binding of pyrene to cyclodextrin polymers

Binding of Pyrene to Cyclodextrin Polymers

T. C. WERNER,* KAREN COLWELL, REZIK A. AGBARIA, and ISIAH M. WARNER Department of Chemistry, Union College, Schenectady, New York (T.C.W., K.C.); and Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803 (R.A.A., I.M.W.)

Polymers containing the three cyclodextrin (CD) molecules, ~-CD, I~-CD, and T-CD, linked by epichlorohydrin (~-CDP, I~-CDP, and 3,-CDP) are highly water-soluble polydisperse mixtures containing CD units joined by repeating glyceryl linkers [-(CH2-CHOH-CH2-),,]. The average n value is 12-15, and gel filtration chromatography analysis indicates that the two major polymer components have molecular weights (MWs) of <2000 (1 CD/polymer chain) and 9- 10,000 (4-5 CDs/polymer chain). We have used fluorescence properties to study the binding of pyrene to the three commercially available CDPs and to dialyzed samples of the CDPs, in which the Iow-MW (<2000, CDPL) and high-MW (9-10,000, CDPH) components have been separated. The pyrene emission I/III ratios for the three polymers are larger and exhibit a smaller range than the I/III ratios for the CD monomers. Moreover, the I/III ratio for the dialyzed polymers, I~-CDPL and I~-CDPH, are, within error, the same as that for 13-CDP. It has been previously shown that additives, such as pentafluoropropanol (PFP), cause a dramatic decrease in the pyrene I/III ratio in the presence of ~-CD. No effect on the pyrene I/III ratio is observed when these additives are added in the presence of the CDPs. The pyrene fluorescence decays in the presence of all three native polymers and the dialyzed I3-CDPs are quite similar but different from the pyrene fluorescence decays in the presence of the three CD monomers. Moreover, the pyrene lifetimes show much greater dependence on iodide quencher concentration in the presence of CDPs than in the presence of I3-CD and 'y-CD. These data suggest that pyrene exists in a more exposed and hydrophilic environment when bound to the CDPs than that observed with the CDs. The agreement of the results for pyrene in the presence of [~-CDP, 13-CDPH, and I3-CDPL would seem to rule out significant cooperative binding from two CD units on a single chain, which has previously been suggested. We conclude that pyrene binding to the CDPs may be largely noninclusional, involving considerable participation of the glyceryl linker units.

Index Headings: Cyclodextrin polymers; Pyrene; Fluorescence

I N T R O D U C T I O N

Many organic molecules have been shown to form inclusion complexes with torroidal-shaped molecules called cyclodextrins. These cyclodextrin (CD) molecules consist of rings of D(+)-g lucopyranose units joined by a-(1,4)- linkages. ~ The three CDs commonly studied are a-CD, I3-CD, and ,~-CD, which contain six, seven and eight glu- copyranose units, respectively.

Organic molecules form inclusion complexes by binding in the CDs ' internal cavities, which are less polar than the surrounding aqueous solution. The great interest in these guest/host complexes arises f rom the variety of po- tential applications of these complexes that have been identified by several investigators. These include solubility enhancement of organics in water, 2 improvement in analytical separations through complex formation with CD-bonded phases, 3 and CD-modif ied mobile phases in

Received 31 July 1995; accepted 4 January 1996. * Author to whom correspondence should be sent.

HPLC, 4 as well as use as modifiers of photochemical be- havior, 5 to control dye aggregation equilibria, 6 and for models of protein complexes. 7

One restriction on the use of cyclodextrins is limited solubility, which is lowest for [3-CD (0.014-0.016 M). As a consequence, attempts have been made to modify [3-CD in order to improve its solubility. These efforts include the synthesis of water-soluble CD polymers. While there are several reported ways to produce these polymers, the most common procedure involves the use of epichlorohydrin. The resulting CD polymers are a polydisperse mixture containing CD units joined by repeating glyceryl linkers [ - (CH2-CHOH-CH2-) , , ] . All three of the CD monomers have been polymerized by this method to produce CD polymers (a -CDR [3-CDR and ~-CDP) which are highly water soluble and commercial ly available.

Fluorescence probes have been extensively employed to study CD binding sites, s-~2 Pyrene is an especially at- tractive probe for this work because its fluorescence in- tensity, its lifetime, and the shape of its emission spectrum all can change when pyrene binds in a CD cavity. 11,12 In particulm; the pyrene I/III vibronic band inten- sity ratio is quite sensitive to the polarity of the pyrene environment. For example, this ratio is about 1.8 in water (band I at 373 nm, band III at 384 nm) but decreases to about 0.6 in the presence of --5 × 10 3 M [3-CD. ~1,~2 The latter value is characteristic of pyrene in a very hydrophobic environment, such as cyclohexane. This fact has been used to suggest that pyrene, which is too large to fit completely into a single [3-CD binding site, forms a 2:1 [3-CD/pyrene complex in which the pyrene is "double capped" by the two [3-CD hosts in a "c lam shell" arrangement? 1,~2 The result is a structure in which pyrene is almost completely submerged in the hydrophobic in- teriors of the two [3-CD molecules. A distinctive feature of this structure is the sensitivity of the bound pyrene spectral properties to the addition of small amounts of alcohols. Nelson and Warner have presented pyrene fluorescence lifetime data showing significant enhancement of the protection of [3-CD-bound pyrene from iodide quenching upon the addition of 1% (v/v) t-butyl alcohol. ~3 In addition, Elliott et al. have reported dramatic decreases in the I /III ratio for pyrene bound to [3-CD when fluorinated alcohols are added. ~4

Xu et al. have also investigated the binding of pyrene to [3-CDR ~2 They present evidence for formation of se- quential 1:1 and 2:1 [3-CD/pyrene complexes in which both [3-CDs can come from the same polymer chain? 2 These authors also propose a clam shell model for the latter complex, even though the I/III ratio ( - 1 . 5 ) is in- dicative of a more hydrophilic environment. They ration-

Volume 50, Number 4, 1996 0003-7028/96/5004-051152.00/0 APPLIED S P E C T R O S C O P Y 511 © 1996 Society for Applied Spectrocopy

alize this result by stating that the glyceryl linkers in the polymer can act as polar cosolvents for bound pyrene. ~2

We have recently reported a systematic comparison of the guest binding ability of all three commercia l ly available CDPs with their respective monomer CDs, using three naphthalene-based fluorescence probes as guests. ~s This work shows that the glyceryl linkers play a major role in the guest/host binding interaction? 5 We describe herein our investigations on the binding of pyrene to these same CDPs. The [3-CDP used in this work differs from the ones described by Xu et al. in having, on the average, a greater number of glyceryl linker groups between CD units. Nonetheless, our pyrene I /III ratio data are in reasonable agreement with data f rom the longer polymers studied by Xu et al. ~2

In aggregate, our data suggest that much more similar pyrene binding sites exist among the three CDPs than among the three monomer CDs and that the linker units play a key role in pyrene binding. Moreover, we believe that our evidence indicates that a closed clam shell arrangement, like the one for ([3-CD)z/pyrene, is unlikely for pyrene bound to these CDPs.

EXPERIMENTAL

Chemicals. The water used in all experiments was deionized, doubly distilled, and passed through a Milli- pore Milli-Q Water System. Pyrene (99+%), potassium iodide, 2,2,2-trifluoroethanol (TFE), and 2,2,3,3,3 penta- f luoro-l-propanol (PFP) were obtained f rom Aldrich Chemical Company, Inc., and the three monomer cyclodextrins (a-CD, [3-CD, and ,y-CD) were obtained f rom American Maize Products Company. The pyrene was re- crystallized twice f rom ethanol, while [3-CD was recrys- tallized f rom water. The t-butyl alcohol was obtained from Fisher Scientific Company.

The three CD polymers were obtained f rom Cyclolab R&D Laboratory Ltd. of Budapest, Hungary. The general formula of the polymers is as shown below:

[ C D - ( C H 2 - C H O H - C H 2 - O ) , X ] v

where CD is c~-, [3-, or 'y-CD; X is H or CD; p is > 1 but < 6 - 8 ; and n is >1 but <18 (a-CD), <21 (~-CD), or <24 ('y-CD). The average n value is 12-15, and the reported % CD is 54 (o~-CDP), 55 ([3-CDP), and 57 ('y-CDP). Gel filtration chromatography (GFC) data f rom Cyclolab indicate that a broad range of molecular weights exists for these polydisperse CD polymers up to a m ax imum of about 11,000. The GFC data show two prominent peaks for [3-CDP at about 2000 and 9-10,000; the former is due to polymers containing a single CD unit per polymer chain, while the latter is f rom polymers containing 4-5 CD units per polymer chain. 16 Cyclolab GFC runs for ~-CDP and ~-CDP show the same two peaks, but the latter is less pronounced than observed with the ~-CDE

Instrumentation. Absorption spectra were recorded with a Hewle t t -Packard 8452A diode array spectropho- tometer, while fluorescence spectra were recorded with a Perk in-Elmer Lambda 5B spectrofluorometer. The latter instrument automatically corrects emission spectra for the wavelength dependence of the emission monochromator and detector combination. Slits were 2.5 nm on both the excitation and emission monochromators .

Fluorescence lifetime data were obtained with an LS 100 fluorescence lifetime system from Photon Technol- ogy International, Inc. This instrument employs a nitro- gen-filled, thyratron-gated flashlamp and an optical box- car detector, which is a special photomultiplier that can be turned on at varying delay times after the lamp is flashed. The excitation wavelength employed was 337 nm and the emission wavelength was 393 nm. A dedicated computer provides analysis of the luminescence data by convoluting the lamp scatter peak with a delta-function- generated decay until an appropriate fit is obtained to the observed fluorescence decay curve. The fitting procedure, which can employ from one to four exponentials in the generated decay curve, uses an iterative procedure based on the Marquardt algorithm. A single exponential fit to the lifetime data was deemed appropriate if a double exponential fit gave no improvement in the ×2 parameter and autocorrelation function. When these conditions were met, the two lifetime values from the double exponential fit were very close to the single exponential fit lifetime.

GFC data were obtained with an HPLC system con- sisting of a Perk in-Elmer 250 LC pump, a Perk in-Elmer Nelson 900 Series interface, a Perk in-Elmer LC-30 re- fractive index detector, an IBM PS/2 computer, and an Epson EX-800 printer. The column was a Perk in-Elmer T S K G2000SW column, which is employed for aqueous GFC of molecules in the 500-100,000 molecular weight range. The mobile phase was water containing 3% meth- anol.

Solutions. Stock solutions of pyrene were prepared by adding a small amount of solid pyrene to water and rap- idly stirring the solution overnight. The resulting stock was then passed through 0.2-tim disposable syringe filters (Anotec). Typically, this procedure resulted in a stock pyrene solution with an absorbance between 0.01 and 0.02 (1 cm cell) at 334 rim, which corresponds to a pyrene concentration of - 2 × 10 -7 M. Pyrene stock solutions were used only on the day they were prepared. The required concentration of CD or CDP was obtained by adding a weighed amount of the solid CD or CDP to an accurately known volume of the pyrene stock solution. All CD and CDP concentrations are expressed as CD molarity, which requires the use of the % CD data f rom Cyclolab (see above) for the CDPs. Solutions were stirred and allowed to stand for up to several hours to ensure equilibration before measurements were made. Additives such as KI or alcohols were delivered by mass (KI) or volume (alcohols) to a known volume of the pyrene stock or of the pyrene stock containing a known concentration of one of the CDs or CDPs. The resulting solutions were stirred for at least one hour before measurements were taken.

Isolation of [3-CDP Molecular Weight Fractions. To see how spectral data would be affected by reducing the heterogeneity of the [3-CDE we dialyzed [~-CDP using dialysis tubing (Spectra/Por CE Molecularporous Dialy- sis Membrane f rom Spectrum) having a molecular weight cutoff of 3500. Several grams of [3-CDP were dissolved in 10-20 m L of water, which was poured into the dialysis tubing and placed in a graduated cylinder containing one liter of water. The contents of the cylinder were magnet- ically stirred. The water in the cylinder was changed seven to nine times over a five- to seven-day period. At the

512 Volume 50, Number 4, 1996

g ~

FIG. 1.

60-

40-

20-

o ~ 1~ 1'5 ~o 25 30

time (min.)

Gel filtration chromatograms of 13-CDP (O) and [3-CDPH (+).

end of this period, the dialyzed polymer was recovered by freeze-drying the solution remaining in the bag. The mass of the dialyzed polymers recovered was about 50% of the original mass of the native polymer. This polymer is designated [3-CDPH to indicate that only the higher- molecular-weight components should be present.

To isolate the low-molecular-weight fraction (MW <3500, [3-CDPL), we repeated the dialysis procedure and collected the initial external solution after one week of stirring. This solution was reduced to 15-20 mL with a rotovap, and the [3-CDPL was recovered by freeze-drying this concentrated solution. About 30% of the original sample was recovered as [3-CDPL.

Gel filtration chromatograms, with the use of a Perkin- Elmer TSK G2000SW column, for [3-CDP and [3-CDPH are shown in Fig. 1. The chromatogram for [3-CDP shows distinct peaks in two regions: 12.7 min and 24.2 rain. This result is as expected, given the GFC data from Cy- clolab (see above). The first peak is the main high-molecular-weight component, while the two unresolved peaks at the longer time are for lower-molecular-weight components. The chromatogram for [3-CDPH shows no peak at the longer retention time, indicating complete re- moval of components below the 3500 molecular weight cutoff of the dialysis tubing. For [3-CDPL, both components are still evident, but the ratio of the lower (24.2 min)- to the higher (12.7 min)-molecular weight components is now 1.3, which is a 3.4-fold increase relative to the ratio observed in the [3-CDP chromatogram (0.37).

The CD concentration for the dialyzed polymers was determined under the assumption of the same % CD obtained for these polymers as reported for the native polymers.-k

t According to Dr. L. Szente of Cyclolab R&D Laboratory Ltd., there is no significant difference in the CD content of fractions with different molecular weights (private communicat ion) .

1,5

1

1/111 Ratio

0.5

®9 [ ]

[ ]

0.602 0.604 0.606

CD Molarity

FIG. 2. The effect of 13-CD concentration on the pyrene IflII ratio in the absence and presence of alcohols. ([~) 13-CD alone; ( 0 ) [3-CD with 2.5 × 10 2 M TFE; (A) [3-CD with 2.5 × 10 -2 M PFP; (+) [3-CDP alone; ( ~ ) 13-CDP with 2.5 X 10 -2 M TFE; (O) I3-CDP with 2.5 X 10 2 M PFR All the [3-CD data are taken from Elliott et al. 14

All measurements were made at room temperature (23 °C).

RESULTS

Pyrene I/III Emission Ratio Data. The dependence of the pyrene I/III emission ratio on increasing CD molarity for [3-CD and ~-CDP in the absence and presence of 2.5 × 10 -2 M TFE or PFP is shown in Fig. 2. The UIII data with ~-CD, taken from Ref. 14, show a marked decrease with increasing [CD], and this trend is signifi- cantly enhanced in the presence of either alcohol, especially PFR By contrast, the I/III ratio shows a much smaller change with increasing CD molarity for [3-CDR Moreover, the change is unaffected by the presence of either alcohol. In Fig. 3, the I/III ratio at a fixed CD molarity (0.0020 M) for [3-CDP is plotted as a function of added alcohol concentration, with the use of TFE, PFP, and t-BuOH. There is essentially no alcohol effect on the I/III ratio up to 4.0% added alcohol, which corresponds to 0.55 M, 0.40 M, and 0.42 M for TFE, PFP, and t-BuOH, respectively.

Plots of I/III data for pyrene against CD molarity for y-CD and y-CDR in the presence and absence of 2.5 × 10 -2 M PFR are given in Fig. 4. The y-CD data are also taken from Ref. 14. The large effect on the I/III ratio of increasing CD molarity observed with y-CD and the small but real additional effect of added PFP are not observed with y-CDR

Limiting values of the pyrene I/III ratio for the CDs and CDPs used in this work are given in Table I. These are limiting values because they do not change signifi-

APPLIED SPECTROSCOPY 513

1.5-

0.5-

1/111 1- Ratio

1/111 Ratio

2-

¢b

1 2 3 4

%(v/v) alcohol

FIG. 3. The effect of selected alcohols on the pyrene I/III ratio in the presence of 2.0 × 10 3 M [3-CDR (+) TFE; ( 0 ) PFP; ((3) t-BuOH.

cantly with increasing CD molarity. What is most striking about these data is the similarity of the I /III ratio (~ 1.55) for all the CDPs, regardless of whether alcohol is present or not. This result is in sharp contrast to the limiting ratios with the CDs, where the ratio is markedly lower and is dependent on both the identity of the CD and the

1.5 TM

o

O

1-

0.5-

0 i

0 0.002 0.(J04 0.006

CD Molarity

FiG. 4. The effect of 'y-CD concentration on the pyrene I/III ratio in the absence and presence of alcohols. ((3) ~-CD alone; ( 0 ) 'y-CD with 2~5 × 10 2 M PFP; (+) 'y-CDP alone; (El) 3/-CDP with 2.5 × 10 2 M PFR All the 3~-CD data are taken from Elliott et al. TM

T A B L E I. L imi t ing pyrene I / I l l ratios, a

CD (alcohol) b I/III ratio (_+0.02)

[3-CD 0.62 c [3-CD (TFE) 0.6 b [3-CD (PFP) 0.38 'L [3-CDP 1.55 [3-CDP (TFE) 1.60 [3-CDP (PFP) 1.56 [3-CDPH 1.55 [3-CDPH (PFP) 1.55 [3-CDPL 1.52 [3-CDPL (PFP) 1.56 -/-CD 0.82 a ~/-CD (PFP) 0.6 a ~-CDP 1.53 ~/-CDP (PFP) 1.55

a Our measured I/III ratio for pyrene in water is 1.75. b Alcohol concentration = 2.5 × 10 -2 M. c See Ref. 11. d See Ref. 14.

presence of alcohol. Note, also, that the two molecular weight fractions of [3-CDP ([3-CDPH and [3-CDPL) produce virtually identical I /III ratios to that of [3-CDE

Pyrene F l u o r e s c e n c e Li fet imes , Fluorescence lifetimes for pyrene in water and in the presence of the three monomer CDs are given in Table II. The c~-CD monomer causes no significant change in the pyrene lifetime, a result which is consistent with the small effect on the I /III ratio with this CD (limiting value is 1.69 vs. 1.75 in water). Little, if any, interaction must occur between pyrene and o~-CD. Lifet ime data in the presence of both [3-CD and ~-CD are better fit by a double exponential than a single exponential decay. The fraction of the total signal f rom the longer component (f2) is calculated f rom the following equation:

f2 = a2"r2/(a~'rl + a2"r2). ( l )

In Eq. 1, % is the longer-lived component and the as are the preexponential factors.

It must be noted that the reliability of the shorter component (-r~) is highly problematic when f2 > 0.9. For example, in the presence of 0.0080 M ~-CD, the f2 value is

T A B L E II. Pyrene l i fet imes with CDs.

CD" ~1 (ns) % (ns) f2

None b 128 None + I 39 a-CD c 132 [3-CD d 141 [3-CD + I ~ 48 [~-CD + I 139 + t-BuOH e ,/-CD 86 [3-CD + I -~ 39 [3-CD + I- 85 +t-BuOH c

363 0.57 328 0.94 388 0.97

269 0.92 25l 0.87 385 0.97

"The concentrations for the CDs are as follows: ~x-CD (0.0085 M), [3- CD (0.010 M), and ~/-CD (0.010 M). No significant change in "r values occurs at higher CD concentration. Where present, the [I--] was 0.015 M and the t-BuOH was 1.0% (v/v).

b XU et al. report 130 ns. ~2 c Xu et al. report pyrene "e value independent of [e~-CD]. ~2 aXu et al. report 130, --300 ns for "e I and "e272

Nelson and Warner have previously reported that [3- and 3'-CD protect pyrene from I quenching and that this protection is enhanced by the addition of t-BuOH.~3


TABLE IlL Pyrene lifetimes with CDPs.

CDP ~ "rl (ns) ~2 (ns) J~

a -CDP 100 250 0.95 a -CDP + I 35 105 0.85 ~-CDP 92 251 0.97 [~-CDP + I 31 106 0.89 [~-CDP + I 61 176 0.72 + t -BuOH '~-CDP 82 246 0.97 "y-CDP + I 35 97 0.87 ~-CDP + I 64 170 0.57 + t -BuOH

"The CD concentrations for the CDPs are as follows: a -CDP (0.0056 M), I3-CDP (0.0020 M), and ~/-CDP (0.0050 M). No significant change in "r values occurs at higher CD concentration. Where present, the [I-] was 0.015 M and the t -BuOH was 1.0% (v/v).

0.70 and "r~ is 128 ns, which is the same -r as that of free pyrene. However, when ['y-CD] is 0.10 M, f2 increases to 0.92 and the ,r~ value is only 86 ns (Table II). In this situation, the relative error in the f~ term will likely be large, leading to large uncertainties in both a~ and 'r~. By contrast, "r2 and f2 values are reasonably reproducible (_+3%) among different samples.

The lifetime data in Table II show that both [3-CD and ",/-CD afford considerable protection to bound pyrene from iodide quenching and that this effect is enhanced by the addition of 1.0% (v/v) t -BuOH--obse rva t ions consistent with those previously reported by Nelson and Warner? 3 It is worth noting that, as with the I/III ratios in Table I, there is a wide range in pyrene lifetime data among the three CDs, signifying substantially different binding interactions for pyrene among the three CDs. This observation can be attributed to the relative sizes of pyrene and the respective CD cavities. Too big to fit ef- ficiently into the c~-CD cavity, pyrene is nicely enveloped in the clam shell arrangement with two 13-CDs, 1~,~2 while the ~-CD cavity is large enough to at least partially contain two pyrene molecules. 9,m

Table III contains pyrene lifetime data in the presence of the CDPs. For all three CDPs without additives, better matches to the observed fluorescence decays are obtained with a two-exponential fit in which the longer component contributes at least 95% of the total fluorescence signal. The most interesting feature of the data in Table III is the remarkable similarity of the lifetime results with the three CDPs, even in the presence of 0.015 M I - and 0.015 M I plus 1.0% (v/v) t-BuOH. This similarity is especially evident when contrasted with the pyrene/CD lifetime data in Table II, which must mean that the extent of exposure of bound pyrene to quenchers, such as O2 and I , does not vary much among the three CDPs. Moreover, this exposure is much greater than with 13- and ,y-CD, given the considerably shorter ~2 values listed in Table III than in Table II.

We also measured pyrene lifetimes in the presence of the three CDPs using a similar PTI LS-100 lifetime system but with photon counting detection. This system gave fluorescence decays which were better fit by three exponentials; however, a long-lived component (~230 ns) was the dominant ( f = 0.8) contributor for all three CDPs, which is not too different from what we report in Table III. The bot tom line is that all three CDPs still produce very similar pyrene lifetime data.

TABLE IV. Pyrene lifetimes with dialyzed [3-CDPs.

CDP" "r I (ns) % (ns) j'~

[3-CDPH 111 250 0.95 !3-CDPL 139 251 0.94

~' [[3-CDPH] and [13-CDPL] = 0.0020 M.

Table IV contains pyrene lifetime data for the dialyzed [3-CDPs. As observed for the I/III ratio data in Table I, the lifetime data are unaffected by dialysis. Thus, remov- al of the higher- or lower-molecular-weight [3-CDP components has no effect on the pyrene spectral data. This observat ion implies that pyrene must bind in a very similar fashion to all the CD polymer components, regardless of their size.

A Comparison of the Pyrene Emission Spectrum in the Presence of 3,-CD and 'y-CDP. The pyrene fluorescence spectra in the presence of ~-CD and ~-CDR shown in Fig. 5, also reveal a difference in the pyrene binding site for these two hosts. The spectra were obtained from solutions containing equal concentrations of pyrene and of "y-CD units (0.0080 M). While the pyrene spectrum in the presence of ~-CD has a significant excimer component about 485 nm, the pyrene spectrum in the presence of ~-CDP has no such component . Several workers have previously reported pyrene and pyrene sulfonate excimer emission in the presence of ~-CD from formation of both 2:1 pyrene/,y-CD and 2:2 pyrene/ 'y-CD complexes, with the former showing high stability. 8-~°,~7 Apparently, nei- ther of these complexes is formed in significant amounts when the ~-CD unit is part of the polymer chain.

100

0 0

0

0

75-

50-

25-

AJ

O - v

360 3;5 .;0 4~s 4;o d2 5;0 2;5 260

wavelength FIG. 5. Fluorescence emission spectrum of pyrene in the presence of 0.0080 M ~-CD (C)) and ~-CDP (+).

APPLIED SPECTROSCOPY 515

D I S C U S S I O N

The pyrene fluorescence data clearly show that much different pyrene binding environments exist for the CDPs than are observed for the CDs. The I /III emission ratios indicate that the pyrene binding sites for the three CDPs are more hydrophilic than those for the CDs. Moreover, lifetime data are consistent with a more exposed bound pyrene with the CDPs than that with the CDs. The similarity of the pyrene binding environments among the CDPs, as expressed by the I/III ratio and lifetime data, is also striking, especially when compared with the range of binding environments among the CDs.

The results with the dialyzed polymers in Tables I and IV indicate that pyrene has virtually the same binding interaction with all po lymer chains regardless of size. This would include the polymer fraction dominated by components having a molecular weight of --2000 and containing a single [3-CD unit. Consequently, the exis- tence of clam-shell structures for pyrene bound to [3-CDR where the two [3-CDs come from the same polymer chain, seems unlikely. Such intramolecular "double capping" probably has an entropy cost that is too high, since the long linker units would have to forfeit considerable mo- tional f reedom to form the clam shell structure. In fact, the formation of such structures by allowing the [3-CDs to come from different chains also is problematic, given the similarity and hydrophilicity of the three CDP binding environments. It does not seem probable that all three CDs would show the same tendency to form a double- capped structure when they are part of a polymer if this is not the case for the CD monomers . Finally, the lack of a significant effect of alcohols on the pyrene I /III ratio with [3-CDP is in sharp contrast to what Elliott et al. observe with the pyrene/[3-CD system, where clam shell binding is well established. TM

The accumulated spectral evidence is consistent with a more open binding environment for pyrene in the polymers, in which the pyrene has extensive contact with the linker chains. The hydroxyl groups on the glyceryl linkers, in addition to included water, may account for the rather hydrophilic nature of the polymer binding sites. The glyceryl linkers of the CDPs must play a prominent role in binding pyrene for the following reasons: First, such a role for the linkers can explain the commonal i ty of the binding sites among the three CDPs. Second, there is no indication, f rom I/III ratio and lifetime data (see Table II), of significant interaction between pyrene and e~-CD, but there is for interaction between pyrene and e~-CDP (see Table III). Presumably, the a - C D cavity is too small for pyrene, so that it must be the linkers that are largely responsible for the pyrene binding with c~-CDP. Finally, the lack of pyrene excimer emission for ~/-CDP, under conditions where it does occur with 3,-CD

(see Fig. 5), suggests that binding with the former involves portions of the polymer other than just the CD units.

In summary, the binding of pyrene to these CDPs, which have long linker chains between CD units, seems to be largely noninclusional. Werner and Warner have recently shown evidence that the glyceryl linker units have a major role in the binding of naphthalene probes to these CDPs, although the binding of these probes also shows some dependence on the identity of the CD unit. ~5 It should be noted that Harada et al. have reported the observat ion of a 2:1 [3-CDP/TNS complex, in which both CD units may come from the same chain? 8 Finally, Xu et al. have presented pyrene I/III ratio data suggesting that a 2:1 [3-CDP/pyrene complex, with clam shell ge- ometry, does result for ~-CD polymers having much shorter glyceryl linkers between [3-CD units than those for the [3-CDPs in this work? 2 It appears, then, that the type of binding that exists between a fluorophore and the CDPs (inclusional, noninclusional, or some hybrid of these two) depends on the ratio of linker units to CD units in the polymer, as well as to the identity of the fluorophore.

A C K N O W L E D G M E N T S

The authors would like to thank the American Maize Products Co. (Hammond, IN) for gifts of the cyclodextrin monomers. T.C.W. acknowledges the Petroleum Research Fund for partial support of this work. I.M.W. acknowledges the National Science Foundation (Grant #CHE-9224177) for partial support of this work.

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(1990). 6. A. Beeby and J. Sodeau, Photochem. Photobiol. 53, 335 (1990). 6. W. G. Herkstroeter, P. A. Martic, and S. Farid, J. Amer. Chem. Soc.

112, 3583 (1990). 7. G. Weber, H. G. Dreckamer, and P. M. Torgerson, Biochem. 18,

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binding of pyrene to cyclodextrin polymers

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