binding. For n = 4,adjacent CD binding is even more facile, and Kin, increases again. The continuing increase for n = 4reflects the greater ease of clam shell binding. Presumably, too long a linkage would reduce the chance of the second CD finding the Py,but we have clearly not yet arrived at that length. We consider the environmental hydrophobicity versus n. Except for the 1E polymer, the binding environment is very hydrophilic and becomes increasingly so with increasing n. CD binding to Py is strongly affected by cosolvents, which can insert themselves into the cavity to form a termolecular c o m p l e ~ . ~ ~ J ~ This can affect both the binding constant and, to a greater extent, the Rs. For the larger n values, the ether linkagesare long enough to insert themselves into the cavity along with the Py, although not necessarily from the linkage connecting the two binding CDs. In addition, there will be dangler chains, which are not terminated on another CD; these will have fewer restrictions on insertion than the interconnectinglinkers. Thus, we suggest that the linkage Figure 7. Molecular models showing the ability of the CD polymers to and danglers are acting as polar cosolventsfor the longer polymers. fold back for clam shell binding from top to bottom: n = 1, n = 3, n = The hydrophilic ether-alcohol linkage presents a polar environ4. ment to Py, increases the effective R, and makes the binding environment more water like. The 2E polymer fits better with a 1:l intramolecular model than the 1 E,but not as well as the remaining polymers. The 2E The unique 1E polymer bears special comment. Most nolinkageis still not long enough to give adjacent CD doublecapping, ticeably, the R, looks more like a cyclohexane environment than but the chain has considerably more conformational flexibility one that has a substantial water content. The molecular models indicate that a single polymer linkage is so short and rigid that and CD double capping from further CDs is feasible. The 2E adjacent CD bindings to the same Py are physically impossible. polymer is probably also a mixed binding case but because of the Thus, it seems likely that a substantial amount of binding of the longer linker, the system has a higher percentageof intramolecular 1 E polymer is sequential with two CDs being supplied by different capping and, thus, more closely conforms to the Figure 6 model. polymer chains. Attempts to fit the data with only the sequential The 3E, 4E, and 8E polymers have ample flexibility to give model do not fit appreciablybetter than the 1 :1 model. We suspect adjacent CD double capping, and all give excellent conformity that the failure of either simple model to quantitatively fit the to the Figure 6 model. data arises from simultaneouscompetitivemodels (single capping, Microencapsulation. It is clear that polymeric CDs are intramolecular double capping, and intermolecular double cappotentially very useful encapsulants. They are much more water ping). This complexity is exacerbated by the fact that the 1E soluble than @-CD, which allows higher carrying capacity. Further, unlike 8- or a-CD, thechelateeffect enormouslyenhances polymer probably has the greatest heterogeneity of chemically different behavior; the difference between an n = 1 and an n = the propensity of CD to double cap the guest. With suitable 2 linkage is larger than for an n = 4and an n = 5 linkage in terms length linkers, the apparent binding constants to hydrophobic of CD binding. substrates are much higher than those for monomeric CDs. This
6554
The Journal of Physical Chemistry, Vol. 97, No. 24, 1993
efficientdoublecappingfurther increasestheabilityofthepolymer to carry large hydrophobicmolecules that would otherwisebe too hydrophobic, even when singly capped, to have reasonable solubility.
Acknowledgment. We gratefully acknowledge support by the National Science Foundation (CHE 88-17809and 91-18034). We thank W. DeMay of Tektronix for the loan of the TDS 540 oscilloscope. We thank J. Quagliano for his assistance in using the Varian Carey 2415 and Dr. Laurie Kelsh for her assistance in acquiring and interpreting our 500-MHzNMR data. We also thank Hewlett-Packard for the gift of the 8452A spectrophotometer and Henry Wilson for his kind assistance. References and Notes
E.J. Inclusion Phenom. 1988,6,537. (2) Otta, K.; Fenyvesi, e.; Zsadon, B.; Szejtli, J.; Tiid&, F. Proc. Int. Symp. Cyclodextrins, 1st 1981,357. (3) (a)Suzuki, M.; Fenyvtsi, E.; Szilasi,M.;Szejtli, J.; Kajtplr, M.; Zsadon, B.; Sasaki, Y . J. Inclusion Phenom. 1984,2, 715. (b) Furue, M.; Harada, A.; Nozahra, S.J. Polym. Sci. 1975,13,357. (c) Harada, A.; Furue, M.; Nozahra, S . Macromolecules 1977,10,676.(d) Saenger, W. Angnu. Chem., Int. Ed. Engl. 1980,19, 344. (4) Demas, J. N.;DeGraff, B. A. Anal. Chem. l991,63,829A. (5) . , (a) Edwards. H. E.: Thomas. J. K. Carbohydr. Res. 1978,65,173. (b) Kalyanasundaram, K.; Thomas, J. K. J . Am. Chem. Soc. 1977,99,2039. (c) Pankasem,S.; Thomas, J. K.J. Phys. Chem. 1991,95,7385.(d) Caminati, G.;Turro, N. J.; Tomalia, D. A. J. Am. Chem. SOC.1990,112,8515. (e) Turro, N. J.; Caminati, C.; Kim, J. Macromolecules 1991,24, 4054. ( 1 ) Fenyvesi,
\
,
Xu et al. (6) Kano, K.; Hashimoto, S.;Imai, A,; Ogawa, T. J. Inclusion Phenom. 1984,2,737. (7) Nakajima, A. J. Lumin. 1977, 15, 277. (8) Sacksteder, L.;Demas, J. N.; DeGraff, B. A. Inorg. Chem. 1989,28, 1787. (9) Leasure,R.M.;SacLsteder,L.;Ntsselrodt,D.;Demas,J.N.;Ddjraff, B. A. Inorg. Chem. 1991,30, 3722. (IO) (a) Demas, J. N. Excited State Lifetime Measurements; Academic: New York,1983. (b) Demas, J. N.; Demas, S.E. Interfacting andScientific Computing on Personal Computers; Allyn & Bacon: New York, 1990. (11) (a) Manor, P. C.; Saenger, W . J. Am. Chem. Soc. 1974,96,3630. (b) Linder, K.; Saenger, W. Carbohydr. Res. 1982,99,103. (12) Mufloz de la Peiia, A.; Ndou, T. T.; Anigbogu, V.C.; Warner, I. M. Anal. Chem. 1991,63,1018. (13) Nelson, G.;Patonay, G.; Warner, I. M. J. Inclusion Phenom. 1988, 6. 277. (14) Muiioz de la PeAa, A.; Ndou, T. T.; Zung, J. B.;Warner, I. M.J. Phys. Chem. 1991,95,3330. (15) Patonay, G.;Shapira, A.; Diam0nd.P.; Warner, I. M. J. Phys. Chem. 1986,90,1963.(16) Kusomoto, Y.Chem. Phys. Lett. 1987,136,535, (17) Hnhtroeter, W.G.;Martic, P. A.; Evans, T. R.; Farid, S . J. Am. Chem. Soc. 1986,108,3275. (18) KusomatoI6has a K1 = 140 M-I which is in excellent agreement with which is a chemically our best fit. His Kz of 83 M-* is about half of our KI, reasonablevalue. However, his rcsults hadconsiderable problems in thequality of the data fitting. We, therefore, believe that the issue of which binding constant is the larger, K1 or Kz,is still unresolved. ( 19) Bergeron, R.J.; Channing, M. A.; Gibeily, G. J.; Pillor, D. M. J . Am. Chem. Soc. 1977, 99, 5146. (20) (a) Bergmark, W. R.; Davis, A.; York,C.; Macintosh, A,; Jonea, G. J. Phys. Chem. 1990,94,5020. (b) Nakajima, A. Bull. Chem. Soc. Jpn. 1984,57, 1143. (c) Nelson, G.;Patonay, G.; Warner, I. M. Anal. Chem. 1988,60,274.
